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What is the effect of host immune response to the viral delivery vector on the efficacy of vaccination? | {
"negative": [
"However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) .",
"Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.",
"2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) .",
"As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen.",
"However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) ."
],
"positive": [
"Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) ."
]
} |
When is a single sample suggested to be sufficient? | {
"negative": [
"Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] .",
"It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing.",
"Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] .",
"Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] .",
"A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV."
],
"positive": [
"Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR."
]
} |
What public event was linked with the outbreak? | {
"negative": [
"Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory (NCL) for Coxiellae and the Robert Koch-Institute (RKI), the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus.",
"The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 .",
"Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep.",
"In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year [10] . In 2001 and 2002, 293 and 191 cases were notified, respectively [11, 12] . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed.",
"The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature [1] . An additional interview with the two cases with early onset (2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" (see Table 3 ). Displayed are attack rates and 95% confidence intervals."
],
"positive": [
"The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases (source 5) were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature [1] ."
]
} |
What does the change in the local airway environment and inflammation promote? | {
"negative": [
"These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) .",
"Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) .",
"In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) .",
"These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses.",
"In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) ."
],
"positive": [
"The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses."
]
} |
How many people have come in contact and how many of these are in observation? | {
"negative": [
"Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19.",
"Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low.",
"Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread.",
"Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff.",
"Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building."
],
"positive": [
"There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19."
]
} |
What viruses were detected? | {
"negative": [
"The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010.",
"Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012.",
"According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season.",
"They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012.",
"During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis."
],
"positive": [
"In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter."
]
} |
What is the incubation period of MERS? | {
"negative": [
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] .",
"(See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] .",
"Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] .",
"6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] ."
],
"positive": [
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula."
]
} |
Can influenza a virus subtype h7n9 be transmit human to human? | {
"negative": [
"The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia.",
"In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days.",
"MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin).",
"Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig.",
"In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] ."
],
"positive": [
"Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] ."
]
} |
What are the 4 structural proteins of the pestivirus polyprotein? | {
"negative": [
"To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ). Cells infected with either the vR26/P-12 or the chimeric vR26_E2gif/P-12 were each detected by the polyclonal anti-pestivirus serum as expected. The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ). In contrast, the anti-BDV-E2 mAb specifically detected infection by the vR26_E2gif/P-12 and did not recognize cells infected with vR26/P-12. Each result is in accord with the structure of the viruses. The 4th passage of vR26 (vR26/P-4) displayed a slower growth rate than the virus obtained after 12 passages (see Figure 5A ).",
"Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC Anti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells. SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] . After 72 h, the cells were fixed and stained with monoclonal antibodies against the NS3 protein (WB103/105, left column), the CSFV E2 protein (WH303 and WH211, middle columns) and the BDV E2 protein (WB166, right column) as indicated and viewed using a fluorescence microscope. structure as determined by NotI digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown).",
"These differences are non-representative variants within the cDNA. Overall, the BAC sequence differed from the cDNA sequence in 18 positions, 9 of these lead to predicted amino acid substitutions within the polyprotein; one in each of N pro , E rns , E1, E2 and NS3 and four amino acid substitutions in NS5B (Table 1) . When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ). To determine the utility of the targeted recombinationmediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC Anti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells.",
"Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the \"Gifhorn\" E2 protein using staining with an anti-BDV mAb ( Figure 2 ). The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells. The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences.",
"Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat antimouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described [27] . The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells."
],
"positive": [
"BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] . Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] . The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years [14] ."
]
} |
What percentage of the world has been infected by tuberculosis? | {
"negative": [
"RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK (p38 MAPK and ERK1/2) and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis (TB) remains a major cause of morbidity and mortality in the world, especially in the developing countries [1] .",
"BACKGROUND: Mycobacterium tuberculosis (MTB) is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α (TNF-α), which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin (BCG), a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase (MAPK) in human blood monocytes. Since MAPK phosphatase-1 (MKP-1) is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.",
"In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity [2] . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) have been implicated as important cellular signaling molecules activated by mycobacteria [3] . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv [4] . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes (PBMo) leading to cytokine expressions via the interaction with PKR [5] .",
"The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit (Pierce, IL, USA). Western blot was done as described [20] . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes (Schleicher & Schuell), and followed by probing with specific antibod-ies for Actin, MKP-1 (Santa Cruz Biotech., USA), phospho-p38 MAPK, phospho-ERK1/2 (Cell Signaling, USA). After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System (Amersham Pharmacia Biotech) as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described [21] .",
"The C T value of the treated cells was compared with that of the untreated or mock-treated cells (ΔΔCT). The relative gene expression of the targeted genes (fold induction) was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit (Pierce, IL, USA). Western blot was done as described [20] ."
],
"positive": [
"CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis (TB) remains a major cause of morbidity and mortality in the world, especially in the developing countries [1] . The disease is caused by Mycobacterium tuberculosis (MTB) and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization (WHO) estimated that there are 9.2 million new TB cases around the world in 2006 [1] . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity [2] ."
]
} |
What is theorized regarding transmission? | {
"negative": [
"Assembly occurs at the cell surface, and the envelope is acquired as the virus buds from the cell and release and maturation almost simultaneous occurred. Replication occurs in the cytoplasm and is very rapid (,4 h) [28, 29] . doi:10.1371/journal.pntd.0000623.g002 www.plosntds.org to gain a more accurate understanding of a potential CHIKV epidemic in the US [32] . During the 7 d preceding birth, no human mother has been reported to transmit the disease vertically. However, about 50% of newborns delivered while the mother was infected with CHIKV contracted the disease from their mother, despite the method of delivery. Furthermore, there have been instances of CHIKV transmission from mother to fetus causing congenital illness and fetal death [33] .",
"aegypti and Ae. albopictus [11] . Transmission in Asia occurs in an urban cycle whereby the mosquito spreads the disease from an infected human to an uninfected human, following an epidemiological pattern similar to dengue fever [12] . The 2005-2006 epidemic of CHIKV in La Reunion islands in the Indian Ocean, spurred the discovery of a new vector species, Ae. albopictus [5] . Wrecking over one-third of the island's population, this epidemic peaked its devastation between January and February 2006, when over 46,000 cases came into light every week, including 284 deaths [5, 13] . Ae.",
"Because of its promising results and the need for a safer vaccine, this consensus DNA vaccine deserves further investigation. Determining longevity of protective effects of the vaccine and persistence of antibody and IFN-c responses could be the next step of investigation. Challenged studies of immunized mice must also be carried out. CHIKV mosquito-borne disease has caused massive outbreaks for at least half a century but is no longer confined to the www.plosntds.org developing nations. It began to encroach into the boundaries of the developing world. As a result, the NIAID has designated CHIKV as a Category C pathogen alongside the influenza and SARS-CoV viruses [3] .",
"With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts. Text: Chikungunya virus (CHIKV), a mosquito-borne pathogen listed by National Institute of Allergy and Infectious Diseases (NIAID) as a Category C Priority Pathogen that causes Chikungunya fever (CHIKF), has been spreading throughout Asia, Africa, and parts of Europe in recent times [1, 2, 3] . CHIKV is an arthropod-borne virus (arbovirus) and is transmitted to humans primarily by Aedes aegypti, the infamous yellow fever propagator [4, 5] . CHIKV infection is marked by severe joint pain, contorting its victims into unusual postures [6] . The disease gets its name from the Kimakonde vernacular language of Tanzania and Mozambique, and the word chikungunya means ''that which contorts or bends up'' and translates in Swahili to ''the illness of the bended walker'' [7, 8, 9] .",
"Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005–07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts."
],
"positive": [
"By mean Day 4 of life, all of the neonates were symptomatic for CHIKV, exhibiting common CHIKF symptoms. Six neonates were confirmed to have contracted CHIKV and developed mengoencephalitis. Of those mothers who, during the La Reunion Island epidemic, were infected long before delivery, only three fetal deaths were reported [12, 33] . Ramful et al. theorized that motherto-child transmission most likely happens transplacentally shortly before delivery [33] . A similar study by Gerardin et al. reported nineteen cases of neonatal infection associated with intrapartum maternal viremia that progressed to develop encephalitis owing to vertical transmission from infected mothers [34] ."
]
} |
When does the influenza vaccination campaign in Reunion Island start? | {
"negative": [
"Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] .",
"In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter.",
"They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012.",
"After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] .",
"Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season."
],
"positive": [
"Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology."
]
} |
What happens when a recipient of a vaccine has immune response to the delivery vector? | {
"negative": [
"This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) .",
"2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) .",
"However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) .",
"Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) .",
"Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review."
],
"positive": [
"As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen."
]
} |
What were the asymptomatic cases tested as? | {
"negative": [
"Additional symptoms reported included headaches (6 cases), sore throat (2), rhinorrhoea (2), shortness of breath (2), myalgia (1), diarrhoea (1) and nausea (1). Fever was reported as the sole symptom for nine cases. In 16 of 29 symptomatic cases, the symptoms at diagnosis were consistent with the case definition for acute respiratory infection [16] , although it is possible that cases presented additional symptoms after diagnosis and these were not reported. Data on pre-existing conditions were reported for seven cases; five had no pre-existing conditions while one was reported to be obese and one had pre-existing cardiac disease. No data on clinical signs e.g. dyspnea etc.",
"The situation has evolved rapidly since then, however, and the number of countries reporting COVID-19 transmission increased rapidly, notably with a large outbreak in northern Italy with 3,089 cases reported as at 5 March [18, 19] . Testing of suspected cases based on geographical risk of importation needs to be complemented with additional approaches to ensure early detection of local circulation of COVID-19, including through testing of severe acute respiratory infections in hospitals irrespectively of travel history as recommended in the WHO case definition updated on 27 February 2020 [21] . The clinical presentation observed in the cases in Europe is that of an acute respiratory infection. However, of the 31 cases with information on symptoms, 20 cases presented with fever and nine cases presented only with fever and no other symptoms. These findings, which are consistent with other published case series, have prompted ECDC to include fever among several clinical signs or symptoms indicative for the suspected case definition. Three cases were aged 65 years or over.",
"The duration of hospitalisation was reported for 16 cases with a median of 13 days (range: 8-23 days). As at 21 February 2020, four cases were still hospitalised. All cases were confirmed according to specific assays targeting at least two separate genes (envelope (E) gene as a screening test and RNA-dependent RNA polymerase (RdRp) gene or nucleoprotein (N) gene for confirmation) [8, 17] . The specimen types tested were reported for 27 cases: 15 had positive nasopharyngeal swabs, nine had positive throat swabs, three cases had positive sputum, two had a positive nasal swab, one case had a positive nasopharyngeal aspirate and one a positive endotracheal aspirate. As at 09:00 on 21 February, few COVID-19 cases had been detected in Europe compared with Asia. However the situation is rapidly developing, with a large outbreak recently identified in northern Italy, with transmission in several municipalities and at least two deaths [18] .",
"Despite this, the finding of older individuals being at higher risk of a severe clinical course is consistent with the evidence from Chinese case series published so far although the majority of infections in China have been mild [22, 23] . This preliminary analysis is based on the first reported cases of COVID-19 cases in the WHO European Region. Given the small sample size, and limited completeness for some variables, all the results presented should be interpreted with caution. With increasing numbers of cases in Europe, data from surveillance and investigations in the region can build on the evidence from countries in Asia experiencing more widespread transmission particularly on disease spectrum and the proportion of infections with severe outcome [22] . Understanding the infection-severity is critical to help plan for the impact on the healthcare system and the wider population. Serological studies are vital to understand the proportion of cases who are asymptomatic.",
"These findings, which are consistent with other published case series, have prompted ECDC to include fever among several clinical signs or symptoms indicative for the suspected case definition. Three cases were aged 65 years or over. All required admission to intensive care and were tourists (imported cases). These findings could reflect the average older age of the tourist population compared with the local contacts exposed to infection in Europe and do not allow us to draw any conclusion on the proportion of severe cases that we could expect in the general population of Europe. Despite this, the finding of older individuals being at higher risk of a severe clinical course is consistent with the evidence from Chinese case series published so far although the majority of infections in China have been mild [22, 23] . This preliminary analysis is based on the first reported cases of COVID-19 cases in the WHO European Region."
],
"positive": [
"Symptoms at the point of diagnosis were reported for 31 cases. Two cases were asymptomatic and remained so until tested negative. The asymptomatic cases were tested as part of screening following repatriation and during contact tracing respectively. Of the remaining 29, 20 reported fever, 14 reported cough and eight reported weakness. Additional symptoms reported included headaches (6 cases), sore throat (2), rhinorrhoea (2), shortness of breath (2), myalgia (1), diarrhoea (1) and nausea (1). Fever was reported as the sole symptom for nine cases."
]
} |
What does the study show? | {
"negative": [
"It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time.",
"A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI.",
"According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season.",
"In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] .",
"It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI)."
],
"positive": [
"Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] ."
]
} |
At what temperatures was the assay completed? | {
"negative": [
"The mixtures of bacteria and compounds were serially diluted 10-fold with SPB and plated on LB plates that were incubated at 37uC overnight. Bacterial colonies were enumerated the following day. After having determined the MICs, bacterial strains from the wells of the microtitre plate with no visible bacterial growth were removed for serial sub cultivation of 2 ml into another new microtitre plate containing 100 ml of broth per well and further incubated for 24 h. The lowest concentration with no visible growth was defined as MBC [28], indicating 99.5% killing of the original inoculum. The absorbance of each well was measured at a wavelength of 620 nm by Microplate Manager 4.0 (Bio-Rad laboratories) and compared with a blank. Solvent (DMSO) was used as a negative control. Three replicates were done for each compound and experiment was repeated two times.",
"DMSO was taken as negative control and Ceftriaxone and clotrimazole were taken as positive control. After incubation at 37uC overnight, the MICs were taken as the lowest inhibitor concentration at which the bacterial growth was inhibited. The average of three values was calculated and that was the MIC for the test material and bacterial strain. For the agar plate count method [27] , 25 mL aliquots of bacteria at 1610 5 CFU/ml in SPB containing 0.03% LB broth were incubated with 25 mL of diluted compounds for 2 h at 37uC. The mixtures of bacteria and compounds were serially diluted 10-fold with SPB and plated on LB plates that were incubated at 37uC overnight. Bacterial colonies were enumerated the following day.",
"The same procedure was used at 32uC and 42uC for thermodynamics studies. The kinetic parameters for the substrate hydrolysis were determined by measuring the initial rate of enzymatic activity. The inhibition constant K i was determined by Dixon method [24] and also by the Lineweaver-Burk equation. The K m value was calculated from the double-reciprocal equation by fitting the data into the computer software Origin 6.1. The Lineweaver-Burk plot was used to determine the types of inhibition. For the kinetic analysis and rate constant determinations, the assays were carried out in triplicate, and the average value was considered throughout this work. Temperature dependence of the inhibition constants was used to determine the thermodynamic parameters.",
"Based on the results of docking studies, ten derivatives of 1pyridylimidazo[1,5-a]pyridine were synthesized according to Siddiqui et al., 2006 [22] which are named as follows: 1- The capacity of the 1-pyridylimidazo[1,5-a]pyridine derivatives to inhibit cysteine proteases was tested using papain as the model enzyme. The proteolytic activity of the reaction mixtures was determined using Bz-DL-Arg-pNA as the chromogenic substrate [23] . To solutions of active papain (final concentration: 0.05 mM) were added concentrated solutions of the different derivatives to final concentrations of 0.2 mM. After incubation for 30 min at 37uC, the substrate solution was added and after a further incubation for 20 min the reaction was stopped by the addition of 5% trichloric acid (TCA) acidified with 2.25% HCl and the absorbance of the reaction mixture was determined at a wavelength of 410 nm by Microplate Manager 4.0 (Bio-Rad laboratories). The same procedure was used at 32uC and 42uC for thermodynamics studies. The kinetic parameters for the substrate hydrolysis were determined by measuring the initial rate of enzymatic activity.",
"The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] . In brief, the bacterial strains (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii)) were grown and diluted to 2610 5 colony-forming units (CFU)/ml in sodium phosphate buffer (SPB) containing 0.03% Luria-Bertani (LB) broth. The synthesized derivatives were dissolved in DMSO and their serial dilution was performed in 50 mL of LB medium in 96-well microtitre plate to achieve the required concentrations (0.1-10 mg/ml) with bacterial inoculums (5610 4 CFU per well). DMSO was taken as negative control and Ceftriaxone and clotrimazole were taken as positive control. After incubation at 37uC overnight, the MICs were taken as the lowest inhibitor concentration at which the bacterial growth was inhibited."
],
"positive": [
"For the kinetic analysis and rate constant determinations, the assays were carried out in triplicate, and the average value was considered throughout this work. Temperature dependence of the inhibition constants was used to determine the thermodynamic parameters. Changes in enthalpy (DH) were determined from the Van't Hoff plots by using the equation, Where DH is enthalpy change, R is gas constant, DS is entropy change and T is the absolute temperature. The entropy change was obtained from the equation, The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme. The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] ."
]
} |
What is sporulation? | {
"negative": [
"Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct (putative) targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Text: Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds.",
"Consistent with a model in which spo0A is positively autoregulated, we noted a sharp increase in the levels of Spo0A as cells approach the stationary growth phase ( Figure 1C) . Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA (Fig. 3B ). All these observations are consistent with direct regulation of these genes by Spo0A in other organisms [5, 45, 49, 62] , and the conservation of the sporulation pathway [2] . Though Spo0A is the key regulator for sporulation in Firmicutes, it regulates numerous other processes in various bacteria. In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology [10, [14] [15] [16] .",
"It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis [14] [15] [16] , virulence factors and stress responses in for instance B. anthracis and B. thuringiensis [17] [18] [19] [20] [21] , and solvent production in Clostridium acetobutylicum [22, 23] . C. difficile is a Gram positive, anaerobic bacterium that is the causative agent of C. difficile infection (CDI) (for recent reviews see [24, 25] ). Though many people are asymptomatically colonized by C. difficile, the bacterium can cause serious health problems, such as pseudomembranous colitis and toxic megacolon, under the influence of risk factors such as age and antibiotic use. As a result, CDI was long regarded a nosocomial infection. Recently, however, an increase in the cases of community acquired CDI can be observed [26] . Outbreaks of CDI have been linked to so called hypervirulent strains, such as PCR ribotypes 027 (BI/ NAP1) and 078 [27, 28] .",
"C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow [24, 25] . There is limited knowledge about the regulation of sporulation in C. difficile. It has been reported that spo0A, as expected, is required for the formation of spores [33] and the gene is required for persistence and transmission in mice [34] . Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A [2] . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A [35] .",
"Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection (CDI), for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630."
],
"positive": [
"These results identify for the first time direct (putative) targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Text: Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds. Spo0A is the key regulator for sporulation [1, 2] . Most of our knowledge about the protein is based on work in Bacilli. Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA [3] [4] [5] . Phosphorylation occurs through the concerted action of several proteins that together form a so called phosphorelay [6] ."
]
} |
What are some risk factors for countries to experience a high prevalence of Acute Respiratory Infections? | {
"negative": [
"This pilot study provides evidence that data from the community can potentially represent different information than that sourced clinically, suggesting the need for community surveillance to enhance public health efforts and scientific understanding of respiratory infections. Text: Acute Respiratory Infections (ARIs) (the cause of both upper respiratory tract infections (URIs) and lower respiratory tract infections (LRIs)) are a major cause of death among children under 5 years old particularly in developing countries where the burden of disease is 2-5 times higher than in developed countries [1] . While these viruses usually cause mild cold-like symptoms and can be self-limiting, in recent years novel coronaviruses such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) have evolved and infected humans, causing severe illness, epidemics and pandemics [2] . Currently, the majority of all infectious disease outbreaks as recorded by the World Health Organization (WHO) occur in the continent of Africa where there is high transmission risk [3, 4] . Further, in developing areas (both rural and urban), there are increasing risk factors such as human-animal interfaces (due to residential-proximity to livestock). These changing epidemiological patterns have resulted in calls for improved ARI surveillance, especially in places of high transmission risk [5] .",
"A total of 33 of the 91 subjects had one or more respiratory tract virus; there were 10 cases of triple infection and 5 of quadruple. Parainfluenza virus 4, respiratory syncytial virus B and enterovirus were the most common viruses in the clinical sample; present in 93.8% (15/16) of clinical subjects, and 6.7% (5/75) of community subjects (significant difference, p < 0.001). Coronavirus OC43 was the most common virus detected in community members (13.3%, 10/75). A different strain, Coronavirus OC 229 E/NL63 was detected among subjects from the clinic (2/16) and not detected in the community. This pilot study provides evidence that data from the community can potentially represent different information than that sourced clinically, suggesting the need for community surveillance to enhance public health efforts and scientific understanding of respiratory infections. Text: Acute Respiratory Infections (ARIs) (the cause of both upper respiratory tract infections (URIs) and lower respiratory tract infections (LRIs)) are a major cause of death among children under 5 years old particularly in developing countries where the burden of disease is 2-5 times higher than in developed countries [1] .",
"For ARI surveillance in particular, infections in the community are those that do not get reported clinically. Clinical data generally represents the most severe cases, and those from locations with access to healthcare institutions. In Nigeria, hospitals are visited only when symptoms are very severe. Thus, it is hypothesized that viral information from clinical sampling is insufficient to either capture disease incidence in general populations or its predictability from symptoms [8] . Efforts worldwide including in East and Southern Africa have been focused on developing community-based participatory disease surveillance methods [9] [10] [11] [12] [13] . Community-based approaches have been shown useful for learning more about emerging respiratory infections such as assessing under-reporting [14] , types of viruses prevalent in communities [10] , and prediction of epidemics [15] .",
"No observations used in this study had any missing data for analyses in this study. Basic participant demographics are summarized in PCR results showed that ten different viruses (influenza A, coronavirus OC 229 E/NL63, RSVA, RSV B, parainfluenza 1-4) were detected. Figure 1 shows how these infections were distributed across virus types as well as in the community versus clinic samples. In sum, a total of 33 of the 91 subjects surveyed had one or more respiratory tract virus (36.3%, 95% CI 26.6-47.0%, Fig. 1 ). Furthermore, 10 of those cases were triple infections and 5 were quadruple infections (illustrated by color of bars in Fig.",
"OBJECTIVE: Recognizing increasing interest in community disease surveillance globally, the goal of this study was to investigate whether respiratory viruses circulating in the community may be represented through clinical (hospital) surveillance in Nigeria. RESULTS: Children were selected via convenience sampling from communities and a tertiary care center (n = 91) during spring 2017 in Ilorin, Nigeria. Nasal swabs were collected and tested using polymerase chain reaction. The majority (79.1%) of subjects were under 6 years old, of whom 46 were infected (63.9%). A total of 33 of the 91 subjects had one or more respiratory tract virus; there were 10 cases of triple infection and 5 of quadruple. Parainfluenza virus 4, respiratory syncytial virus B and enterovirus were the most common viruses in the clinical sample; present in 93.8% (15/16) of clinical subjects, and 6.7% (5/75) of community subjects (significant difference, p < 0.001)."
],
"positive": [
"Further, in developing areas (both rural and urban), there are increasing risk factors such as human-animal interfaces (due to residential-proximity to livestock). These changing epidemiological patterns have resulted in calls for improved ARI surveillance, especially in places of high transmission risk [5] . Nigeria is one such place with high prevalence of many of the risk factors implicated in ARI among children including; age, sex, overcrowding, nutritional status, socio-economic status, and where study of ARIs is currently limited [6] . These broad risk factors alongside limited resources have indicated the need for community-based initiatives for surveillance and interventions [6, 7] . For ARI surveillance in particular, infections in the community are those that do not get reported clinically. Clinical data generally represents the most severe cases, and those from locations with access to healthcare institutions."
]
} |
How was the ELISA-array assay validated? | {
"negative": [
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] .",
"The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] .",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV.",
"Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation."
],
"positive": [
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures."
]
} |
What parameter is used to measure antioxidant levels? | {
"negative": [
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] .",
"The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France).",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77).",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] .",
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05)."
],
"positive": [
"[16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion."
]
} |
How do the authors suggest that ATP1A1 and PHB contribute to the efficient multiplication of mammarenaviruses? | {
"negative": [
"These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody (mAb) to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP [45] . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al.",
"eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system (Fig 5B and S2 Fig) as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM (Fig 8) . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication (S3 Fig). These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses.",
"Here we document the generation of a recombinant LCMV encoding a nucleoprotein (NP) containing an affinity tag (rLCMV/Strep-NP) and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na(+)/K(+) transporting subunit alpha 1 (ATP1A1) and prohibitin (PHB) as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.",
"We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. Text: Introduction Mammarenaviruses (Arenaviridae: Mammarenavirus) cause chronic infections of rodents worldwide [1] . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers (VHFs) in humans and pose important public health problems in their endemic areas [2] [3] [4] [5] [6] . Mammarenaviruses are classified into two main groups, Old World (OW) and New World (NW) [1] .",
"Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication (S5 Fig). The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 (American Type Culture Collection, ATCC, CCL-10), house mouse L929 (ATCC CCL-1), grivet Vero E6 (ATCC CRL-1586), human A549 (ATCC CCL-185), and human HEK 293T (ATCC CRL-3216) cells were grown in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Waltham, MA) containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong (rLCMV ARM) and clone-13 (rLCMV Cl-13) strains, were generated as described [30, 82, 83] ."
],
"positive": [
"These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication (S5 Fig). The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins."
]
} |
What did, In the last century, outbreaks of viral-related diseases that have emerged or re-emerged, involve? | {
"negative": [
"Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] .",
"The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.",
"Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] .",
"In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .",
"A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] ."
],
"positive": [
"During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] ."
]
} |
What is Tamiflu? | {
"negative": [
"Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] . Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] .",
"So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] . The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] .",
"Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] . The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] .",
"Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet.",
"In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics."
],
"positive": [
"Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents. Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light. MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 ."
]
} |
What is a different approach for the detection? | {
"negative": [
"Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies.",
"The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option.",
"[47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection.",
"IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] .",
"Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] ."
],
"positive": [
"A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV."
]
} |
What are the ILI samples wich test negative for influence? | {
"negative": [
"We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory.",
"A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations.",
"Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens.",
"Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season.",
"Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex."
],
"positive": [
"Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012."
]
} |
What is the estimated infection-to-death distribution's mean for coronavirus? | {
"negative": [
"This age-group was chosen as the reference as it had the lowest predicted level of underreporting in previous analyses of data from the Chinese epidemic“. We obtained country-specific estimates of attack rate by age, AR“, for the 11 European countries in our analysis from a previous study which incorporates information on contact between individuals of different ages in countries across Europe.12 We then obtained overall ifr estimates for each country adjusting for both demography and age-specific attack rates. Using estimated epidemiological information from previous studies,“'11 we assume TE to be the sum of two independent random times: the incubation period (infection to onset of symptoms or infection- to-onset) distribution and the time between onset of symptoms and death (onset-to-death). The infection-to-onset distribution is Gamma distributed with mean 5.1 days and coefficient of variation 0.86. The onset-to-death distribution is also Gamma distributed with a mean of 18.8 days and a coefficient of va riation 0.45. ifrm is population averaged over the age structure of a given country. The infection-to-death distribution is therefore given by: um ~ ifrm ~ (Gamma(5.1,0.86) + Gamma(18.8,0.45)) Figure 6 shows the infection-to-death distribution and the resulting survival function that integrates to the infection fatality ratio.",
"Our approach is semi-mechanistic. We propose a plausible structure for the infection process and then estimate parameters empirically. However, many parameters had to be given strong prior distributions or had to be fixed. For these assumptions, we have provided relevant citations to previous studies. As more data become available and better estimates arise, we will update these in weekly reports. Our choice of serial interval distribution strongly influences the prior distribution for starting R0. Our infection fatality ratio, and infection-to-onset-to-death distributions strongly influence the rate of death and hence the estimated number of true underlying cases.",
"Using the probability of death distribution, the expected number of deaths dam, on a given day t, for country, m, is given by the following discrete sum: The number of deaths today is the sum of the past infections weighted by their probability of death, where the probability of death depends on the number of days since infection. 8.2 Infection model The true number of infected individuals, C, is modelled using a discrete renewal process. This approach has been used in numerous previous studies13'16 and has a strong theoretical basis in stochastic individual-based counting processes such as Hawkes process and the Bellman-Harris process.”18 The renewal model is related to the Susceptible-Infected-Recovered model, except the renewal is not expressed in differential form. To model the number ofinfections over time we need to specify a serial interval distribution g with density g(T), (the time between when a person gets infected and when they subsequently infect another other people), which we choose to be Gamma distributed: g ~ Gamma (6.50.62). The serial interval distribution is shown below in Figure 7 and is assumed to be the same for all countries. Figure 7: Serial interval distribution g with a mean of 6.5 days.",
"To mechanistically link ourfunction for deaths to infected cases, we use a previously estimated COVID- 19 infection-fatality-ratio ifr (probability of death given infection)9 together with a distribution oftimes from infection to death TE. The ifr is derived from estimates presented in Verity et al11 which assumed homogeneous attack rates across age-groups. To better match estimates of attack rates by age generated using more detailed information on country and age-specific mixing patterns, we scale these estimates (the unadjusted ifr, referred to here as ifr’) in the following way as in previous work.4 Let Ca be the number of infections generated in age-group a, Na the underlying size of the population in that age group and AR“ 2 Ca/Na the age-group-specific attack rate. The adjusted ifr is then given by: ifra = fififié, where AR50_59 is the predicted attack-rate in the 50-59 year age-group after incorporating country-specific patterns of contact and mixing. This age-group was chosen as the reference as it had the lowest predicted level of underreporting in previous analyses of data from the Chinese epidemic“. We obtained country-specific estimates of attack rate by age, AR“, for the 11 European countries in our analysis from a previous study which incorporates information on contact between individuals of different ages in countries across Europe.12 We then obtained overall ifr estimates for each country adjusting for both demography and age-specific attack rates.",
"Spain has recently seen a large increase in the number of deaths, and given its smaller population, our model estimates that a higher proportion of the population, 15.0% (7.0 [18-19] million people) have been infected to date. Germany is estimated to have one of the lowest attack rates at 0.7% with 600,000 [240,000-1,500,000] people infected. Imperial College COVID-19 Response Team Table l: Posterior model estimates of percentage of total population infected as of 28th March 2020. Country % of total population infected (mean [95% credible intervall) Austria 1.1% [0.36%-3.1%] Belgium 3.7% [1.3%-9.7%] Denmark 1.1% [0.40%-3.1%] France 3.0% [1.1%-7.4%] Germany 0.72% [0.28%-1.8%] Italy 9.8% [3.2%-26%] Norway 0.41% [0.09%-1.2%] Spain 15% [3.7%-41%] Sweden 3.1% [0.85%-8.4%] Switzerland 3.2% [1.3%-7.6%] United Kingdom 2.7% [1.2%-5.4%] 2.2 Reproduction numbers and impact of interventions Averaged across all countries, we estimate initial reproduction numbers of around 3.87 [3.01-4.66], which is in line with other estimates.1'8 These estimates are informed by our choice of serial interval distribution and the initial growth rate of observed deaths. A shorter assumed serial interval results in lower starting reproduction numbers (Appendix 8.4.2, Appendix 8.4.6). The initial reproduction numbers are also uncertain due to (a) importation being the dominant source of new infections early in the epidemic, rather than local transmission (b) possible under-ascertainment in deaths particularly before testing became widespread."
],
"positive": [
"The onset-to-death distribution is also Gamma distributed with a mean of 18.8 days and a coefficient of va riation 0.45. ifrm is population averaged over the age structure of a given country. The infection-to-death distribution is therefore given by: um ~ ifrm ~ (Gamma(5.1,0.86) + Gamma(18.8,0.45)) Figure 6 shows the infection-to-death distribution and the resulting survival function that integrates to the infection fatality ratio. Figure 6: Left, infection-to-death distribution (mean 23.9 days). Right, survival probability of infected individuals per day given the infection fatality ratio (1%) and the infection-to-death distribution on the left. Using the probability of death distribution, the expected number of deaths dam, on a given day t, for country, m, is given by the following discrete sum: The number of deaths today is the sum of the past infections weighted by their probability of death, where the probability of death depends on the number of days since infection. 8.2 Infection model The true number of infected individuals, C, is modelled using a discrete renewal process."
]
} |
What is PPE? | {
"negative": [
"Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness [would have been] needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said: What bothered most of us was how uncomfortable the PPE was and I think that made people nervous: \"How will I manage working in this for hours?\"",
"Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges [35] . Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere [43] . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal.",
"There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness [would have been] needed in the PPE practices'.",
"They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD [31] . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you.",
"Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said: What bothered most of us was how uncomfortable the PPE was and I think that made people nervous: \"How will I manage working in this for hours?\" Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly."
],
"positive": [
"Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland [16, 27] ."
]
} |
How many clinical trials are registered? | {
"negative": [
"In addition, we highlight six ongoing Phase I clinical trials identified in the ClinicalTrials.gov register ( [48, 49] ); Table S4 ) [50] [51] [52] . These trials are all testing the safety and immunogenicity of their respective MERS-CoV vaccine candidates but were excluded as there are no results published yet. The trials are projected to complete in December 2020 (two studies in Russia [50, 51] ) and December 2021 (in Germany [52] ). Existing literature search did not return any results on completed 2019-nCoV trials at the time of writing. Among 23 trials found from the systematic review (Table 5) , there are nine clinical trials registered under the clinical trials registry (ClinicalTrials.gov) for 2019-nCoV therapeutics [53] [54] [55] [56] [57] [58] [59] [60] [61] . Of which five studies on hydroxychloroquine, lopinavir plus ritonavir and arbidol, mesenchymal stem cells, traditional Chinese medicine and glucocorticoid therapy usage have commenced recruitment.",
"While many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir or ribavirin only, there has yet to be well-designed clinical trials investigating their usage. Three completed randomized controlled trials were conducted during the SARS epidemic-3 in China, 1 in Taiwan and 2 in Hong Kong [93] [94] [95] [96] [97] . The studies respectively investigated antibiotic usage involving 190 participants, combination of western and Chinese treatment vs. Chinese treatment in 123 participants, integrative Chinese and Western treatment in 49 patients, usage of a specific Chinese medicine in four participants and early use of corticosteroid in 16 participants. Another notable study was an open non-randomized study investigating ribavirin/lopinavir/ritonavir usage in 152 participants [98] . One randomized controlled trial investigating integrative western and Chinese treatment during the SARS epidemic was excluded as it was a Chinese article [94] . There is only one ongoing randomized controlled trial targeted at MERS therapeutics [99] .",
"The discovered drug targets are said to be more than 95% similar to enzyme targets found on the SARS virus. Researchers note that identified drugs may not be available to address the ongoing outbreak but they hope to make it accessible for future outbreaks. [85] Besides the six completed randomized controlled trials (RCT) selected from the systematic review (Table 6) , there is only one ongoing randomized controlled trial targeted at SARS therapeutics [92] . The studies found from ClinicalTrials.gov have not been updated since 2013. While many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir or ribavirin only, there has yet to be well-designed clinical trials investigating their usage. Three completed randomized controlled trials were conducted during the SARS epidemic-3 in China, 1 in Taiwan and 2 in Hong Kong [93] [94] [95] [96] [97] .",
"It will likely take about a year for most candidates to start phase 1 clinical trials except for those funded by Coalition for Epidemic Preparedness Innovations (CEPI). However, the kit developed by the BGI have passed emergency approval procedure of the National Medical Products Administration, and are currently used in clinical and surveillance centers of China [40] . Of the total of 570 unique studies on 2019-nCoV, SARS CoV or MERS-CoV vaccines screened, only four were eventually included in the review. Most studies on SARS and MERS vaccines were excluded as they were performed in cell or animal models ( Figure 1 ). The four studies included in this review were Phase I clinical trials on SARS or MERS vaccines (Table 4 ) [44] [45] [46] [47] . There were no studies of any population type (cell, animal, human) on the 2019-nCoV at the point of screening.",
"One randomized controlled trial investigating integrative western and Chinese treatment during the SARS epidemic was excluded as it was a Chinese article [94] . There is only one ongoing randomized controlled trial targeted at MERS therapeutics [99] . It investigates the usage of Lopinavir/Ritonavir and Interferon Beta 1B. Likewise, many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir/ribavirin, interferon, and convalescent plasma usage. To date, only one trial has been completed. One phase 1 clinical trial investigating the safety and tolerability of a fully human polyclonal IgG immunoglobulin (SAB-301) was found in available literature [46] ."
],
"positive": [
"Among 23 trials found from the systematic review (Table 5) , there are nine clinical trials registered under the clinical trials registry (ClinicalTrials.gov) for 2019-nCoV therapeutics [53] [54] [55] [56] [57] [58] [59] [60] [61] . Of which five studies on hydroxychloroquine, lopinavir plus ritonavir and arbidol, mesenchymal stem cells, traditional Chinese medicine and glucocorticoid therapy usage have commenced recruitment. The remaining four studies encompass investigation of antivirals, interferon atomization, darunavir and cobicistat, arbidol, and remdesivir usage for 2019-nCoV patients (Table 5) . Seroconversion measured by S1-ELISA occurred in 86% and 94% participants after 2 and 3 doses, respectively, and was maintained in 79% participants up to study end at week 60. Neutralising antibodies were detected in 50% participants at one or more time points during the study, but only 3% maintained neutralisation activity to end of study. T-cell responses were detected in 71% and 76% participants after 2 and 3 doses, respectively."
]
} |
What percentage of people infected with SARS-CoV died? | {
"negative": [
"It is unclear if these differences are due to distinct populations affected by the viruses, because of properties of the virus themselves, or both. Understanding these factors could be a key to increasing host resilience to the infections. MERS-CoV patients had increased morbidity and mortality if they were obese, immunocompromised, diabetic or had cardiac disease [4, 12] . REviEW Jamieson future science group Risk factors for SARS-CoV patients included an older age and male [39] . Immune factors that increased mortality for SARS-CoV were a higher neutrophil count and low T-cell counts [5, 39, 77] . One factor that increased disease for patients infected with SARS-CoV and MERS-CoV was infection with other viruses or bacteria [5, 39] .",
"Recently, other CoVs have been detected in animal populations raising the possibility that we will see a repeat of these types of outbreaks in the near future [11, [16] [17] [18] [19] [20] . Both these zoonotic viruses cause a much more severe disease than what is typically seen for CoVs, making them a global health concern. Both SARS-CoV and MERS-CoV result in severe lung pathology. Many infected patients have acute lung injury (ALI), a condition that is diagnosed based on the presence of pulmonary edema and respiratory failure without a cardiac cause. In some patients there is a progression to the more severe form of ALI, acute respiratory distress syndrome (ARDS) [21] [22] [23] . In order to survive a given infection, a successful host must not only be able to clear the pathogen, but tolerate damage caused by the pathogen itself and also by the host's immune response [24] [25] [26] .",
"Immune factors that increased mortality for SARS-CoV were a higher neutrophil count and low T-cell counts [5, 39, 77] . One factor that increased disease for patients infected with SARS-CoV and MERS-CoV was infection with other viruses or bacteria [5, 39] . This is similar to what is seen with many other respiratory infections. A recent study looking at malaria infections in animal models and human patients demonstrated that resilient hosts can be predicted [28] . Clinical studies have started to correlate specific biomarkers with disease outcomes in ARDS patients [80] . By understanding risk factors for disease severity we can perhaps predict if a host may be nonresilient and tailor the treatment options appropriately.",
"These were often used in combination with IFN therapy [54] . However, retrospective analysis of these data has not led to clear conclusions of the efficacy of these treatment options. Research in this area is still ongoing and it is hoped that we will soon have effective strategies to treat novel CoV [3,36,38,40, [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] . The lack of effective antivirals makes it necessary to examine other potential treatments for SARS-CoV and MERS-CoV. Even if there were effective strategies to decrease viral burden, for these viruses, the potential for new emerging zoonotic CoVs presents additional complications. Vaccines cannot be produced in time to stop the spread of an emerging virus.",
"This review focuses on exploring various host resilience mechanisms that could be exploited for treatment of severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and other respiratory viruses that cause acute lung injury and acute respiratory distress syndrome. Text: The 21st century was heralded with the emergence of two novel coronaviruses (CoV) that have unusually high pathogenicity and mortality [1] [2] [3] [4] [5] . Severe acute respiratory syndrome coronavirus (SARS-Cov) was first identified in 2003 [6] [7] [8] [9] . While there was initially great concern about SARS-CoV, once no new cases emerged, funding and research decreased. However, a decade later Middle East respiratory syndrome coronavirus (MERS-CoV), also known as HCoV-EMC, emerged initially in Saudi Arabia [3, 10] . SARS-CoV infected about 8000 people, and resulted in the deaths of approximately 10% of those infected [11] ."
],
"positive": [
"However, a decade later Middle East respiratory syndrome coronavirus (MERS-CoV), also known as HCoV-EMC, emerged initially in Saudi Arabia [3, 10] . SARS-CoV infected about 8000 people, and resulted in the deaths of approximately 10% of those infected [11] . While MERS-CoV is not as widespread as SARS-CoV, it appears to have an even higher mortality rate, with 35-50% of diagnosed infections resulting in death [3, [12] [13] . These deadly betacoronavirus viruses existed in animal reservoirs [4] [5] 9, [14] [15] . Recently, other CoVs have been detected in animal populations raising the possibility that we will see a repeat of these types of outbreaks in the near future [11, [16] [17] [18] [19] [20] . Both these zoonotic viruses cause a much more severe disease than what is typically seen for CoVs, making them a global health concern."
]
} |
What is the key regulator to sporulation? | {
"negative": [
"The transcription of spo0A in C. difficile is under control of the transition state sigma factor Sigma H [37] , as it is in B. subtilis [61] . Our data indicate that both spo0A and sigH could be targets for direct regulation by Spo0A in C. difficile (Fig. 3A) , raising the possibility of auto-regulation of spo0A. The putative direct regulation of sigH by Spo0A may reflect that the C. difficile genome does not harbor a homolog of the pleiotropic regulator AbrB, which is responsible for the Spo0A-dependent regulation of sigH in B. subtilis [48] . Consistent with a model in which spo0A is positively autoregulated, we noted a sharp increase in the levels of Spo0A as cells approach the stationary growth phase ( Figure 1C) . Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA (Fig.",
"Consistent with a model in which spo0A is positively autoregulated, we noted a sharp increase in the levels of Spo0A as cells approach the stationary growth phase ( Figure 1C) . Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA (Fig. 3B ). All these observations are consistent with direct regulation of these genes by Spo0A in other organisms [5, 45, 49, 62] , and the conservation of the sporulation pathway [2] . Though Spo0A is the key regulator for sporulation in Firmicutes, it regulates numerous other processes in various bacteria. In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology [10, [14] [15] [16] .",
"Smits, unpublished observations). These differences may relate to the much higher AT content of C. difficile compared to B. subtilis (71 vs. 56.5%, respectively), or phosphorylation dependent dimerization, for instance. The initiation of sporulation in B. subtilis is subject to complex regulation (for review see ref [1, 59] ). The activation of Spo0A is controlled by a multi-component phosphorelay that can integrate environmental cues [60] and ensures a gradual increase in the level of phosphorylated Spo0A in the cell [57] . In addition, the transcription of the spo0A gene is controlled by multiple feedback loops. For instance, Spo0A regulates its own transcription by binding to the spo0A promoter [46] , as well as by indirectly stimulating the transcription of sigH, encoding a sigma factor that recognizes the spo0A promoter [48] .",
"Though Spo0A is the key regulator for sporulation in Firmicutes, it regulates numerous other processes in various bacteria. In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology [10, [14] [15] [16] . Importantly, several of these processes are indirectly regulated, through the Spo0A-dependent repression of abrB. Additionally, transcription of abrB responds already to low levels of Spo0A,P [43] . As a result these effects are detectable in late-exponential and early stationary phase, as some Spo0A is present throughout growth in B. subtilis cells. Though abrB is absent from C. difficile, this does not exclude the possibility of indirect transcriptional regulation through Spo0Adependent effects on other regulators.",
"In addition, the transcription of the spo0A gene is controlled by multiple feedback loops. For instance, Spo0A regulates its own transcription by binding to the spo0A promoter [46] , as well as by indirectly stimulating the transcription of sigH, encoding a sigma factor that recognizes the spo0A promoter [48] . In C. difficile, there are some interesting differences and similarities in the regulatory pathways. Most notably, there seems to be no phosphorelay [2] and the phosphorylation state of Spo0A is supposedly controlled by orphan histidine kinases [35] . The transcription of spo0A in C. difficile is under control of the transition state sigma factor Sigma H [37] , as it is in B. subtilis [61] . Our data indicate that both spo0A and sigH could be targets for direct regulation by Spo0A in C. difficile (Fig."
],
"positive": [
"D. Binding of Spo0A-DBD-his6 to the upstream region of spoIIGA is reversed by the addition of PabrB, but not by the addition of PtcdA. E. Binding of Spo0A-DBD-his6 to the upstream region of tcdB is reversed by the addition of PabrB, but not by the addition of PtcdA. F. Binding of Spo0A-DBD-his6 to the upstream region of tcdC is not or moderately affected by the addition of PabrB and/or PtcdA. (TIF) Text S1 Oligonucleotides used in this study and PCR cycling conditions for the EMSA probes. (PDF)"
]
} |
For HCPS, what does that expression favor? | {
"negative": [
"The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.",
"Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: (1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] .",
"There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] .",
"By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.",
"The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] ."
],
"positive": [
"By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs."
]
} |
What is directly related to nuclear condensation? | {
"negative": [
"Mitochondria are the main source for the production of ROS and adenosine triphosphate (ATP) and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu(BrHAP) 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu(BrHAP) 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu(BrHAP) 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway.",
"The result implies that the induction of apoptosis by Cu(II) Schiff base complexes may be associated with the mitochondrial pathway [26, 41, 42] . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP [30] . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets [43] .",
"HT-29 cells treated with Cu (II) complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G(1 ) cell population. At a concentration of 6.25 μg/ml, the Cu(BrHAP)(2 ) compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu (II) complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu(BrHAP)(2 ) compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern [1] .",
"To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu(BrHAP) 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu(BrHAP) 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor-(TNF-). The Cu(BrHAP) 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus (Figure 9 ). Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression [21] .",
"The Cu(BrHAP) 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus (Figure 9 ). Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression [21] . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation [22, 23]. The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence [24] , and agents that can induce apoptosis are known to have potential anticancer effects [25, 26] . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention."
],
"positive": [
"As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu(BrHAP) 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu(BrHAP) 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate (ATP) and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu(BrHAP) 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells."
]
} |
What has epidemiology and research identified the MERS-CoV's cell receptor is? | {
"negative": [
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] . ). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator.",
"A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV.",
"Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern."
],
"positive": [
"Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases."
]
} |
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