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[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]

[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]

[ "BACKGROUND: Coccidiosis due to Eimeria spp. infections in lambs causes increased mortality and substantial production losses, and anticoccidials are important for control of the infection. Anticoccidial resistance has been reported in poultry and swine, and we recently described reduced toltrazuril efficacy in ovine Eimeria spp. in some Norwegian sheep farms using a newly developed faecal oocyst count reduction test FOCRT . The aim of the present study was to use a controlled efficacy trial to assess the efficacy of toltrazuril against a field isolate suspected of being resistant. METHODS: Twenty lambs, 17–22 days old and raised protected against exposure to coccidia, were infected with a field isolate of 100,000 Eimeria spp. oocysts.", "METHODS: Twenty lambs, 17–22 days old and raised protected against exposure to coccidia, were infected with a field isolate of 100,000 Eimeria spp. oocysts. This isolate was obtained from a farm with a previously calculated drug efficacy of 56% 95% confidence interval: -433.9 to 96.6% . At day 7 post-infection, 10 of the lambs were orally treated with 20 mg/kg toltrazuril Baycox Sheep vet., Bayer Animal Health , while the other 10 lambs controls were given physiological saline. Clinical examinations were conducted, and weight gains recorded. Daily faecal samples were scored for diarrhoea on a scale from 1 to 5, and oocyst excretion was determined using a modified McMaster technique.", "Clinical examinations were conducted, and weight gains recorded. Daily faecal samples were scored for diarrhoea on a scale from 1 to 5, and oocyst excretion was determined using a modified McMaster technique. Oocysts were morphologically identified to species level. At 17–24 days post-infection, the lambs were euthanized and necropsied. RESULTS: The tested Eimeria isolate was resistant against toltrazuril, and resistance was seen in both pathogenic and non-pathogenic species. In addition, no significant differences in faecal score, growth, gross pathology or histological changes were identified between the two groups.", "RESULTS: The tested Eimeria isolate was resistant against toltrazuril, and resistance was seen in both pathogenic and non-pathogenic species. In addition, no significant differences in faecal score, growth, gross pathology or histological changes were identified between the two groups. The pathogenic E. ovinoidalis was the dominant species, and no significant difference in the individual prevalence of E. ovinoidalis post-treatment was found between treated 66.9% and control lambs 61.9% . Other species identified included E. crandallis/weybridgensis, E. parva, E. marsica, E. faurei, E. pallida, E. ahsata and E. bakuensis. CONCLUSIONS: This study confirms toltrazuril resistance in ovine Eimeria spp. ; in addition, the data support the use of FOCRT as an appropriate tool for field evaluation of anticoccidial efficacy.", "CONCLUSIONS: This study confirms toltrazuril resistance in ovine Eimeria spp. ; in addition, the data support the use of FOCRT as an appropriate tool for field evaluation of anticoccidial efficacy. Due to limited anticoccidial treatment alternatives, these findings may have important implications for the sheep industry, particularly in northern Europe. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article 10.1186/s13071-018-2976-4 contains supplementary material, which is available to authorized users. Text: Anticoccidial resistance ACR , which develops mainly as a result of intensive long-term use of anticoccidial drugs, occurs widely in poultry production and has also been identified in Cystoisospora suis in piglets . In addition, a field method for the evaluation of reduced anticoccidial efficacy ACE in ovine Eimeria spp., the faecal oocyst count reduction test FOCRT , has recently been developed and indicated that the efficacy of toltrazuril is reduced in some Norwegian sheep flocks .", "Text: Anticoccidial resistance ACR , which develops mainly as a result of intensive long-term use of anticoccidial drugs, occurs widely in poultry production and has also been identified in Cystoisospora suis in piglets . In addition, a field method for the evaluation of reduced anticoccidial efficacy ACE in ovine Eimeria spp., the faecal oocyst count reduction test FOCRT , has recently been developed and indicated that the efficacy of toltrazuril is reduced in some Norwegian sheep flocks . Infections with Eimeria spp. may impact both animal welfare and productivity in the sheep industry, and controlling the infection is important to minimise mortality and morbidity, and to ensure that lamb growth is not compromised . Suggested strategies to control ruminant coccidiosis include pasture management, adequate nutrition, and hygienic measures . However, these measures are often difficult to implement in practice, and the main control approach is often metaphylaxis with anticoccidials .", "Suggested strategies to control ruminant coccidiosis include pasture management, adequate nutrition, and hygienic measures . However, these measures are often difficult to implement in practice, and the main control approach is often metaphylaxis with anticoccidials . Metaphylactic administration of a single oral dose of toltrazuril in the prepatent period has been shown to be effective at reducing clinical signs and maintaining adequate lamb growth rates in different production systems 13, . In contrast, treatment of clinical coccidiosis is considered inefficient due to the extensive intestinal damage already caused by the infection . Loss of sensitivity to toltrazuril, the only anticoccidial registered for use in sheep in the Nordic countries , should therefore be a matter for serious concern for lamb production. The World Association for the Advancement of Veterinary Parasitology guidelines for evaluation of ACE in mammals , states that there is a need for verified methods for evaluation of ACE.", "Loss of sensitivity to toltrazuril, the only anticoccidial registered for use in sheep in the Nordic countries , should therefore be a matter for serious concern for lamb production. The World Association for the Advancement of Veterinary Parasitology guidelines for evaluation of ACE in mammals , states that there is a need for verified methods for evaluation of ACE. Field methods for assessment of drug efficacy, such as the FOCRT and the faecal egg count reduction test used to evaluate anthelmintic efficacy , give only an indication of reduced efficacy, and need verification through controlled efficacy trials CET . In addition, due to the variation in pathogenicity between ovine Eimeria spp., the differentiation of species should be considered separately . The aim of the present study was to perform a CET in order to determine whether different species in a field isolate of ovine Eimeria spp. with suspected ACR, based on the FOCRT , actually demonstrated resistance to toltrazuril.", "The aim of the present study was to perform a CET in order to determine whether different species in a field isolate of ovine Eimeria spp. with suspected ACR, based on the FOCRT , actually demonstrated resistance to toltrazuril. A total of 20 lambs from 8 ewes of the Norwegian White Sheep breed \"Norsk kvit sau\" was included in the study, which was approved by the Norwegian Animal Research Authority ID: 11657 . The ewes were synchronised using Chronogest® CR and PMSG® MSD Animal Health, Buckinghamshire, UK and served by natural mating. Lambs were either snatched at birth n = 16 or delivered by caesarean section n = 4 over a period of 6 days, and thereafter reared artificially. Individual ear tags were used for identification.", "Lambs were either snatched at birth n = 16 or delivered by caesarean section n = 4 over a period of 6 days, and thereafter reared artificially. Individual ear tags were used for identification. Directly after birth, all lambs were washed with Optima pH 4 soap Optima Produkter AS, Norheimsund, Norway and dried before being placed in boxes with expanded metal floors, in groups of four. Infrared heaters were used during the whole trial. An overview of the study groups, including lamb age, birth weight and gender can be found in Additional file 1: Table S1 . Lambs received ovine colostrum from ewes vaccinated against Clostridium spp.", "An overview of the study groups, including lamb age, birth weight and gender can be found in Additional file 1: Table S1 . Lambs received ovine colostrum from ewes vaccinated against Clostridium spp. Covexin-8, Zoetis during the first 30 min of life, followed by colostrum from vaccinated cows Covexin-8, Zoetis during the next 24 h. To avoid cases of haemolytic anaemia, the cow-colostrum had previously been tested on naturally reared lambs. Lambs were then fed ad libitum with a commercial milk replacer Denkamilk, Denkavit, Fiskå, Mølle, Stavanger , using an automatic feeding system Holm & Laue, Godkalven, Figgjo, Norway . The lambs had ad libitum access to water, hay and commercial lamb-starter concentrate FORMEL lam vår, Felleskjøpet, Norway . To ensure that transmission of Eimeria to the lambs via contaminated colostrum and hay could not occur, both were frozen at -75°C for a minimum of 24 h, prior to provision to the lambs.", "The lambs had ad libitum access to water, hay and commercial lamb-starter concentrate FORMEL lam vår, Felleskjøpet, Norway . To ensure that transmission of Eimeria to the lambs via contaminated colostrum and hay could not occur, both were frozen at -75°C for a minimum of 24 h, prior to provision to the lambs. The field isolate of Eimeria spp. was obtained from one of the flocks ID 35 participating in the recent FOCRT study . According to the FOCRT results, toltrazuril had reduced efficacy against Eimeria in two flocks. However, neither of these flocks were available for the CET, due to geographical and practical reasons.", "According to the FOCRT results, toltrazuril had reduced efficacy against Eimeria in two flocks. However, neither of these flocks were available for the CET, due to geographical and practical reasons. Thus, treatment with toltrazuril in the selected flock had been found to have an efficacy of 56.0%, but the results were classified as inconclusive, due to the wide 95% confidence interval CI of -433.9 and 96.6% . To obtain sufficient Eimeria oocysts of this mixed field isolate named \"NMBU ID 35\" for the present study, faecal samples were obtained from 35 lambs in this flock 9 days after toltrazuril treatment Baycox® Sheep vet., Bayer Animal Health, Oslo, Noray . Oocysts were isolated according to Jackson with some modifications. Briefly, faeces were mixed 1:1 with water and filtered.", "Oocysts were isolated according to Jackson with some modifications. Briefly, faeces were mixed 1:1 with water and filtered. The faecal mix filtrate was subsequently mixed 1:1 with saturated sugar-solution density: 1.5 g/l in a plastic container and left to float onto a glass slide. The slide was washed every second hour with deionized water for three consecutive days, and the washings collected. The washings were centrifuged at 2300× g for 20 min, the supernatant discarded and the sediment mixed 1:1 with deionized water in a glass flask with constant aeration. The oocysts in the flask were left to sporulate for 7 days at room temperature. Sporulated oocysts were stored for 18 days at 4°C.", "The oocysts in the flask were left to sporulate for 7 days at room temperature. Sporulated oocysts were stored for 18 days at 4°C. Based on morphology , as seen by light microscopy at 400× magnification see also Faecal samples section , and classification of 300 oocysts, the field isolate consisted of E. parva 32% , E. crandallis/ weybridgensis 25% , E. ovinoidalis 24% , E. faurei 9% , E. marsica 8% , E. pallida 1% , E. ahsata < 1% and E. bakuensis < 1 % . All lambs were infected day 0 at 17-22 days of age, using an oesophageal tube. A dose of approximately 100,000 sporulated oocysts, diluted in water to a total volume of 5 ml, was given to each of the 20 lambs. Then, two randomly selected coin toss lambs from each group of four were orally treated day 7 with 0.4 ml/kg toltrazuril Baycox® Sheep vet.", "A dose of approximately 100,000 sporulated oocysts, diluted in water to a total volume of 5 ml, was given to each of the 20 lambs. Then, two randomly selected coin toss lambs from each group of four were orally treated day 7 with 0.4 ml/kg toltrazuril Baycox® Sheep vet. 50 mg/ml, Bayer Animal Health and the remaining lambs controls were given 0.4 ml/kg of 0.9% NaCl B. Braun Medical AS, Vestskogen, Norway . Clinical examinations were performed daily throughout the trial. Rectal temperature was measured at days 0, 1, 2 and 7, and daily from day 14, and temperatures > 40.5°C were considered as fever. The lambs were weighed once a week using a calibrated weight Kruuse, Drøbak Norway with 0.1 kg sensitivity, until 14 days post-infection, and thereafter three times a week.", "Rectal temperature was measured at days 0, 1, 2 and 7, and daily from day 14, and temperatures > 40.5°C were considered as fever. The lambs were weighed once a week using a calibrated weight Kruuse, Drøbak Norway with 0.1 kg sensitivity, until 14 days post-infection, and thereafter three times a week. Two lambs controls were treated orally with trimethoprim/sulphamethoxasole Bactrim, Roche, Etterstad, Norway during the first three days of life due to suspected Escherichia coli-infection, from which both recovered within 48 h. Six lambs, two controls and four treated with toltrazuril, developed lameness due to interdigital abscessation, and Streptococcus aureus was detected in two lambs. Four lambs recovered without treatment, and two of the lambs recovered after treatment with benzylpenicillinprocaine Penovet vet., Boehringer Ingelheim Vetmedica, Copenhagen, Denmark administered intramuscularly for three days. On clinical examination, special attention was paid to clinical signs associated with Eimeria spp. infections, i.e.", "On clinical examination, special attention was paid to clinical signs associated with Eimeria spp. infections, i.e. dehydration, pyrexia, weakness, anorexia and, in particular, the presence of diarrhoea. Severe haemorrhagic diarrhoea and dehydration in one lamb at day 17, led to euthanasia of that whole group of four lambs. At day 18, another lamb showed signs of haemorrhagic diarrhoea, and all lambs in this group were also euthanized. The remaining three groups were euthanized on days 21, 23, and 24.", "At day 18, another lamb showed signs of haemorrhagic diarrhoea, and all lambs in this group were also euthanized. The remaining three groups were euthanized on days 21, 23, and 24. Blood samples were drawn from v. jugularis using vacuette tubes plain and EDTA-treated; BD, Franklin Lakes, USA at 48 ± 2 h after birth and at days 0, 7 and at euthanasia. Haematology was performed using the ADVIA 120 Haematology system Bayer Diagnostics, Leverkusen, Germany . Dehydration was considered with a haematocrit hct of > 45.0% . Whole blood tubes were centrifuged, and the serum removed and stored at -20°C until further analysis.", "Dehydration was considered with a haematocrit hct of > 45.0% . Whole blood tubes were centrifuged, and the serum removed and stored at -20°C until further analysis. Biochemical analysis was performed by ABX Pentra 400 Horiba, Les Ulis, France , and included analysis of iron, total protein, albumin, urea, creatinine, gamma-glutamyl transferase, glutamate dehydrogenase and beta hydroxybutyric acid. Individual faecal samples from each of the lambs were obtained daily from day 10 of life until the end of the experiment. Visual scoring of faecal consistency was performed on a scale from one to five 1: normal, pelleted; 2: soft; 3: liquid; 4: watery; 5: watery with blood and/or intestinal tissue . A score ≥ 3 was considered as diarrhoea.", "Visual scoring of faecal consistency was performed on a scale from one to five 1: normal, pelleted; 2: soft; 3: liquid; 4: watery; 5: watery with blood and/or intestinal tissue . A score ≥ 3 was considered as diarrhoea. Samples were collected using an in-house \"faecal spoon\" and the faecal samples were put in zip-lock bags, which were vacuum packed Fresh'n'easy, OBH Nordica, Sundbyberg, Sweden , stored at 4°C, and analysed within 37 days. The rate of oocyst excretion was determined using a modified McMaster technique with a theoretical sensitivity of 5 oocysts per gram OPG . One hundred Eimeria oocysts from all samples ≥ 1000 OPG were examined by light microscopy at 400× magnification and identified to species level, using morphological criteria . However, due to their morphological similarity, oocysts of E. crandallis and E. weybridgensis were not differentiated.", "One hundred Eimeria oocysts from all samples ≥ 1000 OPG were examined by light microscopy at 400× magnification and identified to species level, using morphological criteria . However, due to their morphological similarity, oocysts of E. crandallis and E. weybridgensis were not differentiated. Oocyst counts were analysed by the FOCRT , which consists of a two-step procedure. First, timing of treatment and sampling was evaluated, followed by evaluation of treatment efficacy, by comparing post-treatment faecal samples from treated lambs with equivalent samples from untreated controls. Pre-treatment samples sample 1 were obtained on day 7 day of treatment , and post-treatment samples sample 2 were obtained on days 14-18. The FOCRT was then run using the post-treatment oocyst counts for all five possible time intervals 7-11 days between samples 1 and 2.", "Pre-treatment samples sample 1 were obtained on day 7 day of treatment , and post-treatment samples sample 2 were obtained on days 14-18. The FOCRT was then run using the post-treatment oocyst counts for all five possible time intervals 7-11 days between samples 1 and 2. Faecal samples obtained at euthanasia were analysed for rotavirus, coronavirus, Cryptosporidium spp. and general bacteriology. Additional testing for Cryptosporidium spp. was performed in diarrhoeic lambs at the time of infection day 0, n = 10 .", "and general bacteriology. Additional testing for Cryptosporidium spp. was performed in diarrhoeic lambs at the time of infection day 0, n = 10 . Faecal smears were analysed at the Norwegian Veterinary Institute in Oslo for Cryptosporidium by direct immunofluorescence analysis Crypt-a-Glo™, Waterborne Inc., New Orleans, USA , whereas presence of rotavirus and coronavirus were tested by standard diagnostic methods. Samples for bacteriological analyses were obtained from mid-jejunum and the colon spiral, spread on sheep blood agar plates, and incubated under anaerobic and aerobic conditions for 24-48 h at 37°C and 5% CO 2 . In cases of haemorrhagic diarrhoea, additional samples were grown on bromothymol-blue lactose cysteine agar brolactin/CLED agar for potential identification of Salmonella .", "Samples for bacteriological analyses were obtained from mid-jejunum and the colon spiral, spread on sheep blood agar plates, and incubated under anaerobic and aerobic conditions for 24-48 h at 37°C and 5% CO 2 . In cases of haemorrhagic diarrhoea, additional samples were grown on bromothymol-blue lactose cysteine agar brolactin/CLED agar for potential identification of Salmonella . Lambs were euthanized at days 17-24, by intravenous injection with pentobarbital Euthasol vet., Virbac, Sollihøgda, Norway at 140 mg/kg. Standard necropsy was performed immediately thereafter, with emphasis on the intestines. Histological samples were taken from mid-jejunum, proximal and distal ileum, mid and base of caecum, colon spiral, and distal colon, in addition to heart, lung, liver and kidney. The samples were immersion-fixed in 4% formaldehyde, paraffin-embedded, and stained with haematoxylin and eosin HE .", "Histological samples were taken from mid-jejunum, proximal and distal ileum, mid and base of caecum, colon spiral, and distal colon, in addition to heart, lung, liver and kidney. The samples were immersion-fixed in 4% formaldehyde, paraffin-embedded, and stained with haematoxylin and eosin HE . Histological evaluation was performed by light microscopy and a blinded semi-quantitative evaluation single evaluator was done to assess intestinal pathology. Evaluation parameters included changes in: i villi, ii surface epithelium atrophy/attenuation , iii degree of Eimeria-infection, iv hyperaemia, v oedema, vi infiltration of inflammatory cells and vii crypt abscesses, and were scored as follows: 0 = minimal; 1 = little; 2 = moderate; 3 = severe, including half-step grading. In addition, the presence of epithelial necrosis was graded as present . or absent .. A total histology score was calculated for each tissue by summation of all parameters evaluated i-vii .", "In addition, the presence of epithelial necrosis was graded as present . or absent .. A total histology score was calculated for each tissue by summation of all parameters evaluated i-vii . Data were managed in Excel 2013 Microsoft Inc., Redmond, USA , and subsequently analysed in R and Stata 14 Stata Statistical Software: Release 14. Stata-Corp LP, College Station, TX, USA . Evaluation of efficacy was performed according to the FOCRT . For calculations of significance based on means, a t-test was used. P < 0.05 was considered significant. Mean growth rates were above 300 g/day until days 14-16, whereupon mean growth rate decreased to around 0 g/day Fig. 1 .", "P < 0.05 was considered significant. Mean growth rates were above 300 g/day until days 14-16, whereupon mean growth rate decreased to around 0 g/day Fig. 1 . Growth rates increased again from day 21 onwards. The same pattern was observed in both treated and control lambs. From day 15, both treated and control lambs had a mean faecal score of ≥ 3, indicating diarrhoea. The maximum mean faecal score was seen at day 17 3.9 ± 0.2 and day 18 4.4 ± 0.3 in the treated and control groups, respectively.", "From day 15, both treated and control lambs had a mean faecal score of ≥ 3, indicating diarrhoea. The maximum mean faecal score was seen at day 17 3.9 ± 0.2 and day 18 4.4 ± 0.3 in the treated and control groups, respectively. Haemorrhagic diarrhoea was seen from day 14, in two treated and five control lambs, and tenesmus was observed in two control lambs day 17 . An increase in rectal temperature was seen from day 14, with maximum temperatures measured at day 18 40.4 ± 0.4°C and 16 40.9 ± 0.4°C in the treated and control groups, respectively. The mean duration of fever > 40.5°C was 2.3 ± 0.5 days and 1.9 ± 0.4 days for the treated and control groups, respectively. For these parameters, no significant difference between groups were seen at any time.", "The mean duration of fever > 40.5°C was 2.3 ± 0.5 days and 1.9 ± 0.4 days for the treated and control groups, respectively. For these parameters, no significant difference between groups were seen at any time. At euthanasia, the mean hct was 39.2 ± 1.7% and 41.4 ± 1.9% in the treated and control groups, respectively. However, dehydration hct > 45.0% was only seen in 3 lambs, of which one had been treated with toltrazuril. Mean total serum protein decreased in both groups from infection to euthanasia, but no significant differences between the groups were observed. Other biochemical parameters were within normal ranges data not shown .", "Mean total serum protein decreased in both groups from infection to euthanasia, but no significant differences between the groups were observed. Other biochemical parameters were within normal ranges data not shown . Oocyst excretion was first recorded in one treated lamb at day 10 10 OPG , followed by oocyst excretion in all lambs in both groups from day 14 onwards. Peak oocyst excretion was seen in the treated group at day 20 mean OPG: 5,438,500 , and in the control group at day 21 after infection mean OPG: 3,630,850 Fig. 2 . Thereafter, oocyst excretion decreased. There was no significant difference in oocyst excretion and species distribution between the groups at any time.", "2 . Thereafter, oocyst excretion decreased. There was no significant difference in oocyst excretion and species distribution between the groups at any time. All species present in the field isolate were isolated from the faecal samples of all the 20 infected lambs. E. ovinoidalis was the most prevalent species in both treated and control lambs Table 1 . Efficacy, according to the FOCRT, was evaluated with confidence if the slope was ≥ 0.75, and with caution if slope was ≥ 0.5 and < 0.75 . The slope ranged from 1.24 to 1.69 for the total oocyst excretion in the control lambs.", "Efficacy, according to the FOCRT, was evaluated with confidence if the slope was ≥ 0.75, and with caution if slope was ≥ 0.5 and < 0.75 . The slope ranged from 1.24 to 1.69 for the total oocyst excretion in the control lambs. Slopes, maximum likelihood estimates, and 95% CIs for the geometric mean efficacy of all oocysts, E. ovinoidalis, E. crandallis/weybridgensis, and the non-pathogenic Eimeria spp. are presented in Table 2 ; reduced efficacy of toltrazuril is apparent against both pathogenic and non-pathogenic species. The slope was ≥ 0.75 for all time intervals and species, except for four of the five time intervals of E. crandallis/weybridgensis. Samples analysed for Cryptosporidium spp., Salmonella, coronavirus and rotavirus were all negative.", "The slope was ≥ 0.75 for all time intervals and species, except for four of the five time intervals of E. crandallis/weybridgensis. Samples analysed for Cryptosporidium spp., Salmonella, coronavirus and rotavirus were all negative. Bacteriological analyses showed a mixed flora, dominated by coliforms and Enterococcus spp. Gross pathological findings included diffused thickened and folded ileal mucosa 7 treated and 7 controls , and fibrinous ileal content in two lambs one treated and one control . Nodular or plaque-like foci in the ileal mucosa were seen in 4 treated and 6 control lambs Fig. 3a . The regional distal jejunal lymph nodes were moderately increased in size and oedematous in 5 treated and 6 control lambs.", "3a . The regional distal jejunal lymph nodes were moderately increased in size and oedematous in 5 treated and 6 control lambs. Finally, watery abomasal content was seen in > 50 % of the animals in both groups. Microscopy evaluation showed lesions, mainly in the ileum, caecum and colon, with minor lesions in the jejunum Fig. 3b-f . However, there were no significant differences with respect to histological scores between the treated and control groups in any of the intestinal segments. The highest calculated histological score was found in the proximal ileum and at the base of caecum Fig. 4 .", "The highest calculated histological score was found in the proximal ileum and at the base of caecum Fig. 4 . The mean score for each parameter can be found in Additional file 2: Table S2 . Varying quantities of intracellular Eimeria stages were observed in all intestinal segments, except from jejunum, and they were mostly located in the villus epithelium, with fewer parasites in the crypt epithelium and lamina propria, and few in the submucosa and lymphatic vessels. In both treated and control lambs, changes in the intestinal surfaces varied from light atrophy of the jejunal epithelium and blunting of affected ileal villi Fig. 3b , to areas of total flattening, attenuation of surface epithelium Fig.", "In both treated and control lambs, changes in the intestinal surfaces varied from light atrophy of the jejunal epithelium and blunting of affected ileal villi Fig. 3b , to areas of total flattening, attenuation of surface epithelium Fig. 3e and necrosis Fig. 3d . Patches of epithelial necrosis were found in all lambs. Infiltration of inflammatory cells included mostly monocytes and eosinophils, but also neutrophils and macrophages, and was found in both the lamina propria and submucosa. Different degrees of oedema, hyperaemia, and haemorrhage were seen in all tissue sections examined, and in both treated and control lambs. Crypt abscesses Fig.", "Different degrees of oedema, hyperaemia, and haemorrhage were seen in all tissue sections examined, and in both treated and control lambs. Crypt abscesses Fig. 3b were found in varying degree in all lambs, and contained inflammatory cells, debris and different stages of Eimeria spp. As far as we know, this is the first report of experimentally confirmed toltrazuril resistance in a field isolate of ovine Eimeria spp. The results also support the use of FOCRT as a tool to evaluate ACE in the field. Although ten of the 20 lambs experimentally infected with Eimeria were metaphylactically treated with the recommended dose of 20 mg/kg toltrazuril Baycox® Sheep vet., Bayer Animal Health , this treatment did not result in a significant reduction in oocyst excretion in the treated animals, compared with the controls.", "The results also support the use of FOCRT as a tool to evaluate ACE in the field. Although ten of the 20 lambs experimentally infected with Eimeria were metaphylactically treated with the recommended dose of 20 mg/kg toltrazuril Baycox® Sheep vet., Bayer Animal Health , this treatment did not result in a significant reduction in oocyst excretion in the treated animals, compared with the controls. In addition, no significant differences were noted in clinical presentation, gross pathology, and histopathological findings. The speciation data showed that both pathogenic and non-pathogenic species of Eimeria in this isolate were resistant to toltrazuril. The lambs excreted high numbers of oocysts, as has previously been recorded in experimental infections with multiple Eimeria spp. .", "The lambs excreted high numbers of oocysts, as has previously been recorded in experimental infections with multiple Eimeria spp. . Although oocyst excretion decreased from around day 20 after infection, the total duration of excretion could not be determined, as the lambs were euthanized. The excretion pattern noted here, with an exponential increase, a plateau phase, and a decline, has previously been noted in experimental infections . However, due to continuous reinfection under natural field conditions, the duration of oocyst excretion may be longer than observed in the present study. This might also explain why the calculated slope seen for all species in this experimental study is higher than the slopes reported from the preceding field trial .", "However, due to continuous reinfection under natural field conditions, the duration of oocyst excretion may be longer than observed in the present study. This might also explain why the calculated slope seen for all species in this experimental study is higher than the slopes reported from the preceding field trial . Multi-species resistance, as observed here, has also been noted in field isolates of avian Eimeria spp. . Notes: The estimates were based on post-treatment oocyst counts for five time intervals between sample 1 day 7 after infection and sample 2, and was calculated according to the FOCRT . A slope ≥ 0.5 and < 0.75 was evaluated with caution, whereas a slope < 0.5 was interpreted as invalid a Four lambs were euthanized at day 17 Abbreviations: E. ovi, E. ovinoidalis; E. c/w, E. crandallis/weybridgensis; Non-pathogenic, all species except E. ovinoidalis and E. crandallis/weybridgensis Of particular importance in this study is that E. ovinoidalis was the dominant species excreted from infected lambs.", "Notes: The estimates were based on post-treatment oocyst counts for five time intervals between sample 1 day 7 after infection and sample 2, and was calculated according to the FOCRT . A slope ≥ 0.5 and < 0.75 was evaluated with caution, whereas a slope < 0.5 was interpreted as invalid a Four lambs were euthanized at day 17 Abbreviations: E. ovi, E. ovinoidalis; E. c/w, E. crandallis/weybridgensis; Non-pathogenic, all species except E. ovinoidalis and E. crandallis/weybridgensis Of particular importance in this study is that E. ovinoidalis was the dominant species excreted from infected lambs. As this species is one of the most pathogenic Eimeria spp. in sheep , resistance against the most commonly used anticoccidial drug indicates that severe clinical coccidiosis may be expected to occur in resistant flocks. Although E. ovinoidalis was the dominant species excreted, the most prevalent species in the original field-isolate inoculum was E. parva. This could reflect similarities between E. ovinoidalis and E. ninakholyakimovae in goats, the latter of which develops macroschizonts in endothelial cells, resulting in the release of thousands of merozoites .", "Although E. ovinoidalis was the dominant species excreted, the most prevalent species in the original field-isolate inoculum was E. parva. This could reflect similarities between E. ovinoidalis and E. ninakholyakimovae in goats, the latter of which develops macroschizonts in endothelial cells, resulting in the release of thousands of merozoites . Thus, the extent of intracellular multiplication/ replication, which is presumably also related to the extent of pathogenicity associated with this species, is higher for E. ovinoidalis than for the other Eimeria species. For E. crandallis/weybridgensis, the FOCRT calculations showed invalid results from three of the five sampling time points, probably due to the tests being performed too early in the infection. Excretion of E. crandallis/weybridgensis increased predominantly from day 16 onwards, and euthanasia was performed at days 17-24. Thus, the longer prepatent periods for these species compared with E. ovinoidalis probably explain these results.", "Excretion of E. crandallis/weybridgensis increased predominantly from day 16 onwards, and euthanasia was performed at days 17-24. Thus, the longer prepatent periods for these species compared with E. ovinoidalis probably explain these results. This is an important finding, as the number of invalid farms tested in the FOCRT might have been fewer should sample 2 have been collected 10-11 days after sample 1. These findings also highlight the fact that although Eimeria spp. are often considered as a relatively uniform group, they are in fact separate species with potentially important differences in biology and pathogenic potential. Two of the lambs were treated with trimethoprim/ sulpha during their first days of life, preparations that have been shown to be effective in treating ovine coccidiosis .", "are often considered as a relatively uniform group, they are in fact separate species with potentially important differences in biology and pathogenic potential. Two of the lambs were treated with trimethoprim/ sulpha during their first days of life, preparations that have been shown to be effective in treating ovine coccidiosis . However, withdrawal periods for comparable drugs licenced in cattle are 10-15 days for meat , and these lambs were treated > 17 days prior to the experimental infection. In addition, these treated lambs were in the control group, and therefore this treatment should not have affected the results of the study. Similar clinical signs as observed here might be caused by Cryptosporidium spp., coronavirus, rotavirus, and Salmonella spp., but none of these pathogens were detected. In addition, the findings of coliforms and Enterococcus spp.", "Similar clinical signs as observed here might be caused by Cryptosporidium spp., coronavirus, rotavirus, and Salmonella spp., but none of these pathogens were detected. In addition, the findings of coliforms and Enterococcus spp. may be considered as normal intestinal flora of lambs . The observed clinical signs were therefore almost certainly caused by Eimeria spp., particularly the two major pathogenic species, E. ovinoidalis and E. crandallis . Thickened ileal mucosa is often seen in lambs infected with E. ovinoidalis . In addition, the histological changes, such as blunted villi and surface necrosis, as well as the presence of coccidia, hyperaemia, oedema, infiltration of inflammatory cells and crypt abscesses, are also in accordance with previous reports .", "Thickened ileal mucosa is often seen in lambs infected with E. ovinoidalis . In addition, the histological changes, such as blunted villi and surface necrosis, as well as the presence of coccidia, hyperaemia, oedema, infiltration of inflammatory cells and crypt abscesses, are also in accordance with previous reports . To improve our study, an additional group of uninfected lambs might have been advantageous as this would have enabled better comparisons between weight gain and histopathological changes. However, this was not feasible at the time of the study. Furthermore, due to the lack of defined cut-off values for ACE, it might have been advantageous to include an oocyst isolate from a non-suspected farm i.e. a susceptible isolate .", "Furthermore, due to the lack of defined cut-off values for ACE, it might have been advantageous to include an oocyst isolate from a non-suspected farm i.e. a susceptible isolate . This would have enabled comparisons of different parameters, such as oocyst excretion, between treated and control lambs infected with susceptible or resistant Eimeria spp. However, due to lack of tools for selection of such susceptible ovine Eimeria isolates, we therefore chose to restrict our CET to treated and control lambs infected with isolate \"NMBU ID 35\" as a first step in the characterisation of anticoccidial resistance in ovine Eimeria spp. Although the initial efficacy values have not been provided for toltrazuril by the manufacturer, several studies have investigated its effect on oocyst excretion. For example, its efficacy has been found to be 96.9-99.9% in the period from 7 to 98 days after first treatment, in a study in which the lambs were treated every 14 days .", "Although the initial efficacy values have not been provided for toltrazuril by the manufacturer, several studies have investigated its effect on oocyst excretion. For example, its efficacy has been found to be 96.9-99.9% in the period from 7 to 98 days after first treatment, in a study in which the lambs were treated every 14 days . Other studies have shown toltrazuril efficacies either provided in the publication or calculated as 1- mean OPG treated group / mean OPG control group from data in the publication ranging from 90.0 to 100.0% in the period from two to three weeks after treatment 13, 18, 19, . These efficacies are far higher than that calculated in the present study, and therefore the comparative data provides a further clear indication of resistance in the \"NMBU ID 35\" isolate. Toltrazuril has been marketed for anticoccidial treatment in sheep since the 1980s, and its use has increased during recent years, both in Norway and in the UK Dr Gillian Diesel, personal communication . Extensive use of a drug over time may result in decreased efficacy, possibly due to the haploid stages of Eimeria, which immediately select for resistance .", "Toltrazuril has been marketed for anticoccidial treatment in sheep since the 1980s, and its use has increased during recent years, both in Norway and in the UK Dr Gillian Diesel, personal communication . Extensive use of a drug over time may result in decreased efficacy, possibly due to the haploid stages of Eimeria, which immediately select for resistance . Since toltrazuril is the only registered anticoccidial for sheep in several countries, development of resistance in ovine Eimeria species may result in there being few treatment options available for sheep farmers, especially in northern Europe . Diclazuril is an anticoccidial that has been registered for treatment of sheep in several countries, but as it may share a common mode of action to that of toltrazuril , cross-resistance between these two triazine-derivates in ovine Eimeria spp. seems highly likely and should be investigated. Indeed, cross-resistance between diclazuril and toltrazuril was reported for an isolate of avian Eimeria spp.", "seems highly likely and should be investigated. Indeed, cross-resistance between diclazuril and toltrazuril was reported for an isolate of avian Eimeria spp. over 20 years ago . Our results indicate that there is a clear need for tools for evaluating ACE, such that inefficient treatments and, thus, the potential for reduced animal welfare and productivity can be avoided. Such tools are available for poultry, using different metrics, such as oocyst index, body weight gain, relative weight gain, lesion scores and anticoccidial index . However, such methods have not yet been established for use in ruminants , with Fig. 4 Box-and-whisker plots with outliers illustrating the histology score.", "However, such methods have not yet been established for use in ruminants , with Fig. 4 Box-and-whisker plots with outliers illustrating the histology score. The score was a summation of all histological parameters evaluated see text in the 20 Eimeria spp. infected lambs, red: toltrazuril treated, and blue: controls the exception of the newly published FOCRT . Although FOCRT may serve as a tool for field evaluation of ACE, there is a clear requirement for further testing of its use in different settings. Confirmation of the spectre of resistance in ovine Eimeria species increases the urgency of identifying alternative treatments and optimising other control strategies.", "Although FOCRT may serve as a tool for field evaluation of ACE, there is a clear requirement for further testing of its use in different settings. Confirmation of the spectre of resistance in ovine Eimeria species increases the urgency of identifying alternative treatments and optimising other control strategies. The anticoccidial effects of different plants and natural extracts, such as sainfoin Onobrychis viciifolia , carob pods Ceratonia siliqua , pomegranate Punica granatum peel extract, grape seed proanthocyanidin extracts, and different natural antioxidants, have been investigated in vivo and in vitro in different hosts . However, none of these bioactive substances have, as yet, been brought to the market for the prevention of clinical coccidiosis. In addition, there are vaccines available for avian Eimeria spp. , and successful immunisation of goat kids with attenuated Eimeria spp.", "In addition, there are vaccines available for avian Eimeria spp. , and successful immunisation of goat kids with attenuated Eimeria spp. oocysts has been performed . Future studies are necessary in order to develop a commercial vaccine against ovine Eimeria spp. Therefore, current efforts should focus on identifying ACE, and maintaining the efficacy of toltrazuril in susceptible flocks. Management strategies that decrease the need for anticoccidials by reducing the infection pressure, possibly achieved by applying strict hygienic measures, and improved flock and pasture management should be actively encouraged by veterinarians and agricultural policy incentives .", "Therefore, current efforts should focus on identifying ACE, and maintaining the efficacy of toltrazuril in susceptible flocks. Management strategies that decrease the need for anticoccidials by reducing the infection pressure, possibly achieved by applying strict hygienic measures, and improved flock and pasture management should be actively encouraged by veterinarians and agricultural policy incentives . Additionally, farmers should be informed about the importance of correct drenching techniques, including dosage estimation and drench gun calibration, as these have been shown to be inadequate in several farms . To our knowledge, this is the first report of ACR against toltrazuril in an ovine Eimeria field isolate, which included the highly pathogenic species, E. ovinoidalis. The results also support the use of FOCRT for field evaluation of ACE. However, the distribution and prevalence of ACR is unknown and further studies are warranted.", "The results also support the use of FOCRT for field evaluation of ACE. However, the distribution and prevalence of ACR is unknown and further studies are warranted. In the future, difficulties in managing coccidiosis without chemotherapy, due to few available treatment options, may severely affect both animal welfare and the economy of the sheep industry. Additional file 1: Table S1 . Information about the 20 lambs infected with Eimeria spp. at day 0. PDF 22 kb Additional file 2: Table S2 . Histopathological findings from toltrazuril treated lambs and controls euthanized 17-24 days post-infection with 100,000 Eimeria oocysts.", "at day 0. PDF 22 kb Additional file 2: Table S2 . Histopathological findings from toltrazuril treated lambs and controls euthanized 17-24 days post-infection with 100,000 Eimeria oocysts. PDF 118 kb Abbreviations ACE: anticoccidial efficacy; ACR: anticoccidial resistance; CET: controlled efficacy trial; FOCRT: faecal oocyst count reduction test; hct: haematocrit; OPG: oocysts per gram" ]

[ "BACKGROUND: Coccidiosis due to Eimeria spp. infections in lambs causes increased mortality and substantial production losses, and anticoccidials are important for control of the infection. Anticoccidial resistance has been reported in poultry and swine, and we recently described reduced toltrazuril efficacy in ovine Eimeria spp. in some Norwegian sheep farms using a newly developed faecal oocyst count reduction test FOCRT . The aim of the present study was to use a controlled efficacy trial to assess the efficacy of toltrazuril against a field isolate suspected of being resistant. METHODS: Twenty lambs, 17–22 days old and raised protected against exposure to coccidia, were infected with a field isolate of 100,000 Eimeria spp. oocysts.", "METHODS: Twenty lambs, 17–22 days old and raised protected against exposure to coccidia, were infected with a field isolate of 100,000 Eimeria spp. oocysts. This isolate was obtained from a farm with a previously calculated drug efficacy of 56% 95% confidence interval: -433.9 to 96.6% . At day 7 post-infection, 10 of the lambs were orally treated with 20 mg/kg toltrazuril Baycox Sheep vet., Bayer Animal Health , while the other 10 lambs controls were given physiological saline. Clinical examinations were conducted, and weight gains recorded. Daily faecal samples were scored for diarrhoea on a scale from 1 to 5, and oocyst excretion was determined using a modified McMaster technique.", "Clinical examinations were conducted, and weight gains recorded. Daily faecal samples were scored for diarrhoea on a scale from 1 to 5, and oocyst excretion was determined using a modified McMaster technique. Oocysts were morphologically identified to species level. At 17–24 days post-infection, the lambs were euthanized and necropsied. RESULTS: The tested Eimeria isolate was resistant against toltrazuril, and resistance was seen in both pathogenic and non-pathogenic species. In addition, no significant differences in faecal score, growth, gross pathology or histological changes were identified between the two groups.", "RESULTS: The tested Eimeria isolate was resistant against toltrazuril, and resistance was seen in both pathogenic and non-pathogenic species. In addition, no significant differences in faecal score, growth, gross pathology or histological changes were identified between the two groups. The pathogenic E. ovinoidalis was the dominant species, and no significant difference in the individual prevalence of E. ovinoidalis post-treatment was found between treated 66.9% and control lambs 61.9% . Other species identified included E. crandallis/weybridgensis, E. parva, E. marsica, E. faurei, E. pallida, E. ahsata and E. bakuensis. CONCLUSIONS: This study confirms toltrazuril resistance in ovine Eimeria spp. ; in addition, the data support the use of FOCRT as an appropriate tool for field evaluation of anticoccidial efficacy.", "CONCLUSIONS: This study confirms toltrazuril resistance in ovine Eimeria spp. ; in addition, the data support the use of FOCRT as an appropriate tool for field evaluation of anticoccidial efficacy. Due to limited anticoccidial treatment alternatives, these findings may have important implications for the sheep industry, particularly in northern Europe. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article 10.1186/s13071-018-2976-4 contains supplementary material, which is available to authorized users. Text: Anticoccidial resistance ACR , which develops mainly as a result of intensive long-term use of anticoccidial drugs, occurs widely in poultry production and has also been identified in Cystoisospora suis in piglets . In addition, a field method for the evaluation of reduced anticoccidial efficacy ACE in ovine Eimeria spp., the faecal oocyst count reduction test FOCRT , has recently been developed and indicated that the efficacy of toltrazuril is reduced in some Norwegian sheep flocks .", "Text: Anticoccidial resistance ACR , which develops mainly as a result of intensive long-term use of anticoccidial drugs, occurs widely in poultry production and has also been identified in Cystoisospora suis in piglets . In addition, a field method for the evaluation of reduced anticoccidial efficacy ACE in ovine Eimeria spp., the faecal oocyst count reduction test FOCRT , has recently been developed and indicated that the efficacy of toltrazuril is reduced in some Norwegian sheep flocks . Infections with Eimeria spp. may impact both animal welfare and productivity in the sheep industry, and controlling the infection is important to minimise mortality and morbidity, and to ensure that lamb growth is not compromised . Suggested strategies to control ruminant coccidiosis include pasture management, adequate nutrition, and hygienic measures . However, these measures are often difficult to implement in practice, and the main control approach is often metaphylaxis with anticoccidials .", "Suggested strategies to control ruminant coccidiosis include pasture management, adequate nutrition, and hygienic measures . However, these measures are often difficult to implement in practice, and the main control approach is often metaphylaxis with anticoccidials . Metaphylactic administration of a single oral dose of toltrazuril in the prepatent period has been shown to be effective at reducing clinical signs and maintaining adequate lamb growth rates in different production systems 13, . In contrast, treatment of clinical coccidiosis is considered inefficient due to the extensive intestinal damage already caused by the infection . Loss of sensitivity to toltrazuril, the only anticoccidial registered for use in sheep in the Nordic countries , should therefore be a matter for serious concern for lamb production. The World Association for the Advancement of Veterinary Parasitology guidelines for evaluation of ACE in mammals , states that there is a need for verified methods for evaluation of ACE.", "Loss of sensitivity to toltrazuril, the only anticoccidial registered for use in sheep in the Nordic countries , should therefore be a matter for serious concern for lamb production. The World Association for the Advancement of Veterinary Parasitology guidelines for evaluation of ACE in mammals , states that there is a need for verified methods for evaluation of ACE. Field methods for assessment of drug efficacy, such as the FOCRT and the faecal egg count reduction test used to evaluate anthelmintic efficacy , give only an indication of reduced efficacy, and need verification through controlled efficacy trials CET . In addition, due to the variation in pathogenicity between ovine Eimeria spp., the differentiation of species should be considered separately . The aim of the present study was to perform a CET in order to determine whether different species in a field isolate of ovine Eimeria spp. with suspected ACR, based on the FOCRT , actually demonstrated resistance to toltrazuril.", "The aim of the present study was to perform a CET in order to determine whether different species in a field isolate of ovine Eimeria spp. with suspected ACR, based on the FOCRT , actually demonstrated resistance to toltrazuril. A total of 20 lambs from 8 ewes of the Norwegian White Sheep breed \"Norsk kvit sau\" was included in the study, which was approved by the Norwegian Animal Research Authority ID: 11657 . The ewes were synchronised using Chronogest® CR and PMSG® MSD Animal Health, Buckinghamshire, UK and served by natural mating. Lambs were either snatched at birth n = 16 or delivered by caesarean section n = 4 over a period of 6 days, and thereafter reared artificially. Individual ear tags were used for identification.", "Lambs were either snatched at birth n = 16 or delivered by caesarean section n = 4 over a period of 6 days, and thereafter reared artificially. Individual ear tags were used for identification. Directly after birth, all lambs were washed with Optima pH 4 soap Optima Produkter AS, Norheimsund, Norway and dried before being placed in boxes with expanded metal floors, in groups of four. Infrared heaters were used during the whole trial. An overview of the study groups, including lamb age, birth weight and gender can be found in Additional file 1: Table S1 . Lambs received ovine colostrum from ewes vaccinated against Clostridium spp.", "An overview of the study groups, including lamb age, birth weight and gender can be found in Additional file 1: Table S1 . Lambs received ovine colostrum from ewes vaccinated against Clostridium spp. Covexin-8, Zoetis during the first 30 min of life, followed by colostrum from vaccinated cows Covexin-8, Zoetis during the next 24 h. To avoid cases of haemolytic anaemia, the cow-colostrum had previously been tested on naturally reared lambs. Lambs were then fed ad libitum with a commercial milk replacer Denkamilk, Denkavit, Fiskå, Mølle, Stavanger , using an automatic feeding system Holm & Laue, Godkalven, Figgjo, Norway . The lambs had ad libitum access to water, hay and commercial lamb-starter concentrate FORMEL lam vår, Felleskjøpet, Norway . To ensure that transmission of Eimeria to the lambs via contaminated colostrum and hay could not occur, both were frozen at -75°C for a minimum of 24 h, prior to provision to the lambs.", "The lambs had ad libitum access to water, hay and commercial lamb-starter concentrate FORMEL lam vår, Felleskjøpet, Norway . To ensure that transmission of Eimeria to the lambs via contaminated colostrum and hay could not occur, both were frozen at -75°C for a minimum of 24 h, prior to provision to the lambs. The field isolate of Eimeria spp. was obtained from one of the flocks ID 35 participating in the recent FOCRT study . According to the FOCRT results, toltrazuril had reduced efficacy against Eimeria in two flocks. However, neither of these flocks were available for the CET, due to geographical and practical reasons.", "According to the FOCRT results, toltrazuril had reduced efficacy against Eimeria in two flocks. However, neither of these flocks were available for the CET, due to geographical and practical reasons. Thus, treatment with toltrazuril in the selected flock had been found to have an efficacy of 56.0%, but the results were classified as inconclusive, due to the wide 95% confidence interval CI of -433.9 and 96.6% . To obtain sufficient Eimeria oocysts of this mixed field isolate named \"NMBU ID 35\" for the present study, faecal samples were obtained from 35 lambs in this flock 9 days after toltrazuril treatment Baycox® Sheep vet., Bayer Animal Health, Oslo, Noray . Oocysts were isolated according to Jackson with some modifications. Briefly, faeces were mixed 1:1 with water and filtered.", "Oocysts were isolated according to Jackson with some modifications. Briefly, faeces were mixed 1:1 with water and filtered. The faecal mix filtrate was subsequently mixed 1:1 with saturated sugar-solution density: 1.5 g/l in a plastic container and left to float onto a glass slide. The slide was washed every second hour with deionized water for three consecutive days, and the washings collected. The washings were centrifuged at 2300× g for 20 min, the supernatant discarded and the sediment mixed 1:1 with deionized water in a glass flask with constant aeration. The oocysts in the flask were left to sporulate for 7 days at room temperature. Sporulated oocysts were stored for 18 days at 4°C.", "The oocysts in the flask were left to sporulate for 7 days at room temperature. Sporulated oocysts were stored for 18 days at 4°C. Based on morphology , as seen by light microscopy at 400× magnification see also Faecal samples section , and classification of 300 oocysts, the field isolate consisted of E. parva 32% , E. crandallis/ weybridgensis 25% , E. ovinoidalis 24% , E. faurei 9% , E. marsica 8% , E. pallida 1% , E. ahsata < 1% and E. bakuensis < 1 % . All lambs were infected day 0 at 17-22 days of age, using an oesophageal tube. A dose of approximately 100,000 sporulated oocysts, diluted in water to a total volume of 5 ml, was given to each of the 20 lambs. Then, two randomly selected coin toss lambs from each group of four were orally treated day 7 with 0.4 ml/kg toltrazuril Baycox® Sheep vet.", "A dose of approximately 100,000 sporulated oocysts, diluted in water to a total volume of 5 ml, was given to each of the 20 lambs. Then, two randomly selected coin toss lambs from each group of four were orally treated day 7 with 0.4 ml/kg toltrazuril Baycox® Sheep vet. 50 mg/ml, Bayer Animal Health and the remaining lambs controls were given 0.4 ml/kg of 0.9% NaCl B. Braun Medical AS, Vestskogen, Norway . Clinical examinations were performed daily throughout the trial. Rectal temperature was measured at days 0, 1, 2 and 7, and daily from day 14, and temperatures > 40.5°C were considered as fever. The lambs were weighed once a week using a calibrated weight Kruuse, Drøbak Norway with 0.1 kg sensitivity, until 14 days post-infection, and thereafter three times a week.", "Rectal temperature was measured at days 0, 1, 2 and 7, and daily from day 14, and temperatures > 40.5°C were considered as fever. The lambs were weighed once a week using a calibrated weight Kruuse, Drøbak Norway with 0.1 kg sensitivity, until 14 days post-infection, and thereafter three times a week. Two lambs controls were treated orally with trimethoprim/sulphamethoxasole Bactrim, Roche, Etterstad, Norway during the first three days of life due to suspected Escherichia coli-infection, from which both recovered within 48 h. Six lambs, two controls and four treated with toltrazuril, developed lameness due to interdigital abscessation, and Streptococcus aureus was detected in two lambs. Four lambs recovered without treatment, and two of the lambs recovered after treatment with benzylpenicillinprocaine Penovet vet., Boehringer Ingelheim Vetmedica, Copenhagen, Denmark administered intramuscularly for three days. On clinical examination, special attention was paid to clinical signs associated with Eimeria spp. infections, i.e.", "On clinical examination, special attention was paid to clinical signs associated with Eimeria spp. infections, i.e. dehydration, pyrexia, weakness, anorexia and, in particular, the presence of diarrhoea. Severe haemorrhagic diarrhoea and dehydration in one lamb at day 17, led to euthanasia of that whole group of four lambs. At day 18, another lamb showed signs of haemorrhagic diarrhoea, and all lambs in this group were also euthanized. The remaining three groups were euthanized on days 21, 23, and 24.", "At day 18, another lamb showed signs of haemorrhagic diarrhoea, and all lambs in this group were also euthanized. The remaining three groups were euthanized on days 21, 23, and 24. Blood samples were drawn from v. jugularis using vacuette tubes plain and EDTA-treated; BD, Franklin Lakes, USA at 48 ± 2 h after birth and at days 0, 7 and at euthanasia. Haematology was performed using the ADVIA 120 Haematology system Bayer Diagnostics, Leverkusen, Germany . Dehydration was considered with a haematocrit hct of > 45.0% . Whole blood tubes were centrifuged, and the serum removed and stored at -20°C until further analysis.", "Dehydration was considered with a haematocrit hct of > 45.0% . Whole blood tubes were centrifuged, and the serum removed and stored at -20°C until further analysis. Biochemical analysis was performed by ABX Pentra 400 Horiba, Les Ulis, France , and included analysis of iron, total protein, albumin, urea, creatinine, gamma-glutamyl transferase, glutamate dehydrogenase and beta hydroxybutyric acid. Individual faecal samples from each of the lambs were obtained daily from day 10 of life until the end of the experiment. Visual scoring of faecal consistency was performed on a scale from one to five 1: normal, pelleted; 2: soft; 3: liquid; 4: watery; 5: watery with blood and/or intestinal tissue . A score ≥ 3 was considered as diarrhoea.", "Visual scoring of faecal consistency was performed on a scale from one to five 1: normal, pelleted; 2: soft; 3: liquid; 4: watery; 5: watery with blood and/or intestinal tissue . A score ≥ 3 was considered as diarrhoea. Samples were collected using an in-house \"faecal spoon\" and the faecal samples were put in zip-lock bags, which were vacuum packed Fresh'n'easy, OBH Nordica, Sundbyberg, Sweden , stored at 4°C, and analysed within 37 days. The rate of oocyst excretion was determined using a modified McMaster technique with a theoretical sensitivity of 5 oocysts per gram OPG . One hundred Eimeria oocysts from all samples ≥ 1000 OPG were examined by light microscopy at 400× magnification and identified to species level, using morphological criteria . However, due to their morphological similarity, oocysts of E. crandallis and E. weybridgensis were not differentiated.", "One hundred Eimeria oocysts from all samples ≥ 1000 OPG were examined by light microscopy at 400× magnification and identified to species level, using morphological criteria . However, due to their morphological similarity, oocysts of E. crandallis and E. weybridgensis were not differentiated. Oocyst counts were analysed by the FOCRT , which consists of a two-step procedure. First, timing of treatment and sampling was evaluated, followed by evaluation of treatment efficacy, by comparing post-treatment faecal samples from treated lambs with equivalent samples from untreated controls. Pre-treatment samples sample 1 were obtained on day 7 day of treatment , and post-treatment samples sample 2 were obtained on days 14-18. The FOCRT was then run using the post-treatment oocyst counts for all five possible time intervals 7-11 days between samples 1 and 2.", "Pre-treatment samples sample 1 were obtained on day 7 day of treatment , and post-treatment samples sample 2 were obtained on days 14-18. The FOCRT was then run using the post-treatment oocyst counts for all five possible time intervals 7-11 days between samples 1 and 2. Faecal samples obtained at euthanasia were analysed for rotavirus, coronavirus, Cryptosporidium spp. and general bacteriology. Additional testing for Cryptosporidium spp. was performed in diarrhoeic lambs at the time of infection day 0, n = 10 .", "and general bacteriology. Additional testing for Cryptosporidium spp. was performed in diarrhoeic lambs at the time of infection day 0, n = 10 . Faecal smears were analysed at the Norwegian Veterinary Institute in Oslo for Cryptosporidium by direct immunofluorescence analysis Crypt-a-Glo™, Waterborne Inc., New Orleans, USA , whereas presence of rotavirus and coronavirus were tested by standard diagnostic methods. Samples for bacteriological analyses were obtained from mid-jejunum and the colon spiral, spread on sheep blood agar plates, and incubated under anaerobic and aerobic conditions for 24-48 h at 37°C and 5% CO 2 . In cases of haemorrhagic diarrhoea, additional samples were grown on bromothymol-blue lactose cysteine agar brolactin/CLED agar for potential identification of Salmonella .", "Samples for bacteriological analyses were obtained from mid-jejunum and the colon spiral, spread on sheep blood agar plates, and incubated under anaerobic and aerobic conditions for 24-48 h at 37°C and 5% CO 2 . In cases of haemorrhagic diarrhoea, additional samples were grown on bromothymol-blue lactose cysteine agar brolactin/CLED agar for potential identification of Salmonella . Lambs were euthanized at days 17-24, by intravenous injection with pentobarbital Euthasol vet., Virbac, Sollihøgda, Norway at 140 mg/kg. Standard necropsy was performed immediately thereafter, with emphasis on the intestines. Histological samples were taken from mid-jejunum, proximal and distal ileum, mid and base of caecum, colon spiral, and distal colon, in addition to heart, lung, liver and kidney. The samples were immersion-fixed in 4% formaldehyde, paraffin-embedded, and stained with haematoxylin and eosin HE .", "Histological samples were taken from mid-jejunum, proximal and distal ileum, mid and base of caecum, colon spiral, and distal colon, in addition to heart, lung, liver and kidney. The samples were immersion-fixed in 4% formaldehyde, paraffin-embedded, and stained with haematoxylin and eosin HE . Histological evaluation was performed by light microscopy and a blinded semi-quantitative evaluation single evaluator was done to assess intestinal pathology. Evaluation parameters included changes in: i villi, ii surface epithelium atrophy/attenuation , iii degree of Eimeria-infection, iv hyperaemia, v oedema, vi infiltration of inflammatory cells and vii crypt abscesses, and were scored as follows: 0 = minimal; 1 = little; 2 = moderate; 3 = severe, including half-step grading. In addition, the presence of epithelial necrosis was graded as present . or absent .. A total histology score was calculated for each tissue by summation of all parameters evaluated i-vii .", "In addition, the presence of epithelial necrosis was graded as present . or absent .. A total histology score was calculated for each tissue by summation of all parameters evaluated i-vii . Data were managed in Excel 2013 Microsoft Inc., Redmond, USA , and subsequently analysed in R and Stata 14 Stata Statistical Software: Release 14. Stata-Corp LP, College Station, TX, USA . Evaluation of efficacy was performed according to the FOCRT . For calculations of significance based on means, a t-test was used. P < 0.05 was considered significant. Mean growth rates were above 300 g/day until days 14-16, whereupon mean growth rate decreased to around 0 g/day Fig. 1 .", "P < 0.05 was considered significant. Mean growth rates were above 300 g/day until days 14-16, whereupon mean growth rate decreased to around 0 g/day Fig. 1 . Growth rates increased again from day 21 onwards. The same pattern was observed in both treated and control lambs. From day 15, both treated and control lambs had a mean faecal score of ≥ 3, indicating diarrhoea. The maximum mean faecal score was seen at day 17 3.9 ± 0.2 and day 18 4.4 ± 0.3 in the treated and control groups, respectively.", "From day 15, both treated and control lambs had a mean faecal score of ≥ 3, indicating diarrhoea. The maximum mean faecal score was seen at day 17 3.9 ± 0.2 and day 18 4.4 ± 0.3 in the treated and control groups, respectively. Haemorrhagic diarrhoea was seen from day 14, in two treated and five control lambs, and tenesmus was observed in two control lambs day 17 . An increase in rectal temperature was seen from day 14, with maximum temperatures measured at day 18 40.4 ± 0.4°C and 16 40.9 ± 0.4°C in the treated and control groups, respectively. The mean duration of fever > 40.5°C was 2.3 ± 0.5 days and 1.9 ± 0.4 days for the treated and control groups, respectively. For these parameters, no significant difference between groups were seen at any time.", "The mean duration of fever > 40.5°C was 2.3 ± 0.5 days and 1.9 ± 0.4 days for the treated and control groups, respectively. For these parameters, no significant difference between groups were seen at any time. At euthanasia, the mean hct was 39.2 ± 1.7% and 41.4 ± 1.9% in the treated and control groups, respectively. However, dehydration hct > 45.0% was only seen in 3 lambs, of which one had been treated with toltrazuril. Mean total serum protein decreased in both groups from infection to euthanasia, but no significant differences between the groups were observed. Other biochemical parameters were within normal ranges data not shown .", "Mean total serum protein decreased in both groups from infection to euthanasia, but no significant differences between the groups were observed. Other biochemical parameters were within normal ranges data not shown . Oocyst excretion was first recorded in one treated lamb at day 10 10 OPG , followed by oocyst excretion in all lambs in both groups from day 14 onwards. Peak oocyst excretion was seen in the treated group at day 20 mean OPG: 5,438,500 , and in the control group at day 21 after infection mean OPG: 3,630,850 Fig. 2 . Thereafter, oocyst excretion decreased. There was no significant difference in oocyst excretion and species distribution between the groups at any time.", "2 . Thereafter, oocyst excretion decreased. There was no significant difference in oocyst excretion and species distribution between the groups at any time. All species present in the field isolate were isolated from the faecal samples of all the 20 infected lambs. E. ovinoidalis was the most prevalent species in both treated and control lambs Table 1 . Efficacy, according to the FOCRT, was evaluated with confidence if the slope was ≥ 0.75, and with caution if slope was ≥ 0.5 and < 0.75 . The slope ranged from 1.24 to 1.69 for the total oocyst excretion in the control lambs.", "Efficacy, according to the FOCRT, was evaluated with confidence if the slope was ≥ 0.75, and with caution if slope was ≥ 0.5 and < 0.75 . The slope ranged from 1.24 to 1.69 for the total oocyst excretion in the control lambs. Slopes, maximum likelihood estimates, and 95% CIs for the geometric mean efficacy of all oocysts, E. ovinoidalis, E. crandallis/weybridgensis, and the non-pathogenic Eimeria spp. are presented in Table 2 ; reduced efficacy of toltrazuril is apparent against both pathogenic and non-pathogenic species. The slope was ≥ 0.75 for all time intervals and species, except for four of the five time intervals of E. crandallis/weybridgensis. Samples analysed for Cryptosporidium spp., Salmonella, coronavirus and rotavirus were all negative.", "The slope was ≥ 0.75 for all time intervals and species, except for four of the five time intervals of E. crandallis/weybridgensis. Samples analysed for Cryptosporidium spp., Salmonella, coronavirus and rotavirus were all negative. Bacteriological analyses showed a mixed flora, dominated by coliforms and Enterococcus spp. Gross pathological findings included diffused thickened and folded ileal mucosa 7 treated and 7 controls , and fibrinous ileal content in two lambs one treated and one control . Nodular or plaque-like foci in the ileal mucosa were seen in 4 treated and 6 control lambs Fig. 3a . The regional distal jejunal lymph nodes were moderately increased in size and oedematous in 5 treated and 6 control lambs.", "3a . The regional distal jejunal lymph nodes were moderately increased in size and oedematous in 5 treated and 6 control lambs. Finally, watery abomasal content was seen in > 50 % of the animals in both groups. Microscopy evaluation showed lesions, mainly in the ileum, caecum and colon, with minor lesions in the jejunum Fig. 3b-f . However, there were no significant differences with respect to histological scores between the treated and control groups in any of the intestinal segments. The highest calculated histological score was found in the proximal ileum and at the base of caecum Fig. 4 .", "The highest calculated histological score was found in the proximal ileum and at the base of caecum Fig. 4 . The mean score for each parameter can be found in Additional file 2: Table S2 . Varying quantities of intracellular Eimeria stages were observed in all intestinal segments, except from jejunum, and they were mostly located in the villus epithelium, with fewer parasites in the crypt epithelium and lamina propria, and few in the submucosa and lymphatic vessels. In both treated and control lambs, changes in the intestinal surfaces varied from light atrophy of the jejunal epithelium and blunting of affected ileal villi Fig. 3b , to areas of total flattening, attenuation of surface epithelium Fig.", "In both treated and control lambs, changes in the intestinal surfaces varied from light atrophy of the jejunal epithelium and blunting of affected ileal villi Fig. 3b , to areas of total flattening, attenuation of surface epithelium Fig. 3e and necrosis Fig. 3d . Patches of epithelial necrosis were found in all lambs. Infiltration of inflammatory cells included mostly monocytes and eosinophils, but also neutrophils and macrophages, and was found in both the lamina propria and submucosa. Different degrees of oedema, hyperaemia, and haemorrhage were seen in all tissue sections examined, and in both treated and control lambs. Crypt abscesses Fig.", "Different degrees of oedema, hyperaemia, and haemorrhage were seen in all tissue sections examined, and in both treated and control lambs. Crypt abscesses Fig. 3b were found in varying degree in all lambs, and contained inflammatory cells, debris and different stages of Eimeria spp. As far as we know, this is the first report of experimentally confirmed toltrazuril resistance in a field isolate of ovine Eimeria spp. The results also support the use of FOCRT as a tool to evaluate ACE in the field. Although ten of the 20 lambs experimentally infected with Eimeria were metaphylactically treated with the recommended dose of 20 mg/kg toltrazuril Baycox® Sheep vet., Bayer Animal Health , this treatment did not result in a significant reduction in oocyst excretion in the treated animals, compared with the controls.", "The results also support the use of FOCRT as a tool to evaluate ACE in the field. Although ten of the 20 lambs experimentally infected with Eimeria were metaphylactically treated with the recommended dose of 20 mg/kg toltrazuril Baycox® Sheep vet., Bayer Animal Health , this treatment did not result in a significant reduction in oocyst excretion in the treated animals, compared with the controls. In addition, no significant differences were noted in clinical presentation, gross pathology, and histopathological findings. The speciation data showed that both pathogenic and non-pathogenic species of Eimeria in this isolate were resistant to toltrazuril. The lambs excreted high numbers of oocysts, as has previously been recorded in experimental infections with multiple Eimeria spp. .", "The lambs excreted high numbers of oocysts, as has previously been recorded in experimental infections with multiple Eimeria spp. . Although oocyst excretion decreased from around day 20 after infection, the total duration of excretion could not be determined, as the lambs were euthanized. The excretion pattern noted here, with an exponential increase, a plateau phase, and a decline, has previously been noted in experimental infections . However, due to continuous reinfection under natural field conditions, the duration of oocyst excretion may be longer than observed in the present study. This might also explain why the calculated slope seen for all species in this experimental study is higher than the slopes reported from the preceding field trial .", "However, due to continuous reinfection under natural field conditions, the duration of oocyst excretion may be longer than observed in the present study. This might also explain why the calculated slope seen for all species in this experimental study is higher than the slopes reported from the preceding field trial . Multi-species resistance, as observed here, has also been noted in field isolates of avian Eimeria spp. . Notes: The estimates were based on post-treatment oocyst counts for five time intervals between sample 1 day 7 after infection and sample 2, and was calculated according to the FOCRT . A slope ≥ 0.5 and < 0.75 was evaluated with caution, whereas a slope < 0.5 was interpreted as invalid a Four lambs were euthanized at day 17 Abbreviations: E. ovi, E. ovinoidalis; E. c/w, E. crandallis/weybridgensis; Non-pathogenic, all species except E. ovinoidalis and E. crandallis/weybridgensis Of particular importance in this study is that E. ovinoidalis was the dominant species excreted from infected lambs.", "Notes: The estimates were based on post-treatment oocyst counts for five time intervals between sample 1 day 7 after infection and sample 2, and was calculated according to the FOCRT . A slope ≥ 0.5 and < 0.75 was evaluated with caution, whereas a slope < 0.5 was interpreted as invalid a Four lambs were euthanized at day 17 Abbreviations: E. ovi, E. ovinoidalis; E. c/w, E. crandallis/weybridgensis; Non-pathogenic, all species except E. ovinoidalis and E. crandallis/weybridgensis Of particular importance in this study is that E. ovinoidalis was the dominant species excreted from infected lambs. As this species is one of the most pathogenic Eimeria spp. in sheep , resistance against the most commonly used anticoccidial drug indicates that severe clinical coccidiosis may be expected to occur in resistant flocks. Although E. ovinoidalis was the dominant species excreted, the most prevalent species in the original field-isolate inoculum was E. parva. This could reflect similarities between E. ovinoidalis and E. ninakholyakimovae in goats, the latter of which develops macroschizonts in endothelial cells, resulting in the release of thousands of merozoites .", "Although E. ovinoidalis was the dominant species excreted, the most prevalent species in the original field-isolate inoculum was E. parva. This could reflect similarities between E. ovinoidalis and E. ninakholyakimovae in goats, the latter of which develops macroschizonts in endothelial cells, resulting in the release of thousands of merozoites . Thus, the extent of intracellular multiplication/ replication, which is presumably also related to the extent of pathogenicity associated with this species, is higher for E. ovinoidalis than for the other Eimeria species. For E. crandallis/weybridgensis, the FOCRT calculations showed invalid results from three of the five sampling time points, probably due to the tests being performed too early in the infection. Excretion of E. crandallis/weybridgensis increased predominantly from day 16 onwards, and euthanasia was performed at days 17-24. Thus, the longer prepatent periods for these species compared with E. ovinoidalis probably explain these results.", "Excretion of E. crandallis/weybridgensis increased predominantly from day 16 onwards, and euthanasia was performed at days 17-24. Thus, the longer prepatent periods for these species compared with E. ovinoidalis probably explain these results. This is an important finding, as the number of invalid farms tested in the FOCRT might have been fewer should sample 2 have been collected 10-11 days after sample 1. These findings also highlight the fact that although Eimeria spp. are often considered as a relatively uniform group, they are in fact separate species with potentially important differences in biology and pathogenic potential. Two of the lambs were treated with trimethoprim/ sulpha during their first days of life, preparations that have been shown to be effective in treating ovine coccidiosis .", "are often considered as a relatively uniform group, they are in fact separate species with potentially important differences in biology and pathogenic potential. Two of the lambs were treated with trimethoprim/ sulpha during their first days of life, preparations that have been shown to be effective in treating ovine coccidiosis . However, withdrawal periods for comparable drugs licenced in cattle are 10-15 days for meat , and these lambs were treated > 17 days prior to the experimental infection. In addition, these treated lambs were in the control group, and therefore this treatment should not have affected the results of the study. Similar clinical signs as observed here might be caused by Cryptosporidium spp., coronavirus, rotavirus, and Salmonella spp., but none of these pathogens were detected. In addition, the findings of coliforms and Enterococcus spp.", "Similar clinical signs as observed here might be caused by Cryptosporidium spp., coronavirus, rotavirus, and Salmonella spp., but none of these pathogens were detected. In addition, the findings of coliforms and Enterococcus spp. may be considered as normal intestinal flora of lambs . The observed clinical signs were therefore almost certainly caused by Eimeria spp., particularly the two major pathogenic species, E. ovinoidalis and E. crandallis . Thickened ileal mucosa is often seen in lambs infected with E. ovinoidalis . In addition, the histological changes, such as blunted villi and surface necrosis, as well as the presence of coccidia, hyperaemia, oedema, infiltration of inflammatory cells and crypt abscesses, are also in accordance with previous reports .", "Thickened ileal mucosa is often seen in lambs infected with E. ovinoidalis . In addition, the histological changes, such as blunted villi and surface necrosis, as well as the presence of coccidia, hyperaemia, oedema, infiltration of inflammatory cells and crypt abscesses, are also in accordance with previous reports . To improve our study, an additional group of uninfected lambs might have been advantageous as this would have enabled better comparisons between weight gain and histopathological changes. However, this was not feasible at the time of the study. Furthermore, due to the lack of defined cut-off values for ACE, it might have been advantageous to include an oocyst isolate from a non-suspected farm i.e. a susceptible isolate .", "Furthermore, due to the lack of defined cut-off values for ACE, it might have been advantageous to include an oocyst isolate from a non-suspected farm i.e. a susceptible isolate . This would have enabled comparisons of different parameters, such as oocyst excretion, between treated and control lambs infected with susceptible or resistant Eimeria spp. However, due to lack of tools for selection of such susceptible ovine Eimeria isolates, we therefore chose to restrict our CET to treated and control lambs infected with isolate \"NMBU ID 35\" as a first step in the characterisation of anticoccidial resistance in ovine Eimeria spp. Although the initial efficacy values have not been provided for toltrazuril by the manufacturer, several studies have investigated its effect on oocyst excretion. For example, its efficacy has been found to be 96.9-99.9% in the period from 7 to 98 days after first treatment, in a study in which the lambs were treated every 14 days .", "Although the initial efficacy values have not been provided for toltrazuril by the manufacturer, several studies have investigated its effect on oocyst excretion. For example, its efficacy has been found to be 96.9-99.9% in the period from 7 to 98 days after first treatment, in a study in which the lambs were treated every 14 days . Other studies have shown toltrazuril efficacies either provided in the publication or calculated as 1- mean OPG treated group / mean OPG control group from data in the publication ranging from 90.0 to 100.0% in the period from two to three weeks after treatment 13, 18, 19, . These efficacies are far higher than that calculated in the present study, and therefore the comparative data provides a further clear indication of resistance in the \"NMBU ID 35\" isolate. Toltrazuril has been marketed for anticoccidial treatment in sheep since the 1980s, and its use has increased during recent years, both in Norway and in the UK Dr Gillian Diesel, personal communication . Extensive use of a drug over time may result in decreased efficacy, possibly due to the haploid stages of Eimeria, which immediately select for resistance .", "Toltrazuril has been marketed for anticoccidial treatment in sheep since the 1980s, and its use has increased during recent years, both in Norway and in the UK Dr Gillian Diesel, personal communication . Extensive use of a drug over time may result in decreased efficacy, possibly due to the haploid stages of Eimeria, which immediately select for resistance . Since toltrazuril is the only registered anticoccidial for sheep in several countries, development of resistance in ovine Eimeria species may result in there being few treatment options available for sheep farmers, especially in northern Europe . Diclazuril is an anticoccidial that has been registered for treatment of sheep in several countries, but as it may share a common mode of action to that of toltrazuril , cross-resistance between these two triazine-derivates in ovine Eimeria spp. seems highly likely and should be investigated. Indeed, cross-resistance between diclazuril and toltrazuril was reported for an isolate of avian Eimeria spp.", "seems highly likely and should be investigated. Indeed, cross-resistance between diclazuril and toltrazuril was reported for an isolate of avian Eimeria spp. over 20 years ago . Our results indicate that there is a clear need for tools for evaluating ACE, such that inefficient treatments and, thus, the potential for reduced animal welfare and productivity can be avoided. Such tools are available for poultry, using different metrics, such as oocyst index, body weight gain, relative weight gain, lesion scores and anticoccidial index . However, such methods have not yet been established for use in ruminants , with Fig. 4 Box-and-whisker plots with outliers illustrating the histology score.", "However, such methods have not yet been established for use in ruminants , with Fig. 4 Box-and-whisker plots with outliers illustrating the histology score. The score was a summation of all histological parameters evaluated see text in the 20 Eimeria spp. infected lambs, red: toltrazuril treated, and blue: controls the exception of the newly published FOCRT . Although FOCRT may serve as a tool for field evaluation of ACE, there is a clear requirement for further testing of its use in different settings. Confirmation of the spectre of resistance in ovine Eimeria species increases the urgency of identifying alternative treatments and optimising other control strategies.", "Although FOCRT may serve as a tool for field evaluation of ACE, there is a clear requirement for further testing of its use in different settings. Confirmation of the spectre of resistance in ovine Eimeria species increases the urgency of identifying alternative treatments and optimising other control strategies. The anticoccidial effects of different plants and natural extracts, such as sainfoin Onobrychis viciifolia , carob pods Ceratonia siliqua , pomegranate Punica granatum peel extract, grape seed proanthocyanidin extracts, and different natural antioxidants, have been investigated in vivo and in vitro in different hosts . However, none of these bioactive substances have, as yet, been brought to the market for the prevention of clinical coccidiosis. In addition, there are vaccines available for avian Eimeria spp. , and successful immunisation of goat kids with attenuated Eimeria spp.", "In addition, there are vaccines available for avian Eimeria spp. , and successful immunisation of goat kids with attenuated Eimeria spp. oocysts has been performed . Future studies are necessary in order to develop a commercial vaccine against ovine Eimeria spp. Therefore, current efforts should focus on identifying ACE, and maintaining the efficacy of toltrazuril in susceptible flocks. Management strategies that decrease the need for anticoccidials by reducing the infection pressure, possibly achieved by applying strict hygienic measures, and improved flock and pasture management should be actively encouraged by veterinarians and agricultural policy incentives .", "Therefore, current efforts should focus on identifying ACE, and maintaining the efficacy of toltrazuril in susceptible flocks. Management strategies that decrease the need for anticoccidials by reducing the infection pressure, possibly achieved by applying strict hygienic measures, and improved flock and pasture management should be actively encouraged by veterinarians and agricultural policy incentives . Additionally, farmers should be informed about the importance of correct drenching techniques, including dosage estimation and drench gun calibration, as these have been shown to be inadequate in several farms . To our knowledge, this is the first report of ACR against toltrazuril in an ovine Eimeria field isolate, which included the highly pathogenic species, E. ovinoidalis. The results also support the use of FOCRT for field evaluation of ACE. However, the distribution and prevalence of ACR is unknown and further studies are warranted.", "The results also support the use of FOCRT for field evaluation of ACE. However, the distribution and prevalence of ACR is unknown and further studies are warranted. In the future, difficulties in managing coccidiosis without chemotherapy, due to few available treatment options, may severely affect both animal welfare and the economy of the sheep industry. Additional file 1: Table S1 . Information about the 20 lambs infected with Eimeria spp. at day 0. PDF 22 kb Additional file 2: Table S2 . Histopathological findings from toltrazuril treated lambs and controls euthanized 17-24 days post-infection with 100,000 Eimeria oocysts.", "at day 0. PDF 22 kb Additional file 2: Table S2 . Histopathological findings from toltrazuril treated lambs and controls euthanized 17-24 days post-infection with 100,000 Eimeria oocysts. PDF 118 kb Abbreviations ACE: anticoccidial efficacy; ACR: anticoccidial resistance; CET: controlled efficacy trial; FOCRT: faecal oocyst count reduction test; hct: haematocrit; OPG: oocysts per gram" ]

[ "In order to improve clinical management and prevention of viral infections in hospitalised children improved etiological insight is needed. The aim of the present study was to assess the spectrum of respiratory viral pathogens in children admitted to hospital with acute respiratory tract infections in Cyprus. For this purpose nasopharyngeal swab samples from 424 children less than 12 years of age with acute respiratory tract infections were collected over three epidemic seasons and were analysed for the presence of the most common 15 respiratory viruses. A viral pathogen was identified in 86% of the samples, with multiple infections being observed in almost 20% of the samples. The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. While RSV and PIV3 incidence decreased significantly with age, the opposite was observed for influenza A and B as well as adenovirus infections. The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections. Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood .", "Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood . Illnesses caused by respiratory viruses include, among others, common colds, pharyngitis, croup, bronchiolitis, viral pneumonia and otitis media. Rapid diagnosis is important not only for timely therapeutic intervention but also for the identification of a beginning influenza epidemic and the avoidance of unnecessary antibiotic treatment . RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children.", "RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children. Respiratory infections cluster during winter and early spring months. The leading viral agents include respiratory syncytial virus RSV , influenza A and B INF-A, INF-B viruses, parainfluenza viruses PIVs , and human adenoviruses HAdVs . In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization.", "In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization. URTIs cause non-severe but widespread epidemics that are responsible for continuous circulation of pathogens in the community. LRTIs have been classified as frank pneumonia and bronchiolitis with clinical, radiological and etiological features that usually overlap . Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons.", "Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons. The study was approved by the Cyprus National Bioethics Committee. Accordingly, written informed consent was obtained from parents prior to sample taking. Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded.", "Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded. Nasal swab samples were collected using the BD Universal Viral Transport Collection Kit. Viral RNA/DNA was extracted from 400 μl sample using the iPrep PureLink Virus Kit on an iPrep purification instrument Invitrogen . A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations.", "A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations. For this purpose, all available complete genome sequences were obtained for each virus from GenBank, imported into the BioEdit Sequence Alignment Editor v7.1.7 and aligned using ClustalX. In case of mismatches between published primers/probe and target sequences, modifications were applied, as indicated in Table 1 . The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing.", "The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing. In order to assess the sensitivity and specificity of the assays, the laboratory enrolled for two consecutive years in Quality Control for Molecular Diagnostics QCMD external quality assessment schemes for all viruses, except Bocavirus, which was unavailable. In summary, the established assays were able to correctly identify all viruses tested, proving their suitability for diagnostic application. A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed.", "A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed. The same statistical tests were used to compare the frequency of subjects with single or multiple infections between age groups. In addition, Pearson correlation was used to examine co-infections of different viruses. All statistical analyses were performed using StataSE 12 StatCorp. 2007. College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus.", "College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus. The median age of the children was 15 months range: 0-140 months with 243 being male and 181 female male/ female ratio 1.34 . The age distribution is shown in Fig 1. Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections.", "Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections. With moderate frequency have been detected HAdV in 31 7.3% patients, influenza A in 28 6.6% , HBoV in 24 5.7% , enteroviruses and PIV 3 in 23 5.4% of patients respectively, and Influenza B in 21 5.0% . A low frequency was exhibited by HMPV with 16 3.8% positive samples, human coronavirus OC43 with 13 3.1% , PIV 1 with 12 2.8% , PIV 4 with 9 2.1% , PIV 2 with 7 1.7% and HCoV NL63 with 6 1.4% . Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 .", "Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 . Dual infections accounted for 17% of all positive samples and three viruses were detected in 2.7% of samples . A single patient sample displayed a quadruple infection being simultaneously positive for RSV, rhinovirus, HBoV and influenza B. Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus.", "Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus. As a percentage, however, the virus appearing most often in co- infections was HBoV, which was found in more than 70% of cases together with another virus, followed by coronaviruses HCoV OC43 and HCoV NL63 with 61% and 67%, respectively. On the other hand, the viruses most rarely seen in co-infections were influenza viruses A and B as well as RSV. Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table.", "Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table. Significant correlation P-value < 0.05 was seen mostly for co-infections with RSV, however correlations were very weak r<0.3 and negative. This finding can probably be explained by the fact that RSV infections occurred predominantly in the very young, where co-infections were less frequently observed. On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. However, as more than 100 different rhinovirus strains have been identified to be circulating worldwide in parallel and successively, a potential seasonality of individual rhinovirus serotypes may be masked by overlapping patterns . The data was further analysed with regard to the age distribution of virus infection see Table 2 . In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV.", "In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV. Rhinoviruses, HBoV and enteroviruses are most frequently observed in children from 4 months to 3 years of age. The age dependency of the virus incidence is visualized in Fig 3 for the seven most frequently observed viruses. The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children.", "The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children. Regarding multiple infections, children less than 3 month of age and those older than 4 years had a significantly smaller risk to present with multiple infections as compared to the other two age groups p-value = 0.014 . A reason for this could be that very young children have limited contact to others reducing thereby the chance for a co-infection, whereas children older than 3 years already established immunity to an increasing number of viruses encountered previously. This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way.", "This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way. The high rate of positive samples 85.8% is evidence of the high sensitivity of the Multiplex-assays used and that the range of viruses included in the analysis is comprehensive. Many previous studies have shown detection rates ranging from below 50% to 75% . The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, .", "The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, . Our data support the results of similar studies performed in the Middle East region. A recently published study found that RSV was the most commonly detected virus in nasopharyngeal swabs from children presenting symptoms of RTIs and in addition to that it also showed that RSV infections follow a similar circulation pattern peaking from December to March . Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%.", "Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%. HBoV, HCoV and EV were found most frequently in co-infections. All three subtypes of HCoV were co-detected with several other viruses, while HBoV was co-detected mainly with HRV and RSV. In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere .", "In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere . Even though this study did not allow for investigating a possible association between multiple infections and disease severity, a review of the literature shows that such a potential association is still subject to controversy, since there are reports showing no relationship of multiple virus infection with respiratoty illness severity on one hand or a significant association on the other. Studies have shown that viral co-infection was significantly associated with longer duration of illness symptoms, but with a decreased severity in hospitalized children regarding oxygen requirement and intensive care unit admission, whereas the findings of other studies have indicated that severe clinical phenotypes were more prevalent in co-infection patients, especially in RSV co-infections that may increase the severity of RSV associated disease in children 25, . Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures .", "Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures . The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections." ]

[ "In order to improve clinical management and prevention of viral infections in hospitalised children improved etiological insight is needed. The aim of the present study was to assess the spectrum of respiratory viral pathogens in children admitted to hospital with acute respiratory tract infections in Cyprus. For this purpose nasopharyngeal swab samples from 424 children less than 12 years of age with acute respiratory tract infections were collected over three epidemic seasons and were analysed for the presence of the most common 15 respiratory viruses. A viral pathogen was identified in 86% of the samples, with multiple infections being observed in almost 20% of the samples. The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. While RSV and PIV3 incidence decreased significantly with age, the opposite was observed for influenza A and B as well as adenovirus infections. The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections. Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood .", "Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood . Illnesses caused by respiratory viruses include, among others, common colds, pharyngitis, croup, bronchiolitis, viral pneumonia and otitis media. Rapid diagnosis is important not only for timely therapeutic intervention but also for the identification of a beginning influenza epidemic and the avoidance of unnecessary antibiotic treatment . RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children.", "RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children. Respiratory infections cluster during winter and early spring months. The leading viral agents include respiratory syncytial virus RSV , influenza A and B INF-A, INF-B viruses, parainfluenza viruses PIVs , and human adenoviruses HAdVs . In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization.", "In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization. URTIs cause non-severe but widespread epidemics that are responsible for continuous circulation of pathogens in the community. LRTIs have been classified as frank pneumonia and bronchiolitis with clinical, radiological and etiological features that usually overlap . Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons.", "Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons. The study was approved by the Cyprus National Bioethics Committee. Accordingly, written informed consent was obtained from parents prior to sample taking. Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded.", "Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded. Nasal swab samples were collected using the BD Universal Viral Transport Collection Kit. Viral RNA/DNA was extracted from 400 μl sample using the iPrep PureLink Virus Kit on an iPrep purification instrument Invitrogen . A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations.", "A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations. For this purpose, all available complete genome sequences were obtained for each virus from GenBank, imported into the BioEdit Sequence Alignment Editor v7.1.7 and aligned using ClustalX. In case of mismatches between published primers/probe and target sequences, modifications were applied, as indicated in Table 1 . The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing.", "The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing. In order to assess the sensitivity and specificity of the assays, the laboratory enrolled for two consecutive years in Quality Control for Molecular Diagnostics QCMD external quality assessment schemes for all viruses, except Bocavirus, which was unavailable. In summary, the established assays were able to correctly identify all viruses tested, proving their suitability for diagnostic application. A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed.", "A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed. The same statistical tests were used to compare the frequency of subjects with single or multiple infections between age groups. In addition, Pearson correlation was used to examine co-infections of different viruses. All statistical analyses were performed using StataSE 12 StatCorp. 2007. College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus.", "College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus. The median age of the children was 15 months range: 0-140 months with 243 being male and 181 female male/ female ratio 1.34 . The age distribution is shown in Fig 1. Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections.", "Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections. With moderate frequency have been detected HAdV in 31 7.3% patients, influenza A in 28 6.6% , HBoV in 24 5.7% , enteroviruses and PIV 3 in 23 5.4% of patients respectively, and Influenza B in 21 5.0% . A low frequency was exhibited by HMPV with 16 3.8% positive samples, human coronavirus OC43 with 13 3.1% , PIV 1 with 12 2.8% , PIV 4 with 9 2.1% , PIV 2 with 7 1.7% and HCoV NL63 with 6 1.4% . Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 .", "Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 . Dual infections accounted for 17% of all positive samples and three viruses were detected in 2.7% of samples . A single patient sample displayed a quadruple infection being simultaneously positive for RSV, rhinovirus, HBoV and influenza B. Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus.", "Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus. As a percentage, however, the virus appearing most often in co- infections was HBoV, which was found in more than 70% of cases together with another virus, followed by coronaviruses HCoV OC43 and HCoV NL63 with 61% and 67%, respectively. On the other hand, the viruses most rarely seen in co-infections were influenza viruses A and B as well as RSV. Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table.", "Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table. Significant correlation P-value < 0.05 was seen mostly for co-infections with RSV, however correlations were very weak r<0.3 and negative. This finding can probably be explained by the fact that RSV infections occurred predominantly in the very young, where co-infections were less frequently observed. On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. However, as more than 100 different rhinovirus strains have been identified to be circulating worldwide in parallel and successively, a potential seasonality of individual rhinovirus serotypes may be masked by overlapping patterns . The data was further analysed with regard to the age distribution of virus infection see Table 2 . In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV.", "In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV. Rhinoviruses, HBoV and enteroviruses are most frequently observed in children from 4 months to 3 years of age. The age dependency of the virus incidence is visualized in Fig 3 for the seven most frequently observed viruses. The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children.", "The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children. Regarding multiple infections, children less than 3 month of age and those older than 4 years had a significantly smaller risk to present with multiple infections as compared to the other two age groups p-value = 0.014 . A reason for this could be that very young children have limited contact to others reducing thereby the chance for a co-infection, whereas children older than 3 years already established immunity to an increasing number of viruses encountered previously. This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way.", "This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way. The high rate of positive samples 85.8% is evidence of the high sensitivity of the Multiplex-assays used and that the range of viruses included in the analysis is comprehensive. Many previous studies have shown detection rates ranging from below 50% to 75% . The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, .", "The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, . Our data support the results of similar studies performed in the Middle East region. A recently published study found that RSV was the most commonly detected virus in nasopharyngeal swabs from children presenting symptoms of RTIs and in addition to that it also showed that RSV infections follow a similar circulation pattern peaking from December to March . Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%.", "Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%. HBoV, HCoV and EV were found most frequently in co-infections. All three subtypes of HCoV were co-detected with several other viruses, while HBoV was co-detected mainly with HRV and RSV. In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere .", "In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere . Even though this study did not allow for investigating a possible association between multiple infections and disease severity, a review of the literature shows that such a potential association is still subject to controversy, since there are reports showing no relationship of multiple virus infection with respiratoty illness severity on one hand or a significant association on the other. Studies have shown that viral co-infection was significantly associated with longer duration of illness symptoms, but with a decreased severity in hospitalized children regarding oxygen requirement and intensive care unit admission, whereas the findings of other studies have indicated that severe clinical phenotypes were more prevalent in co-infection patients, especially in RSV co-infections that may increase the severity of RSV associated disease in children 25, . Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures .", "Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures . The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections." ]

[ "In order to improve clinical management and prevention of viral infections in hospitalised children improved etiological insight is needed. The aim of the present study was to assess the spectrum of respiratory viral pathogens in children admitted to hospital with acute respiratory tract infections in Cyprus. For this purpose nasopharyngeal swab samples from 424 children less than 12 years of age with acute respiratory tract infections were collected over three epidemic seasons and were analysed for the presence of the most common 15 respiratory viruses. A viral pathogen was identified in 86% of the samples, with multiple infections being observed in almost 20% of the samples. The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. While RSV and PIV3 incidence decreased significantly with age, the opposite was observed for influenza A and B as well as adenovirus infections. The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections. Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood .", "Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood . Illnesses caused by respiratory viruses include, among others, common colds, pharyngitis, croup, bronchiolitis, viral pneumonia and otitis media. Rapid diagnosis is important not only for timely therapeutic intervention but also for the identification of a beginning influenza epidemic and the avoidance of unnecessary antibiotic treatment . RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children.", "RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children. Respiratory infections cluster during winter and early spring months. The leading viral agents include respiratory syncytial virus RSV , influenza A and B INF-A, INF-B viruses, parainfluenza viruses PIVs , and human adenoviruses HAdVs . In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization.", "In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization. URTIs cause non-severe but widespread epidemics that are responsible for continuous circulation of pathogens in the community. LRTIs have been classified as frank pneumonia and bronchiolitis with clinical, radiological and etiological features that usually overlap . Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons.", "Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons. The study was approved by the Cyprus National Bioethics Committee. Accordingly, written informed consent was obtained from parents prior to sample taking. Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded.", "Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded. Nasal swab samples were collected using the BD Universal Viral Transport Collection Kit. Viral RNA/DNA was extracted from 400 μl sample using the iPrep PureLink Virus Kit on an iPrep purification instrument Invitrogen . A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations.", "A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations. For this purpose, all available complete genome sequences were obtained for each virus from GenBank, imported into the BioEdit Sequence Alignment Editor v7.1.7 and aligned using ClustalX. In case of mismatches between published primers/probe and target sequences, modifications were applied, as indicated in Table 1 . The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing.", "The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing. In order to assess the sensitivity and specificity of the assays, the laboratory enrolled for two consecutive years in Quality Control for Molecular Diagnostics QCMD external quality assessment schemes for all viruses, except Bocavirus, which was unavailable. In summary, the established assays were able to correctly identify all viruses tested, proving their suitability for diagnostic application. A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed.", "A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed. The same statistical tests were used to compare the frequency of subjects with single or multiple infections between age groups. In addition, Pearson correlation was used to examine co-infections of different viruses. All statistical analyses were performed using StataSE 12 StatCorp. 2007. College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus.", "College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus. The median age of the children was 15 months range: 0-140 months with 243 being male and 181 female male/ female ratio 1.34 . The age distribution is shown in Fig 1. Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections.", "Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections. With moderate frequency have been detected HAdV in 31 7.3% patients, influenza A in 28 6.6% , HBoV in 24 5.7% , enteroviruses and PIV 3 in 23 5.4% of patients respectively, and Influenza B in 21 5.0% . A low frequency was exhibited by HMPV with 16 3.8% positive samples, human coronavirus OC43 with 13 3.1% , PIV 1 with 12 2.8% , PIV 4 with 9 2.1% , PIV 2 with 7 1.7% and HCoV NL63 with 6 1.4% . Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 .", "Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 . Dual infections accounted for 17% of all positive samples and three viruses were detected in 2.7% of samples . A single patient sample displayed a quadruple infection being simultaneously positive for RSV, rhinovirus, HBoV and influenza B. Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus.", "Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus. As a percentage, however, the virus appearing most often in co- infections was HBoV, which was found in more than 70% of cases together with another virus, followed by coronaviruses HCoV OC43 and HCoV NL63 with 61% and 67%, respectively. On the other hand, the viruses most rarely seen in co-infections were influenza viruses A and B as well as RSV. Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table.", "Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table. Significant correlation P-value < 0.05 was seen mostly for co-infections with RSV, however correlations were very weak r<0.3 and negative. This finding can probably be explained by the fact that RSV infections occurred predominantly in the very young, where co-infections were less frequently observed. On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. However, as more than 100 different rhinovirus strains have been identified to be circulating worldwide in parallel and successively, a potential seasonality of individual rhinovirus serotypes may be masked by overlapping patterns . The data was further analysed with regard to the age distribution of virus infection see Table 2 . In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV.", "In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV. Rhinoviruses, HBoV and enteroviruses are most frequently observed in children from 4 months to 3 years of age. The age dependency of the virus incidence is visualized in Fig 3 for the seven most frequently observed viruses. The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children.", "The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children. Regarding multiple infections, children less than 3 month of age and those older than 4 years had a significantly smaller risk to present with multiple infections as compared to the other two age groups p-value = 0.014 . A reason for this could be that very young children have limited contact to others reducing thereby the chance for a co-infection, whereas children older than 3 years already established immunity to an increasing number of viruses encountered previously. This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way.", "This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way. The high rate of positive samples 85.8% is evidence of the high sensitivity of the Multiplex-assays used and that the range of viruses included in the analysis is comprehensive. Many previous studies have shown detection rates ranging from below 50% to 75% . The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, .", "The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, . Our data support the results of similar studies performed in the Middle East region. A recently published study found that RSV was the most commonly detected virus in nasopharyngeal swabs from children presenting symptoms of RTIs and in addition to that it also showed that RSV infections follow a similar circulation pattern peaking from December to March . Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%.", "Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%. HBoV, HCoV and EV were found most frequently in co-infections. All three subtypes of HCoV were co-detected with several other viruses, while HBoV was co-detected mainly with HRV and RSV. In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere .", "In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere . Even though this study did not allow for investigating a possible association between multiple infections and disease severity, a review of the literature shows that such a potential association is still subject to controversy, since there are reports showing no relationship of multiple virus infection with respiratoty illness severity on one hand or a significant association on the other. Studies have shown that viral co-infection was significantly associated with longer duration of illness symptoms, but with a decreased severity in hospitalized children regarding oxygen requirement and intensive care unit admission, whereas the findings of other studies have indicated that severe clinical phenotypes were more prevalent in co-infection patients, especially in RSV co-infections that may increase the severity of RSV associated disease in children 25, . Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures .", "Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures . The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections." ]

[ "In order to improve clinical management and prevention of viral infections in hospitalised children improved etiological insight is needed. The aim of the present study was to assess the spectrum of respiratory viral pathogens in children admitted to hospital with acute respiratory tract infections in Cyprus. For this purpose nasopharyngeal swab samples from 424 children less than 12 years of age with acute respiratory tract infections were collected over three epidemic seasons and were analysed for the presence of the most common 15 respiratory viruses. A viral pathogen was identified in 86% of the samples, with multiple infections being observed in almost 20% of the samples. The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. While RSV and PIV3 incidence decreased significantly with age, the opposite was observed for influenza A and B as well as adenovirus infections. The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections. Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood .", "Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood . Illnesses caused by respiratory viruses include, among others, common colds, pharyngitis, croup, bronchiolitis, viral pneumonia and otitis media. Rapid diagnosis is important not only for timely therapeutic intervention but also for the identification of a beginning influenza epidemic and the avoidance of unnecessary antibiotic treatment . RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children.", "RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children. Respiratory infections cluster during winter and early spring months. The leading viral agents include respiratory syncytial virus RSV , influenza A and B INF-A, INF-B viruses, parainfluenza viruses PIVs , and human adenoviruses HAdVs . In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization.", "In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization. URTIs cause non-severe but widespread epidemics that are responsible for continuous circulation of pathogens in the community. LRTIs have been classified as frank pneumonia and bronchiolitis with clinical, radiological and etiological features that usually overlap . Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons.", "Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons. The study was approved by the Cyprus National Bioethics Committee. Accordingly, written informed consent was obtained from parents prior to sample taking. Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded.", "Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded. Nasal swab samples were collected using the BD Universal Viral Transport Collection Kit. Viral RNA/DNA was extracted from 400 μl sample using the iPrep PureLink Virus Kit on an iPrep purification instrument Invitrogen . A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations.", "A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations. For this purpose, all available complete genome sequences were obtained for each virus from GenBank, imported into the BioEdit Sequence Alignment Editor v7.1.7 and aligned using ClustalX. In case of mismatches between published primers/probe and target sequences, modifications were applied, as indicated in Table 1 . The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing.", "The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing. In order to assess the sensitivity and specificity of the assays, the laboratory enrolled for two consecutive years in Quality Control for Molecular Diagnostics QCMD external quality assessment schemes for all viruses, except Bocavirus, which was unavailable. In summary, the established assays were able to correctly identify all viruses tested, proving their suitability for diagnostic application. A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed.", "A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed. The same statistical tests were used to compare the frequency of subjects with single or multiple infections between age groups. In addition, Pearson correlation was used to examine co-infections of different viruses. All statistical analyses were performed using StataSE 12 StatCorp. 2007. College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus.", "College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus. The median age of the children was 15 months range: 0-140 months with 243 being male and 181 female male/ female ratio 1.34 . The age distribution is shown in Fig 1. Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections.", "Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections. With moderate frequency have been detected HAdV in 31 7.3% patients, influenza A in 28 6.6% , HBoV in 24 5.7% , enteroviruses and PIV 3 in 23 5.4% of patients respectively, and Influenza B in 21 5.0% . A low frequency was exhibited by HMPV with 16 3.8% positive samples, human coronavirus OC43 with 13 3.1% , PIV 1 with 12 2.8% , PIV 4 with 9 2.1% , PIV 2 with 7 1.7% and HCoV NL63 with 6 1.4% . Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 .", "Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 . Dual infections accounted for 17% of all positive samples and three viruses were detected in 2.7% of samples . A single patient sample displayed a quadruple infection being simultaneously positive for RSV, rhinovirus, HBoV and influenza B. Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus.", "Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus. As a percentage, however, the virus appearing most often in co- infections was HBoV, which was found in more than 70% of cases together with another virus, followed by coronaviruses HCoV OC43 and HCoV NL63 with 61% and 67%, respectively. On the other hand, the viruses most rarely seen in co-infections were influenza viruses A and B as well as RSV. Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table.", "Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table. Significant correlation P-value < 0.05 was seen mostly for co-infections with RSV, however correlations were very weak r<0.3 and negative. This finding can probably be explained by the fact that RSV infections occurred predominantly in the very young, where co-infections were less frequently observed. On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. However, as more than 100 different rhinovirus strains have been identified to be circulating worldwide in parallel and successively, a potential seasonality of individual rhinovirus serotypes may be masked by overlapping patterns . The data was further analysed with regard to the age distribution of virus infection see Table 2 . In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV.", "In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV. Rhinoviruses, HBoV and enteroviruses are most frequently observed in children from 4 months to 3 years of age. The age dependency of the virus incidence is visualized in Fig 3 for the seven most frequently observed viruses. The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children.", "The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children. Regarding multiple infections, children less than 3 month of age and those older than 4 years had a significantly smaller risk to present with multiple infections as compared to the other two age groups p-value = 0.014 . A reason for this could be that very young children have limited contact to others reducing thereby the chance for a co-infection, whereas children older than 3 years already established immunity to an increasing number of viruses encountered previously. This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way.", "This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way. The high rate of positive samples 85.8% is evidence of the high sensitivity of the Multiplex-assays used and that the range of viruses included in the analysis is comprehensive. Many previous studies have shown detection rates ranging from below 50% to 75% . The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, .", "The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, . Our data support the results of similar studies performed in the Middle East region. A recently published study found that RSV was the most commonly detected virus in nasopharyngeal swabs from children presenting symptoms of RTIs and in addition to that it also showed that RSV infections follow a similar circulation pattern peaking from December to March . Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%.", "Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%. HBoV, HCoV and EV were found most frequently in co-infections. All three subtypes of HCoV were co-detected with several other viruses, while HBoV was co-detected mainly with HRV and RSV. In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere .", "In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere . Even though this study did not allow for investigating a possible association between multiple infections and disease severity, a review of the literature shows that such a potential association is still subject to controversy, since there are reports showing no relationship of multiple virus infection with respiratoty illness severity on one hand or a significant association on the other. Studies have shown that viral co-infection was significantly associated with longer duration of illness symptoms, but with a decreased severity in hospitalized children regarding oxygen requirement and intensive care unit admission, whereas the findings of other studies have indicated that severe clinical phenotypes were more prevalent in co-infection patients, especially in RSV co-infections that may increase the severity of RSV associated disease in children 25, . Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures .", "Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures . The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections." ]

[ "In order to improve clinical management and prevention of viral infections in hospitalised children improved etiological insight is needed. The aim of the present study was to assess the spectrum of respiratory viral pathogens in children admitted to hospital with acute respiratory tract infections in Cyprus. For this purpose nasopharyngeal swab samples from 424 children less than 12 years of age with acute respiratory tract infections were collected over three epidemic seasons and were analysed for the presence of the most common 15 respiratory viruses. A viral pathogen was identified in 86% of the samples, with multiple infections being observed in almost 20% of the samples. The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. While RSV and PIV3 incidence decreased significantly with age, the opposite was observed for influenza A and B as well as adenovirus infections. The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections. Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood .", "Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood . Illnesses caused by respiratory viruses include, among others, common colds, pharyngitis, croup, bronchiolitis, viral pneumonia and otitis media. Rapid diagnosis is important not only for timely therapeutic intervention but also for the identification of a beginning influenza epidemic and the avoidance of unnecessary antibiotic treatment . RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children.", "RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children. Respiratory infections cluster during winter and early spring months. The leading viral agents include respiratory syncytial virus RSV , influenza A and B INF-A, INF-B viruses, parainfluenza viruses PIVs , and human adenoviruses HAdVs . In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization.", "In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization. URTIs cause non-severe but widespread epidemics that are responsible for continuous circulation of pathogens in the community. LRTIs have been classified as frank pneumonia and bronchiolitis with clinical, radiological and etiological features that usually overlap . Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons.", "Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons. The study was approved by the Cyprus National Bioethics Committee. Accordingly, written informed consent was obtained from parents prior to sample taking. Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded.", "Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded. Nasal swab samples were collected using the BD Universal Viral Transport Collection Kit. Viral RNA/DNA was extracted from 400 μl sample using the iPrep PureLink Virus Kit on an iPrep purification instrument Invitrogen . A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations.", "A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations. For this purpose, all available complete genome sequences were obtained for each virus from GenBank, imported into the BioEdit Sequence Alignment Editor v7.1.7 and aligned using ClustalX. In case of mismatches between published primers/probe and target sequences, modifications were applied, as indicated in Table 1 . The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing.", "The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing. In order to assess the sensitivity and specificity of the assays, the laboratory enrolled for two consecutive years in Quality Control for Molecular Diagnostics QCMD external quality assessment schemes for all viruses, except Bocavirus, which was unavailable. In summary, the established assays were able to correctly identify all viruses tested, proving their suitability for diagnostic application. A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed.", "A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed. The same statistical tests were used to compare the frequency of subjects with single or multiple infections between age groups. In addition, Pearson correlation was used to examine co-infections of different viruses. All statistical analyses were performed using StataSE 12 StatCorp. 2007. College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus.", "College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus. The median age of the children was 15 months range: 0-140 months with 243 being male and 181 female male/ female ratio 1.34 . The age distribution is shown in Fig 1. Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections.", "Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections. With moderate frequency have been detected HAdV in 31 7.3% patients, influenza A in 28 6.6% , HBoV in 24 5.7% , enteroviruses and PIV 3 in 23 5.4% of patients respectively, and Influenza B in 21 5.0% . A low frequency was exhibited by HMPV with 16 3.8% positive samples, human coronavirus OC43 with 13 3.1% , PIV 1 with 12 2.8% , PIV 4 with 9 2.1% , PIV 2 with 7 1.7% and HCoV NL63 with 6 1.4% . Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 .", "Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 . Dual infections accounted for 17% of all positive samples and three viruses were detected in 2.7% of samples . A single patient sample displayed a quadruple infection being simultaneously positive for RSV, rhinovirus, HBoV and influenza B. Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus.", "Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus. As a percentage, however, the virus appearing most often in co- infections was HBoV, which was found in more than 70% of cases together with another virus, followed by coronaviruses HCoV OC43 and HCoV NL63 with 61% and 67%, respectively. On the other hand, the viruses most rarely seen in co-infections were influenza viruses A and B as well as RSV. Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table.", "Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table. Significant correlation P-value < 0.05 was seen mostly for co-infections with RSV, however correlations were very weak r<0.3 and negative. This finding can probably be explained by the fact that RSV infections occurred predominantly in the very young, where co-infections were less frequently observed. On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. However, as more than 100 different rhinovirus strains have been identified to be circulating worldwide in parallel and successively, a potential seasonality of individual rhinovirus serotypes may be masked by overlapping patterns . The data was further analysed with regard to the age distribution of virus infection see Table 2 . In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV.", "In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV. Rhinoviruses, HBoV and enteroviruses are most frequently observed in children from 4 months to 3 years of age. The age dependency of the virus incidence is visualized in Fig 3 for the seven most frequently observed viruses. The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children.", "The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children. Regarding multiple infections, children less than 3 month of age and those older than 4 years had a significantly smaller risk to present with multiple infections as compared to the other two age groups p-value = 0.014 . A reason for this could be that very young children have limited contact to others reducing thereby the chance for a co-infection, whereas children older than 3 years already established immunity to an increasing number of viruses encountered previously. This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way.", "This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way. The high rate of positive samples 85.8% is evidence of the high sensitivity of the Multiplex-assays used and that the range of viruses included in the analysis is comprehensive. Many previous studies have shown detection rates ranging from below 50% to 75% . The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, .", "The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, . Our data support the results of similar studies performed in the Middle East region. A recently published study found that RSV was the most commonly detected virus in nasopharyngeal swabs from children presenting symptoms of RTIs and in addition to that it also showed that RSV infections follow a similar circulation pattern peaking from December to March . Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%.", "Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%. HBoV, HCoV and EV were found most frequently in co-infections. All three subtypes of HCoV were co-detected with several other viruses, while HBoV was co-detected mainly with HRV and RSV. In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere .", "In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere . Even though this study did not allow for investigating a possible association between multiple infections and disease severity, a review of the literature shows that such a potential association is still subject to controversy, since there are reports showing no relationship of multiple virus infection with respiratoty illness severity on one hand or a significant association on the other. Studies have shown that viral co-infection was significantly associated with longer duration of illness symptoms, but with a decreased severity in hospitalized children regarding oxygen requirement and intensive care unit admission, whereas the findings of other studies have indicated that severe clinical phenotypes were more prevalent in co-infection patients, especially in RSV co-infections that may increase the severity of RSV associated disease in children 25, . Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures .", "Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures . The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections." ]

[ "In order to improve clinical management and prevention of viral infections in hospitalised children improved etiological insight is needed. The aim of the present study was to assess the spectrum of respiratory viral pathogens in children admitted to hospital with acute respiratory tract infections in Cyprus. For this purpose nasopharyngeal swab samples from 424 children less than 12 years of age with acute respiratory tract infections were collected over three epidemic seasons and were analysed for the presence of the most common 15 respiratory viruses. A viral pathogen was identified in 86% of the samples, with multiple infections being observed in almost 20% of the samples. The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. While RSV and PIV3 incidence decreased significantly with age, the opposite was observed for influenza A and B as well as adenovirus infections. The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections. Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood .", "Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood . Illnesses caused by respiratory viruses include, among others, common colds, pharyngitis, croup, bronchiolitis, viral pneumonia and otitis media. Rapid diagnosis is important not only for timely therapeutic intervention but also for the identification of a beginning influenza epidemic and the avoidance of unnecessary antibiotic treatment . RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children.", "RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children. Respiratory infections cluster during winter and early spring months. The leading viral agents include respiratory syncytial virus RSV , influenza A and B INF-A, INF-B viruses, parainfluenza viruses PIVs , and human adenoviruses HAdVs . In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization.", "In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization. URTIs cause non-severe but widespread epidemics that are responsible for continuous circulation of pathogens in the community. LRTIs have been classified as frank pneumonia and bronchiolitis with clinical, radiological and etiological features that usually overlap . Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons.", "Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons. The study was approved by the Cyprus National Bioethics Committee. Accordingly, written informed consent was obtained from parents prior to sample taking. Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded.", "Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded. Nasal swab samples were collected using the BD Universal Viral Transport Collection Kit. Viral RNA/DNA was extracted from 400 μl sample using the iPrep PureLink Virus Kit on an iPrep purification instrument Invitrogen . A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations.", "A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations. For this purpose, all available complete genome sequences were obtained for each virus from GenBank, imported into the BioEdit Sequence Alignment Editor v7.1.7 and aligned using ClustalX. In case of mismatches between published primers/probe and target sequences, modifications were applied, as indicated in Table 1 . The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing.", "The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing. In order to assess the sensitivity and specificity of the assays, the laboratory enrolled for two consecutive years in Quality Control for Molecular Diagnostics QCMD external quality assessment schemes for all viruses, except Bocavirus, which was unavailable. In summary, the established assays were able to correctly identify all viruses tested, proving their suitability for diagnostic application. A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed.", "A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed. The same statistical tests were used to compare the frequency of subjects with single or multiple infections between age groups. In addition, Pearson correlation was used to examine co-infections of different viruses. All statistical analyses were performed using StataSE 12 StatCorp. 2007. College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus.", "College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus. The median age of the children was 15 months range: 0-140 months with 243 being male and 181 female male/ female ratio 1.34 . The age distribution is shown in Fig 1. Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections.", "Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections. With moderate frequency have been detected HAdV in 31 7.3% patients, influenza A in 28 6.6% , HBoV in 24 5.7% , enteroviruses and PIV 3 in 23 5.4% of patients respectively, and Influenza B in 21 5.0% . A low frequency was exhibited by HMPV with 16 3.8% positive samples, human coronavirus OC43 with 13 3.1% , PIV 1 with 12 2.8% , PIV 4 with 9 2.1% , PIV 2 with 7 1.7% and HCoV NL63 with 6 1.4% . Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 .", "Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 . Dual infections accounted for 17% of all positive samples and three viruses were detected in 2.7% of samples . A single patient sample displayed a quadruple infection being simultaneously positive for RSV, rhinovirus, HBoV and influenza B. Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus.", "Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus. As a percentage, however, the virus appearing most often in co- infections was HBoV, which was found in more than 70% of cases together with another virus, followed by coronaviruses HCoV OC43 and HCoV NL63 with 61% and 67%, respectively. On the other hand, the viruses most rarely seen in co-infections were influenza viruses A and B as well as RSV. Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table.", "Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table. Significant correlation P-value < 0.05 was seen mostly for co-infections with RSV, however correlations were very weak r<0.3 and negative. This finding can probably be explained by the fact that RSV infections occurred predominantly in the very young, where co-infections were less frequently observed. On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. However, as more than 100 different rhinovirus strains have been identified to be circulating worldwide in parallel and successively, a potential seasonality of individual rhinovirus serotypes may be masked by overlapping patterns . The data was further analysed with regard to the age distribution of virus infection see Table 2 . In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV.", "In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV. Rhinoviruses, HBoV and enteroviruses are most frequently observed in children from 4 months to 3 years of age. The age dependency of the virus incidence is visualized in Fig 3 for the seven most frequently observed viruses. The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children.", "The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children. Regarding multiple infections, children less than 3 month of age and those older than 4 years had a significantly smaller risk to present with multiple infections as compared to the other two age groups p-value = 0.014 . A reason for this could be that very young children have limited contact to others reducing thereby the chance for a co-infection, whereas children older than 3 years already established immunity to an increasing number of viruses encountered previously. This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way.", "This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way. The high rate of positive samples 85.8% is evidence of the high sensitivity of the Multiplex-assays used and that the range of viruses included in the analysis is comprehensive. Many previous studies have shown detection rates ranging from below 50% to 75% . The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, .", "The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, . Our data support the results of similar studies performed in the Middle East region. A recently published study found that RSV was the most commonly detected virus in nasopharyngeal swabs from children presenting symptoms of RTIs and in addition to that it also showed that RSV infections follow a similar circulation pattern peaking from December to March . Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%.", "Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%. HBoV, HCoV and EV were found most frequently in co-infections. All three subtypes of HCoV were co-detected with several other viruses, while HBoV was co-detected mainly with HRV and RSV. In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere .", "In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere . Even though this study did not allow for investigating a possible association between multiple infections and disease severity, a review of the literature shows that such a potential association is still subject to controversy, since there are reports showing no relationship of multiple virus infection with respiratoty illness severity on one hand or a significant association on the other. Studies have shown that viral co-infection was significantly associated with longer duration of illness symptoms, but with a decreased severity in hospitalized children regarding oxygen requirement and intensive care unit admission, whereas the findings of other studies have indicated that severe clinical phenotypes were more prevalent in co-infection patients, especially in RSV co-infections that may increase the severity of RSV associated disease in children 25, . Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures .", "Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures . The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections." ]

[ "In order to improve clinical management and prevention of viral infections in hospitalised children improved etiological insight is needed. The aim of the present study was to assess the spectrum of respiratory viral pathogens in children admitted to hospital with acute respiratory tract infections in Cyprus. For this purpose nasopharyngeal swab samples from 424 children less than 12 years of age with acute respiratory tract infections were collected over three epidemic seasons and were analysed for the presence of the most common 15 respiratory viruses. A viral pathogen was identified in 86% of the samples, with multiple infections being observed in almost 20% of the samples. The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. While RSV and PIV3 incidence decreased significantly with age, the opposite was observed for influenza A and B as well as adenovirus infections. The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections. Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood .", "Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood . Illnesses caused by respiratory viruses include, among others, common colds, pharyngitis, croup, bronchiolitis, viral pneumonia and otitis media. Rapid diagnosis is important not only for timely therapeutic intervention but also for the identification of a beginning influenza epidemic and the avoidance of unnecessary antibiotic treatment . RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children.", "RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children. Respiratory infections cluster during winter and early spring months. The leading viral agents include respiratory syncytial virus RSV , influenza A and B INF-A, INF-B viruses, parainfluenza viruses PIVs , and human adenoviruses HAdVs . In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization.", "In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization. URTIs cause non-severe but widespread epidemics that are responsible for continuous circulation of pathogens in the community. LRTIs have been classified as frank pneumonia and bronchiolitis with clinical, radiological and etiological features that usually overlap . Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons.", "Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons. The study was approved by the Cyprus National Bioethics Committee. Accordingly, written informed consent was obtained from parents prior to sample taking. Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded.", "Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded. Nasal swab samples were collected using the BD Universal Viral Transport Collection Kit. Viral RNA/DNA was extracted from 400 μl sample using the iPrep PureLink Virus Kit on an iPrep purification instrument Invitrogen . A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations.", "A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations. For this purpose, all available complete genome sequences were obtained for each virus from GenBank, imported into the BioEdit Sequence Alignment Editor v7.1.7 and aligned using ClustalX. In case of mismatches between published primers/probe and target sequences, modifications were applied, as indicated in Table 1 . The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing.", "The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing. In order to assess the sensitivity and specificity of the assays, the laboratory enrolled for two consecutive years in Quality Control for Molecular Diagnostics QCMD external quality assessment schemes for all viruses, except Bocavirus, which was unavailable. In summary, the established assays were able to correctly identify all viruses tested, proving their suitability for diagnostic application. A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed.", "A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed. The same statistical tests were used to compare the frequency of subjects with single or multiple infections between age groups. In addition, Pearson correlation was used to examine co-infections of different viruses. All statistical analyses were performed using StataSE 12 StatCorp. 2007. College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus.", "College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus. The median age of the children was 15 months range: 0-140 months with 243 being male and 181 female male/ female ratio 1.34 . The age distribution is shown in Fig 1. Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections.", "Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections. With moderate frequency have been detected HAdV in 31 7.3% patients, influenza A in 28 6.6% , HBoV in 24 5.7% , enteroviruses and PIV 3 in 23 5.4% of patients respectively, and Influenza B in 21 5.0% . A low frequency was exhibited by HMPV with 16 3.8% positive samples, human coronavirus OC43 with 13 3.1% , PIV 1 with 12 2.8% , PIV 4 with 9 2.1% , PIV 2 with 7 1.7% and HCoV NL63 with 6 1.4% . Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 .", "Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 . Dual infections accounted for 17% of all positive samples and three viruses were detected in 2.7% of samples . A single patient sample displayed a quadruple infection being simultaneously positive for RSV, rhinovirus, HBoV and influenza B. Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus.", "Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus. As a percentage, however, the virus appearing most often in co- infections was HBoV, which was found in more than 70% of cases together with another virus, followed by coronaviruses HCoV OC43 and HCoV NL63 with 61% and 67%, respectively. On the other hand, the viruses most rarely seen in co-infections were influenza viruses A and B as well as RSV. Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table.", "Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table. Significant correlation P-value < 0.05 was seen mostly for co-infections with RSV, however correlations were very weak r<0.3 and negative. This finding can probably be explained by the fact that RSV infections occurred predominantly in the very young, where co-infections were less frequently observed. On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. However, as more than 100 different rhinovirus strains have been identified to be circulating worldwide in parallel and successively, a potential seasonality of individual rhinovirus serotypes may be masked by overlapping patterns . The data was further analysed with regard to the age distribution of virus infection see Table 2 . In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV.", "In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV. Rhinoviruses, HBoV and enteroviruses are most frequently observed in children from 4 months to 3 years of age. The age dependency of the virus incidence is visualized in Fig 3 for the seven most frequently observed viruses. The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children.", "The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children. Regarding multiple infections, children less than 3 month of age and those older than 4 years had a significantly smaller risk to present with multiple infections as compared to the other two age groups p-value = 0.014 . A reason for this could be that very young children have limited contact to others reducing thereby the chance for a co-infection, whereas children older than 3 years already established immunity to an increasing number of viruses encountered previously. This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way.", "This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way. The high rate of positive samples 85.8% is evidence of the high sensitivity of the Multiplex-assays used and that the range of viruses included in the analysis is comprehensive. Many previous studies have shown detection rates ranging from below 50% to 75% . The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, .", "The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, . Our data support the results of similar studies performed in the Middle East region. A recently published study found that RSV was the most commonly detected virus in nasopharyngeal swabs from children presenting symptoms of RTIs and in addition to that it also showed that RSV infections follow a similar circulation pattern peaking from December to March . Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%.", "Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%. HBoV, HCoV and EV were found most frequently in co-infections. All three subtypes of HCoV were co-detected with several other viruses, while HBoV was co-detected mainly with HRV and RSV. In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere .", "In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere . Even though this study did not allow for investigating a possible association between multiple infections and disease severity, a review of the literature shows that such a potential association is still subject to controversy, since there are reports showing no relationship of multiple virus infection with respiratoty illness severity on one hand or a significant association on the other. Studies have shown that viral co-infection was significantly associated with longer duration of illness symptoms, but with a decreased severity in hospitalized children regarding oxygen requirement and intensive care unit admission, whereas the findings of other studies have indicated that severe clinical phenotypes were more prevalent in co-infection patients, especially in RSV co-infections that may increase the severity of RSV associated disease in children 25, . Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures .", "Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures . The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections." ]

[ "In order to improve clinical management and prevention of viral infections in hospitalised children improved etiological insight is needed. The aim of the present study was to assess the spectrum of respiratory viral pathogens in children admitted to hospital with acute respiratory tract infections in Cyprus. For this purpose nasopharyngeal swab samples from 424 children less than 12 years of age with acute respiratory tract infections were collected over three epidemic seasons and were analysed for the presence of the most common 15 respiratory viruses. A viral pathogen was identified in 86% of the samples, with multiple infections being observed in almost 20% of the samples. The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "The most frequently detected viruses were RSV 30.4% and Rhinovirus 27.4% . RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. While RSV and PIV3 incidence decreased significantly with age, the opposite was observed for influenza A and B as well as adenovirus infections. The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections. Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood .", "Text: Viral Respiratory tract infections RTI represent a major public health problem because of their world-wide occurrence, ease of transmission and considerable morbidity and mortality effecting people of all ages. Children are on average infected two to three times more frequently than adults, with acute RTIs being the most common infection in childhood . Illnesses caused by respiratory viruses include, among others, common colds, pharyngitis, croup, bronchiolitis, viral pneumonia and otitis media. Rapid diagnosis is important not only for timely therapeutic intervention but also for the identification of a beginning influenza epidemic and the avoidance of unnecessary antibiotic treatment . RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children.", "RTIs are a major cause of morbidity and mortality worldwide. Acute RTI is most common in children under five years of age, and represents 30-50% of the paediatric medical admissions, as well as 20-40% of hospitalizations in children. Respiratory infections cluster during winter and early spring months. The leading viral agents include respiratory syncytial virus RSV , influenza A and B INF-A, INF-B viruses, parainfluenza viruses PIVs , and human adenoviruses HAdVs . In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization.", "In addition, there is a continuously increasing list of new respiratory viruses that contribute significantly to the burden of acute respiratory infections, such as the recently identified human metapneumovirus HMPV and human Bocavirus HBoV . Acute RTIs are classified as upper UTRIs and lower RTI LRTIs , according to the involved anatomic localization. URTIs cause non-severe but widespread epidemics that are responsible for continuous circulation of pathogens in the community. LRTIs have been classified as frank pneumonia and bronchiolitis with clinical, radiological and etiological features that usually overlap . Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons.", "Viruses are again the foremost agents of LRTIs often misdiagnosed as bacterial in origin and hence treated with antibiotics unnecessarily . The main aim of this study was to determine the aetiology of acute respiratory tract infections in Cypriot children and assess the epidemiology of the identified viral pathogens over three epidemic seasons. The study was approved by the Cyprus National Bioethics Committee. Accordingly, written informed consent was obtained from parents prior to sample taking. Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded.", "Between November 2010 and October 2013, 485 nasopharyngeal swab samples were collected from children up to 12 years of age, who had been hospitalized with acute respiratory tract infection at the Archbishop Makarios III hospital, Nicosia. Clinical and demographic information including symptoms, duration of hospitalisation, diagnosis and treatment were recorded. Nasal swab samples were collected using the BD Universal Viral Transport Collection Kit. Viral RNA/DNA was extracted from 400 μl sample using the iPrep PureLink Virus Kit on an iPrep purification instrument Invitrogen . A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations.", "A set of four multiplex Real-Time RT-PCR assays was established and validated for the detection of the 15 most common respiratory viruses as follows: assay 1: influenzaviruses A and B, RSV, assay 2: parainfluenzaviruses 1-4, assay 3: HAdV, enteroviruses, HMPV and HBoV and assay 4: rhinoviruses and the human coronaviruses OC43, NL63 and 229E Table 1 . Published primer and probe sets were used as a basis for designing the assays, however, all primer/probe sequences were checked against newly build sequence alignments of all viruses tested and were modified, if necessary, to account for possible sequence variations. For this purpose, all available complete genome sequences were obtained for each virus from GenBank, imported into the BioEdit Sequence Alignment Editor v7.1.7 and aligned using ClustalX. In case of mismatches between published primers/probe and target sequences, modifications were applied, as indicated in Table 1 . The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing.", "The alignments for the viruses, which necessitated changes to the primers/probe are available in Fasta-Format as supplement S1-S4 Files. Primer concentrations and reaction conditions for the four assays were subsequently optimised for multiplexing. In order to assess the sensitivity and specificity of the assays, the laboratory enrolled for two consecutive years in Quality Control for Molecular Diagnostics QCMD external quality assessment schemes for all viruses, except Bocavirus, which was unavailable. In summary, the established assays were able to correctly identify all viruses tested, proving their suitability for diagnostic application. A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed.", "A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher's exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed. The same statistical tests were used to compare the frequency of subjects with single or multiple infections between age groups. In addition, Pearson correlation was used to examine co-infections of different viruses. All statistical analyses were performed using StataSE 12 StatCorp. 2007. College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus.", "College Station, TX, USA . The present study was a prospective investigation of children hospitalized with acute respiratory tract infections between November 2010 and October 2013 in Cyprus. The median age of the children was 15 months range: 0-140 months with 243 being male and 181 female male/ female ratio 1.34 . The age distribution is shown in Fig 1. Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections.", "Out of the 424 samples analysed, 364 85.8% were positive for one or more viruses. Results are summarized in Table 2 .The most commonly detected viruses were RSV, which was found in 129 30.4% patients and rhinoviruses in 116 27.4% accounting together for almost 60% of all detections. With moderate frequency have been detected HAdV in 31 7.3% patients, influenza A in 28 6.6% , HBoV in 24 5.7% , enteroviruses and PIV 3 in 23 5.4% of patients respectively, and Influenza B in 21 5.0% . A low frequency was exhibited by HMPV with 16 3.8% positive samples, human coronavirus OC43 with 13 3.1% , PIV 1 with 12 2.8% , PIV 4 with 9 2.1% , PIV 2 with 7 1.7% and HCoV NL63 with 6 1.4% . Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 .", "Coronavirus 229E could be detected only in a single sample. Co-infections with two or more viruses were observed in 84 out of the 364 positive samples see Table 2 . Dual infections accounted for 17% of all positive samples and three viruses were detected in 2.7% of samples . A single patient sample displayed a quadruple infection being simultaneously positive for RSV, rhinovirus, HBoV and influenza B. Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus.", "Table 3 summarizes the frequency of each virus in single vs. multiple infections as well as the number of co-occurrences of viruses for each possible virus combination. In absolute terms the most common combination observed was RSV/rhinovirus. As a percentage, however, the virus appearing most often in co- infections was HBoV, which was found in more than 70% of cases together with another virus, followed by coronaviruses HCoV OC43 and HCoV NL63 with 61% and 67%, respectively. On the other hand, the viruses most rarely seen in co-infections were influenza viruses A and B as well as RSV. Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table.", "Pearson correlation coefficients were calculated to examine the likelihood of co-infections of different viruses. The results of the analysis are summarized in Table 1 in S1 Table. Significant correlation P-value < 0.05 was seen mostly for co-infections with RSV, however correlations were very weak r<0.3 and negative. This finding can probably be explained by the fact that RSV infections occurred predominantly in the very young, where co-infections were less frequently observed. On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year.", "On the other hand, a significant positive correlation was observed for enterovirus and rhinovirus co-infection hinting maybe at similarities in circulation patterns and/or transmission modes. Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses A and B combined Fig 2 , with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. However, as more than 100 different rhinovirus strains have been identified to be circulating worldwide in parallel and successively, a potential seasonality of individual rhinovirus serotypes may be masked by overlapping patterns . The data was further analysed with regard to the age distribution of virus infection see Table 2 . In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV.", "In infants up to 3 months old, RSV was by far the most common pathogen 58.1% , followed by rhinovirus 20.3% and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age p-value < 0.0001 dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV. Rhinoviruses, HBoV and enteroviruses are most frequently observed in children from 4 months to 3 years of age. The age dependency of the virus incidence is visualized in Fig 3 for the seven most frequently observed viruses. The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children.", "The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4-12 years old p-value = 0.020 . This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children. Regarding multiple infections, children less than 3 month of age and those older than 4 years had a significantly smaller risk to present with multiple infections as compared to the other two age groups p-value = 0.014 . A reason for this could be that very young children have limited contact to others reducing thereby the chance for a co-infection, whereas children older than 3 years already established immunity to an increasing number of viruses encountered previously. This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way.", "This study for the first time examined the aetiology of acute respiratory tract infections in hospitalised children in Cyprus. Four multiplex Real-Time RT-PCR assays were developed in order to detect the most common respiratory viral pathogens in a fast and cost-effective way. The high rate of positive samples 85.8% is evidence of the high sensitivity of the Multiplex-assays used and that the range of viruses included in the analysis is comprehensive. Many previous studies have shown detection rates ranging from below 50% to 75% . The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, .", "The most common viruses detected were RSV and rhinovirus accounting for almost 60% of all cases. Both viruses were reported previously by others as the major aetiology for respiratory viral infections in young children with rhinoviruses being recognized increasingly for their role in lower respiratory tract infections 20, . Our data support the results of similar studies performed in the Middle East region. A recently published study found that RSV was the most commonly detected virus in nasopharyngeal swabs from children presenting symptoms of RTIs and in addition to that it also showed that RSV infections follow a similar circulation pattern peaking from December to March . Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%.", "Another study has revealed that RSV and PIV3 incidence decreases significantly with age, whereas the opposite is observed for influenza and adenovirus infections, a trend that was also observed in our study . Mixed infections were observed in approximately 20% of all samples, which is in the middle of previously reported rates ranging from 10 to almost 40%. HBoV, HCoV and EV were found most frequently in co-infections. All three subtypes of HCoV were co-detected with several other viruses, while HBoV was co-detected mainly with HRV and RSV. In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere .", "In the case of EV infections, EV were almost predominantly associated with HRV. The rare presence of InfA and InfB viruses in multiple infections witnessed in our study was also observed elsewhere . Even though this study did not allow for investigating a possible association between multiple infections and disease severity, a review of the literature shows that such a potential association is still subject to controversy, since there are reports showing no relationship of multiple virus infection with respiratoty illness severity on one hand or a significant association on the other. Studies have shown that viral co-infection was significantly associated with longer duration of illness symptoms, but with a decreased severity in hospitalized children regarding oxygen requirement and intensive care unit admission, whereas the findings of other studies have indicated that severe clinical phenotypes were more prevalent in co-infection patients, especially in RSV co-infections that may increase the severity of RSV associated disease in children 25, . Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures .", "Viral respiratory infections continue to be a worldwide health concern. As the clinical symptoms of patients with acute respiratory tract infections do usually not allow a discrimination of viral or bacterial aetiology, rapid and reliable diagnostic tools are required for better antibiotic stewardship and the implementation of appropriate infection control measures . The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections." ]

[ "The objective of this study was to investigate the presence of equine coronavirus ECoV in clinical samples submitted to a diagnostic laboratory in Ireland. A total of 424 clinical samples were examined from equids with enteric disease in 24 Irish counties between 2011 and 2015. A real-time reverse transcription polymerase chain reaction was used to detect ECoV RNA. Nucleocapsid, spike and the region from the p4.7 to p12.7 genes of positive samples were sequenced, and sequence and phylogenetic analyses were conducted. Five samples 1.2% collected in 2011 and 2013 tested positive for ECoV. Positive samples were collected from adult horses, Thoroughbred foals and a donkey foal.", "Five samples 1.2% collected in 2011 and 2013 tested positive for ECoV. Positive samples were collected from adult horses, Thoroughbred foals and a donkey foal. Sequence and/or phylogenetic analysis showed that nucleocapsid, spike and p12.7 genes were highly conserved and were closely related to ECoVs identified in other countries. In contrast, the region from p4.7 and the non-coding region following the p4.7 gene had deletions or insertions. The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the Irish viruses were distinguishable from those circulating in other countries. This is the first report of ECoV detected in both foals and adult horses in Ireland.", "The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the Irish viruses were distinguishable from those circulating in other countries. This is the first report of ECoV detected in both foals and adult horses in Ireland. Text: Equine coronavirus ECoV is a positive-stranded RNA virus and belongs to the species Betacoronavirus 1 in the genus Betacoronavirus . The clinical signs associated with ECoV infection during outbreaks in the USA and Japan were fever, anorexia, lethargy and diarrhoea. The same clinical signs were also recorded in an experimental challenge study using Japanese draft horses . The main transmission route is considered to be faecal-oral and ECoV is usually detected in faecal samples.", "The same clinical signs were also recorded in an experimental challenge study using Japanese draft horses . The main transmission route is considered to be faecal-oral and ECoV is usually detected in faecal samples. However, the molecular detection of ECoV in faeces from horses with diarrhoea, does not prove causation. Coronaviruses can cause both enteric and respiratory disease in many avian and mammalian species but ECoV is less likely to be found in respiratory secretions than in faeces . Both molecular and seroepidemiology studies suggest that ECoV may be more prevalent in the USA than in other countries . ECoV was detected in samples collected from equids in 48 states of the USA .", "Both molecular and seroepidemiology studies suggest that ECoV may be more prevalent in the USA than in other countries . ECoV was detected in samples collected from equids in 48 states of the USA . In central Kentucky, approximately 30% of both healthy and diarrheic Thoroughbred foals were infected with ECoV . All of the qPCR positive foals with diarrhoea were co-infected with other pathogens such as rotavirus or Clostridium perfringens, suggesting that there was potential for ECoV to be over-diagnosed as a causative agent in complex diseases. In contrast in Japan, although an outbreak of diarrhoea occurred among ECoV-infected draft horses at one racecourse , there have been no similar outbreaks subsequently, and all rectal swabs collected from diarrheic Thoroughbred foals were negative. Furthermore, only 2.5% of the rectal swabs collected from healthy foals in the largest Thoroughbred horse breeding region in Japan were positive for ECoV .", "In contrast in Japan, although an outbreak of diarrhoea occurred among ECoV-infected draft horses at one racecourse , there have been no similar outbreaks subsequently, and all rectal swabs collected from diarrheic Thoroughbred foals were negative. Furthermore, only 2.5% of the rectal swabs collected from healthy foals in the largest Thoroughbred horse breeding region in Japan were positive for ECoV . In France, 2.8% of 395 faecal samples and 0.5% of 200 respiratory samples collected in 58 counties tested positive for ECoV . Similar to the reports from Japan and France, a low prevalence of ECoV was also observed in the UK , Saudi Arabia and Oman . The objective of this study was to investigate the presence of ECoV in clinical samples submitted to a diagnostic laboratory in Ireland. The samples were tested by real-time reverse transcription polymerase chain reaction rRT-PCR as it has been shown to be the most sensitive diagnostic method for ECoV and is routinely employed as an alternative to virus isolation in diagnostic laboratories worldwide, both for timely diagnosis and in epidemiological studies .", "The objective of this study was to investigate the presence of ECoV in clinical samples submitted to a diagnostic laboratory in Ireland. The samples were tested by real-time reverse transcription polymerase chain reaction rRT-PCR as it has been shown to be the most sensitive diagnostic method for ECoV and is routinely employed as an alternative to virus isolation in diagnostic laboratories worldwide, both for timely diagnosis and in epidemiological studies . Virus isolation and biological characterisation were beyond the capacity of this study, which was similar in scope to that of the studies in horse populations in the USA, Europe and Asia . The rRT-PCR assay was performed as previously described using a primer set targeting the nucleocapsid N gene ECoV-380f, ECoV-522r and ECoV-436p Table 1 and AgPath-ID One-Step RT-PCR Kit Thermo Fisher Scientific, MA, USA according to the manufacturer's instructions. To prove that the extraction was successful and that there was no inhibition during rRT-PCR amplification, an internal positive control primer/probe PrimerDesign, Southampton, UK was added to the master mix. Thermal cycling conditions were; 48 • C for 10 min and 95 • C for 10 min, followed by 40 cycles at 94 • C for 15 s and 60 • C for 45 s. The SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity Thermo Fisher Scientific, MA, USA was used for sequencing analysis of two of the five ECoV samples identified.", "To prove that the extraction was successful and that there was no inhibition during rRT-PCR amplification, an internal positive control primer/probe PrimerDesign, Southampton, UK was added to the master mix. Thermal cycling conditions were; 48 • C for 10 min and 95 • C for 10 min, followed by 40 cycles at 94 • C for 15 s and 60 • C for 45 s. The SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity Thermo Fisher Scientific, MA, USA was used for sequencing analysis of two of the five ECoV samples identified. There was inadequate viral nucleic acid in the other three samples for sequencing. The primer sets used to amplify the nucleocapsid N gene , the partial spike S gene , and the region from the p4.7 to p12.7 genes of non-structural proteins Oue, personal communication are shown in Table 1 . The RT-PCR products were sequenced commercially by GATC Biotech Cologne, Germany . Sequence analysis was performed using the BLAST and CLUSTALW programs, and Vector NTI Advance 11.5 software Thermo Fisher Scientific, MA, USA .", "The RT-PCR products were sequenced commercially by GATC Biotech Cologne, Germany . Sequence analysis was performed using the BLAST and CLUSTALW programs, and Vector NTI Advance 11.5 software Thermo Fisher Scientific, MA, USA . Phylogenetic analysis of nucleotide sequences was conducted with MEGA software Version 5.2 . A phylogenetic tree was constructed based on nucleotide sequences of the K2+G N gene and TN93 S gene using the maximum likelihood method. MEGA software was used to select the optimal substitution models. Statistical analysis of the tree was performed with the bootstrap test 1000 replicates for multiple alignments.", "MEGA software was used to select the optimal substitution models. Statistical analysis of the tree was performed with the bootstrap test 1000 replicates for multiple alignments. The complete genome sequences of NC99 EF446615 , Tokachi09 LC061272 , Obihiro12-1 LC061273 and Obihiro12-2 LC061274 , the N AB671298 and S AB671299 genes of Obihiro2004, the N gene of Hidaka-No.61/2012 LC054263 and Hidaka-No.119/2012 LC054264 , the S gene of ECoV_FRA_2011/1 KC178705 , ECoV_FRA_2011/2 KC178704 , ECoV_FRA_2012/1 KC178703 , ECoV_FRA_2012/2 KC178702 and ECoV_FRA_2012/3 KC178701 were used in sequence and/or phylogenetic analysis. The accession numbers registered in GenBank/EMBL/DDBJ are as follows: the complete sequences of the N gene; 11V11708/IRL LC149485 and 13V08313/IRL LC149486 , the partial sequences of the S gene; 11V11708/IRL LC149487 and13V08313/IRL LC149488 and the complete sequences from the p4.7 to p12.7 genes; 11V11708/IRL LC149489 and13V08313/IRL LC149490 . One six-week-old foal was the only clinical case on a public Thoroughbred stud farm with approximately 30 mares when it presented with diarrhoea. Recovery took over three weeks during which it received fluid therapy, probiotics, antiulcer medication and antibiotics.", "One six-week-old foal was the only clinical case on a public Thoroughbred stud farm with approximately 30 mares when it presented with diarrhoea. Recovery took over three weeks during which it received fluid therapy, probiotics, antiulcer medication and antibiotics. The second foal was a 14-day-old filly, which had been hospitalised with diarrhoea two days prior to sample collection. The foal responded well to supportive treatment and at the time of sample collection, the diarrhoea had resolved. The five ECoV positive samples tested negative for equine rotavirus. The nucleotide sequences of the complete N gene, the partial S gene and the region from the p4.7 to p12.7 genes of two positive samples 11V11708/IRL/2011 and 13V08313/IRL/2013 were determined.", "The five ECoV positive samples tested negative for equine rotavirus. The nucleotide sequences of the complete N gene, the partial S gene and the region from the p4.7 to p12.7 genes of two positive samples 11V11708/IRL/2011 and 13V08313/IRL/2013 were determined. The nucleotide identities of the N and S genes of the two Irish ECoVs were 99.8% 1338/1341 nucleotides and 99.5% 650/653 nucleotides , respectively. The nucleotide identities of the N gene of the two Irish ECoVs and the ECoVs from other continents are summarised in Table 2 . Phylogenetic analysis was performed for the nucleotide sequences of the complete N and partial S genes Figure 1 . The analysis for the N gene showed that Irish ECoVs were independently clustered although they were closely related to Japanese viruses identified after 2009.", "Phylogenetic analysis was performed for the nucleotide sequences of the complete N and partial S genes Figure 1 . The analysis for the N gene showed that Irish ECoVs were independently clustered although they were closely related to Japanese viruses identified after 2009. In the phylogenetic tree of the S gene, Irish ECoVs were closely related to all other ECoVs analysed. The length of the region from the p4.7 to p12.7 genes in the two viruses was 544 base pairs. Compared with NC99, Irish ECoVs, had a total of 37 nucleotide deletions within p4.7 and the non-coding region following the p4.7 gene. Compared with Obihiro 12-1 and 12-2, Irish ECoVs had a three-nucleotide insertion.", "Compared with NC99, Irish ECoVs, had a total of 37 nucleotide deletions within p4.7 and the non-coding region following the p4.7 gene. Compared with Obihiro 12-1 and 12-2, Irish ECoVs had a three-nucleotide insertion. When compared with Tokachi09, the Irish ECoVs had a 148-nucleotide insertion see Figure S1 . The p12.7 gene of the two Irish ECoVs did not have deletions or insertions, and the nucleotide identities were 98.8-99.7% between these viruses and the other ECoVs NC99, Tokachi09, Obihiro12-1 and Obihiro12-2 . This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease. However, detection of ECoV in faeces samples from horses with enteric disease does not prove This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease.", "This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease. However, detection of ECoV in faeces samples from horses with enteric disease does not prove This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease. However, detection of ECoV in faeces samples from horses with enteric disease does not prove causation. In this study, 424 samples collected between 2011 and 2015 from equids with enteric disease were tested, and only five samples 1.2% were positive for ECoV. The inclusion of an internal positive control in the rRT-PCR eliminated the possibility of false negative results due to the presence of PCR inhibitors but the high content of nucleases associated with faeces samples may have caused some RNA degradation. However, this low prevalence of ECoV is similar to that identified in France and among Thoroughbred foals in Japan .", "The inclusion of an internal positive control in the rRT-PCR eliminated the possibility of false negative results due to the presence of PCR inhibitors but the high content of nucleases associated with faeces samples may have caused some RNA degradation. However, this low prevalence of ECoV is similar to that identified in France and among Thoroughbred foals in Japan . Although ECoV has been identified on three continents, little is known about the genetic and pathogenic diversity in field viruses. In this study, sequence and phylogenetic analysis Figure 1 demonstrated a high level of homology between viruses detected in a donkey and a horse in two provinces in Ireland in different years. This suggests that Irish ECoVs may have low genetic diversity. Compared with the ECoVs of other countries, the N, S and p12.7 genes of the two Irish viruses were highly conserved.", "This suggests that Irish ECoVs may have low genetic diversity. Compared with the ECoVs of other countries, the N, S and p12.7 genes of the two Irish viruses were highly conserved. In contrast, the region from p4.7 and the non-coding region following the p4.7 gene had deletions or insertions Figure S1 . Because of polymorphism in this region, this region could be useful for epidemiological investigation . The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the viruses in Ireland may be distinguishable from those circulating in other countries. The positive samples were collected in November ., March . and April . in this study.", "The positive samples were collected in November ., March . and April . in this study. Higher case numbers are identified in the USA during the colder months October to April , and our results were consistent with the circulation period in USA. It has been reported that outbreaks mainly occurred among adult riding, racing and show horses in USA . The choice of cases to include in the current study may not have been optimal for detection of ECoV as the majority of samples were from foals. However, two positive samples were collected from adult horses in a combined riding school/show jumping yard in the West of Ireland.", "The choice of cases to include in the current study may not have been optimal for detection of ECoV as the majority of samples were from foals. However, two positive samples were collected from adult horses in a combined riding school/show jumping yard in the West of Ireland. At the time of sample collection in April 2013, the monthly mean temperatures were below long-term average and in parts of the West, were the coldest in 24 years . Cold weather may have been a predisposing factor to the ECoV infection on the farm. Two positive samples were collected from Thoroughbred foals. A faeces sample collected from one foal with severe watery diarrhoea and inappetance was positive for ECoV but a sample collected three days later tested negative.", "Two positive samples were collected from Thoroughbred foals. A faeces sample collected from one foal with severe watery diarrhoea and inappetance was positive for ECoV but a sample collected three days later tested negative. A potential difficulty in detecting ECoV from naturally infected horses has been noted previously as serial samples from seven sick horses in the USA suggested that ECoV only persisted for three to nine days in faeces . In both cases, the diarrhoea may have been caused by other unidentified coinfecting pathogens as has been suggested by investigators in the USA . This is the first report of ECoV detection in faeces samples from both foals and adult horses in Ireland. The viruses identified in Ireland are genetically closely related to the Japanese viruses and the results of this study give no indication of significant genetic or phenotypic diversity.", "This is the first report of ECoV detection in faeces samples from both foals and adult horses in Ireland. The viruses identified in Ireland are genetically closely related to the Japanese viruses and the results of this study give no indication of significant genetic or phenotypic diversity. In recent years, there has been an increase in awareness and testing for ECoV in the USA and elsewhere . Horse breeding and racing activities in Ireland are the most prominent and important of any country on a per capita basis. There are over 50 Thoroughbred horses per 10,000 of population in Ireland, compared to between three and five for Great Britain, France and the USA . Thus, an investigation of ECoV in Ireland is pertinent not only to increase awareness nationally of the epidemiology of the virus and promote discussion on its clinical importance, but also to inform the industry globally of the health status of Irish horses.", "There are over 50 Thoroughbred horses per 10,000 of population in Ireland, compared to between three and five for Great Britain, France and the USA . Thus, an investigation of ECoV in Ireland is pertinent not only to increase awareness nationally of the epidemiology of the virus and promote discussion on its clinical importance, but also to inform the industry globally of the health status of Irish horses. Ireland exports horses all over the world. By illustration, in 2016 the country was the second biggest seller of bloodstock at public auctions second only to the USA . Many questions remain with regard to the clinical significance of ECoV. The outbreak at a draft-horse racetrack in Japan in 2009 affected 132 of approximately 600 horses and resulted in non-starters and the implementation of movement restrictions .", "Many questions remain with regard to the clinical significance of ECoV. The outbreak at a draft-horse racetrack in Japan in 2009 affected 132 of approximately 600 horses and resulted in non-starters and the implementation of movement restrictions . However, draft horses appear to have a higher infection rate than other breeds and an outbreak of similar severity has not been reported in Thoroughbred racehorses . The much higher incidence of ECoV positive Thoroughbred foals identified in Kentucky compared to similar populations internationally suggests an increased susceptibility to ECoV infection in that population. In the past, specific environmental factors were associated with extensive reproductive loss in the Kentucky area and to a lesser extent in other states , but predisposing regional factors such as differences in management, environment or husbandry have not been identified for ECoV. It has been suggested that ECoV is a coinfecting agent in foals with diarrhoea and clinical infections have predominantly been reported in adult horses with a mono-infection with EcoV .", "In the past, specific environmental factors were associated with extensive reproductive loss in the Kentucky area and to a lesser extent in other states , but predisposing regional factors such as differences in management, environment or husbandry have not been identified for ECoV. It has been suggested that ECoV is a coinfecting agent in foals with diarrhoea and clinical infections have predominantly been reported in adult horses with a mono-infection with EcoV . There was no indication from the results of this study that coronavirus is a major cause of diarrhoea in Irish horses but the introduction of rRT-PCR as a routine diagnostic test will assist in elucidating the significance of this virus to the Irish breeding, racing and sports industries. The primary focus in future will be on testing adult horses that present with anorexia, lethargy, fever and changes in faecal character as a significant association has been demonstrated between this clinical status and molecular detection of ECoV in faeces ." ]

[ "The objective of this study was to investigate the presence of equine coronavirus ECoV in clinical samples submitted to a diagnostic laboratory in Ireland. A total of 424 clinical samples were examined from equids with enteric disease in 24 Irish counties between 2011 and 2015. A real-time reverse transcription polymerase chain reaction was used to detect ECoV RNA. Nucleocapsid, spike and the region from the p4.7 to p12.7 genes of positive samples were sequenced, and sequence and phylogenetic analyses were conducted. Five samples 1.2% collected in 2011 and 2013 tested positive for ECoV. Positive samples were collected from adult horses, Thoroughbred foals and a donkey foal.", "Five samples 1.2% collected in 2011 and 2013 tested positive for ECoV. Positive samples were collected from adult horses, Thoroughbred foals and a donkey foal. Sequence and/or phylogenetic analysis showed that nucleocapsid, spike and p12.7 genes were highly conserved and were closely related to ECoVs identified in other countries. In contrast, the region from p4.7 and the non-coding region following the p4.7 gene had deletions or insertions. The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the Irish viruses were distinguishable from those circulating in other countries. This is the first report of ECoV detected in both foals and adult horses in Ireland.", "The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the Irish viruses were distinguishable from those circulating in other countries. This is the first report of ECoV detected in both foals and adult horses in Ireland. Text: Equine coronavirus ECoV is a positive-stranded RNA virus and belongs to the species Betacoronavirus 1 in the genus Betacoronavirus . The clinical signs associated with ECoV infection during outbreaks in the USA and Japan were fever, anorexia, lethargy and diarrhoea. The same clinical signs were also recorded in an experimental challenge study using Japanese draft horses . The main transmission route is considered to be faecal-oral and ECoV is usually detected in faecal samples.", "The same clinical signs were also recorded in an experimental challenge study using Japanese draft horses . The main transmission route is considered to be faecal-oral and ECoV is usually detected in faecal samples. However, the molecular detection of ECoV in faeces from horses with diarrhoea, does not prove causation. Coronaviruses can cause both enteric and respiratory disease in many avian and mammalian species but ECoV is less likely to be found in respiratory secretions than in faeces . Both molecular and seroepidemiology studies suggest that ECoV may be more prevalent in the USA than in other countries . ECoV was detected in samples collected from equids in 48 states of the USA .", "Both molecular and seroepidemiology studies suggest that ECoV may be more prevalent in the USA than in other countries . ECoV was detected in samples collected from equids in 48 states of the USA . In central Kentucky, approximately 30% of both healthy and diarrheic Thoroughbred foals were infected with ECoV . All of the qPCR positive foals with diarrhoea were co-infected with other pathogens such as rotavirus or Clostridium perfringens, suggesting that there was potential for ECoV to be over-diagnosed as a causative agent in complex diseases. In contrast in Japan, although an outbreak of diarrhoea occurred among ECoV-infected draft horses at one racecourse , there have been no similar outbreaks subsequently, and all rectal swabs collected from diarrheic Thoroughbred foals were negative. Furthermore, only 2.5% of the rectal swabs collected from healthy foals in the largest Thoroughbred horse breeding region in Japan were positive for ECoV .", "In contrast in Japan, although an outbreak of diarrhoea occurred among ECoV-infected draft horses at one racecourse , there have been no similar outbreaks subsequently, and all rectal swabs collected from diarrheic Thoroughbred foals were negative. Furthermore, only 2.5% of the rectal swabs collected from healthy foals in the largest Thoroughbred horse breeding region in Japan were positive for ECoV . In France, 2.8% of 395 faecal samples and 0.5% of 200 respiratory samples collected in 58 counties tested positive for ECoV . Similar to the reports from Japan and France, a low prevalence of ECoV was also observed in the UK , Saudi Arabia and Oman . The objective of this study was to investigate the presence of ECoV in clinical samples submitted to a diagnostic laboratory in Ireland. The samples were tested by real-time reverse transcription polymerase chain reaction rRT-PCR as it has been shown to be the most sensitive diagnostic method for ECoV and is routinely employed as an alternative to virus isolation in diagnostic laboratories worldwide, both for timely diagnosis and in epidemiological studies .", "The objective of this study was to investigate the presence of ECoV in clinical samples submitted to a diagnostic laboratory in Ireland. The samples were tested by real-time reverse transcription polymerase chain reaction rRT-PCR as it has been shown to be the most sensitive diagnostic method for ECoV and is routinely employed as an alternative to virus isolation in diagnostic laboratories worldwide, both for timely diagnosis and in epidemiological studies . Virus isolation and biological characterisation were beyond the capacity of this study, which was similar in scope to that of the studies in horse populations in the USA, Europe and Asia . The rRT-PCR assay was performed as previously described using a primer set targeting the nucleocapsid N gene ECoV-380f, ECoV-522r and ECoV-436p Table 1 and AgPath-ID One-Step RT-PCR Kit Thermo Fisher Scientific, MA, USA according to the manufacturer's instructions. To prove that the extraction was successful and that there was no inhibition during rRT-PCR amplification, an internal positive control primer/probe PrimerDesign, Southampton, UK was added to the master mix. Thermal cycling conditions were; 48 • C for 10 min and 95 • C for 10 min, followed by 40 cycles at 94 • C for 15 s and 60 • C for 45 s. The SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity Thermo Fisher Scientific, MA, USA was used for sequencing analysis of two of the five ECoV samples identified.", "To prove that the extraction was successful and that there was no inhibition during rRT-PCR amplification, an internal positive control primer/probe PrimerDesign, Southampton, UK was added to the master mix. Thermal cycling conditions were; 48 • C for 10 min and 95 • C for 10 min, followed by 40 cycles at 94 • C for 15 s and 60 • C for 45 s. The SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity Thermo Fisher Scientific, MA, USA was used for sequencing analysis of two of the five ECoV samples identified. There was inadequate viral nucleic acid in the other three samples for sequencing. The primer sets used to amplify the nucleocapsid N gene , the partial spike S gene , and the region from the p4.7 to p12.7 genes of non-structural proteins Oue, personal communication are shown in Table 1 . The RT-PCR products were sequenced commercially by GATC Biotech Cologne, Germany . Sequence analysis was performed using the BLAST and CLUSTALW programs, and Vector NTI Advance 11.5 software Thermo Fisher Scientific, MA, USA .", "The RT-PCR products were sequenced commercially by GATC Biotech Cologne, Germany . Sequence analysis was performed using the BLAST and CLUSTALW programs, and Vector NTI Advance 11.5 software Thermo Fisher Scientific, MA, USA . Phylogenetic analysis of nucleotide sequences was conducted with MEGA software Version 5.2 . A phylogenetic tree was constructed based on nucleotide sequences of the K2+G N gene and TN93 S gene using the maximum likelihood method. MEGA software was used to select the optimal substitution models. Statistical analysis of the tree was performed with the bootstrap test 1000 replicates for multiple alignments.", "MEGA software was used to select the optimal substitution models. Statistical analysis of the tree was performed with the bootstrap test 1000 replicates for multiple alignments. The complete genome sequences of NC99 EF446615 , Tokachi09 LC061272 , Obihiro12-1 LC061273 and Obihiro12-2 LC061274 , the N AB671298 and S AB671299 genes of Obihiro2004, the N gene of Hidaka-No.61/2012 LC054263 and Hidaka-No.119/2012 LC054264 , the S gene of ECoV_FRA_2011/1 KC178705 , ECoV_FRA_2011/2 KC178704 , ECoV_FRA_2012/1 KC178703 , ECoV_FRA_2012/2 KC178702 and ECoV_FRA_2012/3 KC178701 were used in sequence and/or phylogenetic analysis. The accession numbers registered in GenBank/EMBL/DDBJ are as follows: the complete sequences of the N gene; 11V11708/IRL LC149485 and 13V08313/IRL LC149486 , the partial sequences of the S gene; 11V11708/IRL LC149487 and13V08313/IRL LC149488 and the complete sequences from the p4.7 to p12.7 genes; 11V11708/IRL LC149489 and13V08313/IRL LC149490 . One six-week-old foal was the only clinical case on a public Thoroughbred stud farm with approximately 30 mares when it presented with diarrhoea. Recovery took over three weeks during which it received fluid therapy, probiotics, antiulcer medication and antibiotics.", "One six-week-old foal was the only clinical case on a public Thoroughbred stud farm with approximately 30 mares when it presented with diarrhoea. Recovery took over three weeks during which it received fluid therapy, probiotics, antiulcer medication and antibiotics. The second foal was a 14-day-old filly, which had been hospitalised with diarrhoea two days prior to sample collection. The foal responded well to supportive treatment and at the time of sample collection, the diarrhoea had resolved. The five ECoV positive samples tested negative for equine rotavirus. The nucleotide sequences of the complete N gene, the partial S gene and the region from the p4.7 to p12.7 genes of two positive samples 11V11708/IRL/2011 and 13V08313/IRL/2013 were determined.", "The five ECoV positive samples tested negative for equine rotavirus. The nucleotide sequences of the complete N gene, the partial S gene and the region from the p4.7 to p12.7 genes of two positive samples 11V11708/IRL/2011 and 13V08313/IRL/2013 were determined. The nucleotide identities of the N and S genes of the two Irish ECoVs were 99.8% 1338/1341 nucleotides and 99.5% 650/653 nucleotides , respectively. The nucleotide identities of the N gene of the two Irish ECoVs and the ECoVs from other continents are summarised in Table 2 . Phylogenetic analysis was performed for the nucleotide sequences of the complete N and partial S genes Figure 1 . The analysis for the N gene showed that Irish ECoVs were independently clustered although they were closely related to Japanese viruses identified after 2009.", "Phylogenetic analysis was performed for the nucleotide sequences of the complete N and partial S genes Figure 1 . The analysis for the N gene showed that Irish ECoVs were independently clustered although they were closely related to Japanese viruses identified after 2009. In the phylogenetic tree of the S gene, Irish ECoVs were closely related to all other ECoVs analysed. The length of the region from the p4.7 to p12.7 genes in the two viruses was 544 base pairs. Compared with NC99, Irish ECoVs, had a total of 37 nucleotide deletions within p4.7 and the non-coding region following the p4.7 gene. Compared with Obihiro 12-1 and 12-2, Irish ECoVs had a three-nucleotide insertion.", "Compared with NC99, Irish ECoVs, had a total of 37 nucleotide deletions within p4.7 and the non-coding region following the p4.7 gene. Compared with Obihiro 12-1 and 12-2, Irish ECoVs had a three-nucleotide insertion. When compared with Tokachi09, the Irish ECoVs had a 148-nucleotide insertion see Figure S1 . The p12.7 gene of the two Irish ECoVs did not have deletions or insertions, and the nucleotide identities were 98.8-99.7% between these viruses and the other ECoVs NC99, Tokachi09, Obihiro12-1 and Obihiro12-2 . This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease. However, detection of ECoV in faeces samples from horses with enteric disease does not prove This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease.", "This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease. However, detection of ECoV in faeces samples from horses with enteric disease does not prove This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease. However, detection of ECoV in faeces samples from horses with enteric disease does not prove causation. In this study, 424 samples collected between 2011 and 2015 from equids with enteric disease were tested, and only five samples 1.2% were positive for ECoV. The inclusion of an internal positive control in the rRT-PCR eliminated the possibility of false negative results due to the presence of PCR inhibitors but the high content of nucleases associated with faeces samples may have caused some RNA degradation. However, this low prevalence of ECoV is similar to that identified in France and among Thoroughbred foals in Japan .", "The inclusion of an internal positive control in the rRT-PCR eliminated the possibility of false negative results due to the presence of PCR inhibitors but the high content of nucleases associated with faeces samples may have caused some RNA degradation. However, this low prevalence of ECoV is similar to that identified in France and among Thoroughbred foals in Japan . Although ECoV has been identified on three continents, little is known about the genetic and pathogenic diversity in field viruses. In this study, sequence and phylogenetic analysis Figure 1 demonstrated a high level of homology between viruses detected in a donkey and a horse in two provinces in Ireland in different years. This suggests that Irish ECoVs may have low genetic diversity. Compared with the ECoVs of other countries, the N, S and p12.7 genes of the two Irish viruses were highly conserved.", "This suggests that Irish ECoVs may have low genetic diversity. Compared with the ECoVs of other countries, the N, S and p12.7 genes of the two Irish viruses were highly conserved. In contrast, the region from p4.7 and the non-coding region following the p4.7 gene had deletions or insertions Figure S1 . Because of polymorphism in this region, this region could be useful for epidemiological investigation . The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the viruses in Ireland may be distinguishable from those circulating in other countries. The positive samples were collected in November ., March . and April . in this study.", "The positive samples were collected in November ., March . and April . in this study. Higher case numbers are identified in the USA during the colder months October to April , and our results were consistent with the circulation period in USA. It has been reported that outbreaks mainly occurred among adult riding, racing and show horses in USA . The choice of cases to include in the current study may not have been optimal for detection of ECoV as the majority of samples were from foals. However, two positive samples were collected from adult horses in a combined riding school/show jumping yard in the West of Ireland.", "The choice of cases to include in the current study may not have been optimal for detection of ECoV as the majority of samples were from foals. However, two positive samples were collected from adult horses in a combined riding school/show jumping yard in the West of Ireland. At the time of sample collection in April 2013, the monthly mean temperatures were below long-term average and in parts of the West, were the coldest in 24 years . Cold weather may have been a predisposing factor to the ECoV infection on the farm. Two positive samples were collected from Thoroughbred foals. A faeces sample collected from one foal with severe watery diarrhoea and inappetance was positive for ECoV but a sample collected three days later tested negative.", "Two positive samples were collected from Thoroughbred foals. A faeces sample collected from one foal with severe watery diarrhoea and inappetance was positive for ECoV but a sample collected three days later tested negative. A potential difficulty in detecting ECoV from naturally infected horses has been noted previously as serial samples from seven sick horses in the USA suggested that ECoV only persisted for three to nine days in faeces . In both cases, the diarrhoea may have been caused by other unidentified coinfecting pathogens as has been suggested by investigators in the USA . This is the first report of ECoV detection in faeces samples from both foals and adult horses in Ireland. The viruses identified in Ireland are genetically closely related to the Japanese viruses and the results of this study give no indication of significant genetic or phenotypic diversity.", "This is the first report of ECoV detection in faeces samples from both foals and adult horses in Ireland. The viruses identified in Ireland are genetically closely related to the Japanese viruses and the results of this study give no indication of significant genetic or phenotypic diversity. In recent years, there has been an increase in awareness and testing for ECoV in the USA and elsewhere . Horse breeding and racing activities in Ireland are the most prominent and important of any country on a per capita basis. There are over 50 Thoroughbred horses per 10,000 of population in Ireland, compared to between three and five for Great Britain, France and the USA . Thus, an investigation of ECoV in Ireland is pertinent not only to increase awareness nationally of the epidemiology of the virus and promote discussion on its clinical importance, but also to inform the industry globally of the health status of Irish horses.", "There are over 50 Thoroughbred horses per 10,000 of population in Ireland, compared to between three and five for Great Britain, France and the USA . Thus, an investigation of ECoV in Ireland is pertinent not only to increase awareness nationally of the epidemiology of the virus and promote discussion on its clinical importance, but also to inform the industry globally of the health status of Irish horses. Ireland exports horses all over the world. By illustration, in 2016 the country was the second biggest seller of bloodstock at public auctions second only to the USA . Many questions remain with regard to the clinical significance of ECoV. The outbreak at a draft-horse racetrack in Japan in 2009 affected 132 of approximately 600 horses and resulted in non-starters and the implementation of movement restrictions .", "Many questions remain with regard to the clinical significance of ECoV. The outbreak at a draft-horse racetrack in Japan in 2009 affected 132 of approximately 600 horses and resulted in non-starters and the implementation of movement restrictions . However, draft horses appear to have a higher infection rate than other breeds and an outbreak of similar severity has not been reported in Thoroughbred racehorses . The much higher incidence of ECoV positive Thoroughbred foals identified in Kentucky compared to similar populations internationally suggests an increased susceptibility to ECoV infection in that population. In the past, specific environmental factors were associated with extensive reproductive loss in the Kentucky area and to a lesser extent in other states , but predisposing regional factors such as differences in management, environment or husbandry have not been identified for ECoV. It has been suggested that ECoV is a coinfecting agent in foals with diarrhoea and clinical infections have predominantly been reported in adult horses with a mono-infection with EcoV .", "In the past, specific environmental factors were associated with extensive reproductive loss in the Kentucky area and to a lesser extent in other states , but predisposing regional factors such as differences in management, environment or husbandry have not been identified for ECoV. It has been suggested that ECoV is a coinfecting agent in foals with diarrhoea and clinical infections have predominantly been reported in adult horses with a mono-infection with EcoV . There was no indication from the results of this study that coronavirus is a major cause of diarrhoea in Irish horses but the introduction of rRT-PCR as a routine diagnostic test will assist in elucidating the significance of this virus to the Irish breeding, racing and sports industries. The primary focus in future will be on testing adult horses that present with anorexia, lethargy, fever and changes in faecal character as a significant association has been demonstrated between this clinical status and molecular detection of ECoV in faeces ." ]

[ "The objective of this study was to investigate the presence of equine coronavirus ECoV in clinical samples submitted to a diagnostic laboratory in Ireland. A total of 424 clinical samples were examined from equids with enteric disease in 24 Irish counties between 2011 and 2015. A real-time reverse transcription polymerase chain reaction was used to detect ECoV RNA. Nucleocapsid, spike and the region from the p4.7 to p12.7 genes of positive samples were sequenced, and sequence and phylogenetic analyses were conducted. Five samples 1.2% collected in 2011 and 2013 tested positive for ECoV. Positive samples were collected from adult horses, Thoroughbred foals and a donkey foal.", "Five samples 1.2% collected in 2011 and 2013 tested positive for ECoV. Positive samples were collected from adult horses, Thoroughbred foals and a donkey foal. Sequence and/or phylogenetic analysis showed that nucleocapsid, spike and p12.7 genes were highly conserved and were closely related to ECoVs identified in other countries. In contrast, the region from p4.7 and the non-coding region following the p4.7 gene had deletions or insertions. The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the Irish viruses were distinguishable from those circulating in other countries. This is the first report of ECoV detected in both foals and adult horses in Ireland.", "The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the Irish viruses were distinguishable from those circulating in other countries. This is the first report of ECoV detected in both foals and adult horses in Ireland. Text: Equine coronavirus ECoV is a positive-stranded RNA virus and belongs to the species Betacoronavirus 1 in the genus Betacoronavirus . The clinical signs associated with ECoV infection during outbreaks in the USA and Japan were fever, anorexia, lethargy and diarrhoea. The same clinical signs were also recorded in an experimental challenge study using Japanese draft horses . The main transmission route is considered to be faecal-oral and ECoV is usually detected in faecal samples.", "The same clinical signs were also recorded in an experimental challenge study using Japanese draft horses . The main transmission route is considered to be faecal-oral and ECoV is usually detected in faecal samples. However, the molecular detection of ECoV in faeces from horses with diarrhoea, does not prove causation. Coronaviruses can cause both enteric and respiratory disease in many avian and mammalian species but ECoV is less likely to be found in respiratory secretions than in faeces . Both molecular and seroepidemiology studies suggest that ECoV may be more prevalent in the USA than in other countries . ECoV was detected in samples collected from equids in 48 states of the USA .", "Both molecular and seroepidemiology studies suggest that ECoV may be more prevalent in the USA than in other countries . ECoV was detected in samples collected from equids in 48 states of the USA . In central Kentucky, approximately 30% of both healthy and diarrheic Thoroughbred foals were infected with ECoV . All of the qPCR positive foals with diarrhoea were co-infected with other pathogens such as rotavirus or Clostridium perfringens, suggesting that there was potential for ECoV to be over-diagnosed as a causative agent in complex diseases. In contrast in Japan, although an outbreak of diarrhoea occurred among ECoV-infected draft horses at one racecourse , there have been no similar outbreaks subsequently, and all rectal swabs collected from diarrheic Thoroughbred foals were negative. Furthermore, only 2.5% of the rectal swabs collected from healthy foals in the largest Thoroughbred horse breeding region in Japan were positive for ECoV .", "In contrast in Japan, although an outbreak of diarrhoea occurred among ECoV-infected draft horses at one racecourse , there have been no similar outbreaks subsequently, and all rectal swabs collected from diarrheic Thoroughbred foals were negative. Furthermore, only 2.5% of the rectal swabs collected from healthy foals in the largest Thoroughbred horse breeding region in Japan were positive for ECoV . In France, 2.8% of 395 faecal samples and 0.5% of 200 respiratory samples collected in 58 counties tested positive for ECoV . Similar to the reports from Japan and France, a low prevalence of ECoV was also observed in the UK , Saudi Arabia and Oman . The objective of this study was to investigate the presence of ECoV in clinical samples submitted to a diagnostic laboratory in Ireland. The samples were tested by real-time reverse transcription polymerase chain reaction rRT-PCR as it has been shown to be the most sensitive diagnostic method for ECoV and is routinely employed as an alternative to virus isolation in diagnostic laboratories worldwide, both for timely diagnosis and in epidemiological studies .", "The objective of this study was to investigate the presence of ECoV in clinical samples submitted to a diagnostic laboratory in Ireland. The samples were tested by real-time reverse transcription polymerase chain reaction rRT-PCR as it has been shown to be the most sensitive diagnostic method for ECoV and is routinely employed as an alternative to virus isolation in diagnostic laboratories worldwide, both for timely diagnosis and in epidemiological studies . Virus isolation and biological characterisation were beyond the capacity of this study, which was similar in scope to that of the studies in horse populations in the USA, Europe and Asia . The rRT-PCR assay was performed as previously described using a primer set targeting the nucleocapsid N gene ECoV-380f, ECoV-522r and ECoV-436p Table 1 and AgPath-ID One-Step RT-PCR Kit Thermo Fisher Scientific, MA, USA according to the manufacturer's instructions. To prove that the extraction was successful and that there was no inhibition during rRT-PCR amplification, an internal positive control primer/probe PrimerDesign, Southampton, UK was added to the master mix. Thermal cycling conditions were; 48 • C for 10 min and 95 • C for 10 min, followed by 40 cycles at 94 • C for 15 s and 60 • C for 45 s. The SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity Thermo Fisher Scientific, MA, USA was used for sequencing analysis of two of the five ECoV samples identified.", "To prove that the extraction was successful and that there was no inhibition during rRT-PCR amplification, an internal positive control primer/probe PrimerDesign, Southampton, UK was added to the master mix. Thermal cycling conditions were; 48 • C for 10 min and 95 • C for 10 min, followed by 40 cycles at 94 • C for 15 s and 60 • C for 45 s. The SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity Thermo Fisher Scientific, MA, USA was used for sequencing analysis of two of the five ECoV samples identified. There was inadequate viral nucleic acid in the other three samples for sequencing. The primer sets used to amplify the nucleocapsid N gene , the partial spike S gene , and the region from the p4.7 to p12.7 genes of non-structural proteins Oue, personal communication are shown in Table 1 . The RT-PCR products were sequenced commercially by GATC Biotech Cologne, Germany . Sequence analysis was performed using the BLAST and CLUSTALW programs, and Vector NTI Advance 11.5 software Thermo Fisher Scientific, MA, USA .", "The RT-PCR products were sequenced commercially by GATC Biotech Cologne, Germany . Sequence analysis was performed using the BLAST and CLUSTALW programs, and Vector NTI Advance 11.5 software Thermo Fisher Scientific, MA, USA . Phylogenetic analysis of nucleotide sequences was conducted with MEGA software Version 5.2 . A phylogenetic tree was constructed based on nucleotide sequences of the K2+G N gene and TN93 S gene using the maximum likelihood method. MEGA software was used to select the optimal substitution models. Statistical analysis of the tree was performed with the bootstrap test 1000 replicates for multiple alignments.", "MEGA software was used to select the optimal substitution models. Statistical analysis of the tree was performed with the bootstrap test 1000 replicates for multiple alignments. The complete genome sequences of NC99 EF446615 , Tokachi09 LC061272 , Obihiro12-1 LC061273 and Obihiro12-2 LC061274 , the N AB671298 and S AB671299 genes of Obihiro2004, the N gene of Hidaka-No.61/2012 LC054263 and Hidaka-No.119/2012 LC054264 , the S gene of ECoV_FRA_2011/1 KC178705 , ECoV_FRA_2011/2 KC178704 , ECoV_FRA_2012/1 KC178703 , ECoV_FRA_2012/2 KC178702 and ECoV_FRA_2012/3 KC178701 were used in sequence and/or phylogenetic analysis. The accession numbers registered in GenBank/EMBL/DDBJ are as follows: the complete sequences of the N gene; 11V11708/IRL LC149485 and 13V08313/IRL LC149486 , the partial sequences of the S gene; 11V11708/IRL LC149487 and13V08313/IRL LC149488 and the complete sequences from the p4.7 to p12.7 genes; 11V11708/IRL LC149489 and13V08313/IRL LC149490 . One six-week-old foal was the only clinical case on a public Thoroughbred stud farm with approximately 30 mares when it presented with diarrhoea. Recovery took over three weeks during which it received fluid therapy, probiotics, antiulcer medication and antibiotics.", "One six-week-old foal was the only clinical case on a public Thoroughbred stud farm with approximately 30 mares when it presented with diarrhoea. Recovery took over three weeks during which it received fluid therapy, probiotics, antiulcer medication and antibiotics. The second foal was a 14-day-old filly, which had been hospitalised with diarrhoea two days prior to sample collection. The foal responded well to supportive treatment and at the time of sample collection, the diarrhoea had resolved. The five ECoV positive samples tested negative for equine rotavirus. The nucleotide sequences of the complete N gene, the partial S gene and the region from the p4.7 to p12.7 genes of two positive samples 11V11708/IRL/2011 and 13V08313/IRL/2013 were determined.", "The five ECoV positive samples tested negative for equine rotavirus. The nucleotide sequences of the complete N gene, the partial S gene and the region from the p4.7 to p12.7 genes of two positive samples 11V11708/IRL/2011 and 13V08313/IRL/2013 were determined. The nucleotide identities of the N and S genes of the two Irish ECoVs were 99.8% 1338/1341 nucleotides and 99.5% 650/653 nucleotides , respectively. The nucleotide identities of the N gene of the two Irish ECoVs and the ECoVs from other continents are summarised in Table 2 . Phylogenetic analysis was performed for the nucleotide sequences of the complete N and partial S genes Figure 1 . The analysis for the N gene showed that Irish ECoVs were independently clustered although they were closely related to Japanese viruses identified after 2009.", "Phylogenetic analysis was performed for the nucleotide sequences of the complete N and partial S genes Figure 1 . The analysis for the N gene showed that Irish ECoVs were independently clustered although they were closely related to Japanese viruses identified after 2009. In the phylogenetic tree of the S gene, Irish ECoVs were closely related to all other ECoVs analysed. The length of the region from the p4.7 to p12.7 genes in the two viruses was 544 base pairs. Compared with NC99, Irish ECoVs, had a total of 37 nucleotide deletions within p4.7 and the non-coding region following the p4.7 gene. Compared with Obihiro 12-1 and 12-2, Irish ECoVs had a three-nucleotide insertion.", "Compared with NC99, Irish ECoVs, had a total of 37 nucleotide deletions within p4.7 and the non-coding region following the p4.7 gene. Compared with Obihiro 12-1 and 12-2, Irish ECoVs had a three-nucleotide insertion. When compared with Tokachi09, the Irish ECoVs had a 148-nucleotide insertion see Figure S1 . The p12.7 gene of the two Irish ECoVs did not have deletions or insertions, and the nucleotide identities were 98.8-99.7% between these viruses and the other ECoVs NC99, Tokachi09, Obihiro12-1 and Obihiro12-2 . This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease. However, detection of ECoV in faeces samples from horses with enteric disease does not prove This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease.", "This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease. However, detection of ECoV in faeces samples from horses with enteric disease does not prove This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease. However, detection of ECoV in faeces samples from horses with enteric disease does not prove causation. In this study, 424 samples collected between 2011 and 2015 from equids with enteric disease were tested, and only five samples 1.2% were positive for ECoV. The inclusion of an internal positive control in the rRT-PCR eliminated the possibility of false negative results due to the presence of PCR inhibitors but the high content of nucleases associated with faeces samples may have caused some RNA degradation. However, this low prevalence of ECoV is similar to that identified in France and among Thoroughbred foals in Japan .", "The inclusion of an internal positive control in the rRT-PCR eliminated the possibility of false negative results due to the presence of PCR inhibitors but the high content of nucleases associated with faeces samples may have caused some RNA degradation. However, this low prevalence of ECoV is similar to that identified in France and among Thoroughbred foals in Japan . Although ECoV has been identified on three continents, little is known about the genetic and pathogenic diversity in field viruses. In this study, sequence and phylogenetic analysis Figure 1 demonstrated a high level of homology between viruses detected in a donkey and a horse in two provinces in Ireland in different years. This suggests that Irish ECoVs may have low genetic diversity. Compared with the ECoVs of other countries, the N, S and p12.7 genes of the two Irish viruses were highly conserved.", "This suggests that Irish ECoVs may have low genetic diversity. Compared with the ECoVs of other countries, the N, S and p12.7 genes of the two Irish viruses were highly conserved. In contrast, the region from p4.7 and the non-coding region following the p4.7 gene had deletions or insertions Figure S1 . Because of polymorphism in this region, this region could be useful for epidemiological investigation . The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the viruses in Ireland may be distinguishable from those circulating in other countries. The positive samples were collected in November ., March . and April . in this study.", "The positive samples were collected in November ., March . and April . in this study. Higher case numbers are identified in the USA during the colder months October to April , and our results were consistent with the circulation period in USA. It has been reported that outbreaks mainly occurred among adult riding, racing and show horses in USA . The choice of cases to include in the current study may not have been optimal for detection of ECoV as the majority of samples were from foals. However, two positive samples were collected from adult horses in a combined riding school/show jumping yard in the West of Ireland.", "The choice of cases to include in the current study may not have been optimal for detection of ECoV as the majority of samples were from foals. However, two positive samples were collected from adult horses in a combined riding school/show jumping yard in the West of Ireland. At the time of sample collection in April 2013, the monthly mean temperatures were below long-term average and in parts of the West, were the coldest in 24 years . Cold weather may have been a predisposing factor to the ECoV infection on the farm. Two positive samples were collected from Thoroughbred foals. A faeces sample collected from one foal with severe watery diarrhoea and inappetance was positive for ECoV but a sample collected three days later tested negative.", "Two positive samples were collected from Thoroughbred foals. A faeces sample collected from one foal with severe watery diarrhoea and inappetance was positive for ECoV but a sample collected three days later tested negative. A potential difficulty in detecting ECoV from naturally infected horses has been noted previously as serial samples from seven sick horses in the USA suggested that ECoV only persisted for three to nine days in faeces . In both cases, the diarrhoea may have been caused by other unidentified coinfecting pathogens as has been suggested by investigators in the USA . This is the first report of ECoV detection in faeces samples from both foals and adult horses in Ireland. The viruses identified in Ireland are genetically closely related to the Japanese viruses and the results of this study give no indication of significant genetic or phenotypic diversity.", "This is the first report of ECoV detection in faeces samples from both foals and adult horses in Ireland. The viruses identified in Ireland are genetically closely related to the Japanese viruses and the results of this study give no indication of significant genetic or phenotypic diversity. In recent years, there has been an increase in awareness and testing for ECoV in the USA and elsewhere . Horse breeding and racing activities in Ireland are the most prominent and important of any country on a per capita basis. There are over 50 Thoroughbred horses per 10,000 of population in Ireland, compared to between three and five for Great Britain, France and the USA . Thus, an investigation of ECoV in Ireland is pertinent not only to increase awareness nationally of the epidemiology of the virus and promote discussion on its clinical importance, but also to inform the industry globally of the health status of Irish horses.", "There are over 50 Thoroughbred horses per 10,000 of population in Ireland, compared to between three and five for Great Britain, France and the USA . Thus, an investigation of ECoV in Ireland is pertinent not only to increase awareness nationally of the epidemiology of the virus and promote discussion on its clinical importance, but also to inform the industry globally of the health status of Irish horses. Ireland exports horses all over the world. By illustration, in 2016 the country was the second biggest seller of bloodstock at public auctions second only to the USA . Many questions remain with regard to the clinical significance of ECoV. The outbreak at a draft-horse racetrack in Japan in 2009 affected 132 of approximately 600 horses and resulted in non-starters and the implementation of movement restrictions .", "Many questions remain with regard to the clinical significance of ECoV. The outbreak at a draft-horse racetrack in Japan in 2009 affected 132 of approximately 600 horses and resulted in non-starters and the implementation of movement restrictions . However, draft horses appear to have a higher infection rate than other breeds and an outbreak of similar severity has not been reported in Thoroughbred racehorses . The much higher incidence of ECoV positive Thoroughbred foals identified in Kentucky compared to similar populations internationally suggests an increased susceptibility to ECoV infection in that population. In the past, specific environmental factors were associated with extensive reproductive loss in the Kentucky area and to a lesser extent in other states , but predisposing regional factors such as differences in management, environment or husbandry have not been identified for ECoV. It has been suggested that ECoV is a coinfecting agent in foals with diarrhoea and clinical infections have predominantly been reported in adult horses with a mono-infection with EcoV .", "In the past, specific environmental factors were associated with extensive reproductive loss in the Kentucky area and to a lesser extent in other states , but predisposing regional factors such as differences in management, environment or husbandry have not been identified for ECoV. It has been suggested that ECoV is a coinfecting agent in foals with diarrhoea and clinical infections have predominantly been reported in adult horses with a mono-infection with EcoV . There was no indication from the results of this study that coronavirus is a major cause of diarrhoea in Irish horses but the introduction of rRT-PCR as a routine diagnostic test will assist in elucidating the significance of this virus to the Irish breeding, racing and sports industries. The primary focus in future will be on testing adult horses that present with anorexia, lethargy, fever and changes in faecal character as a significant association has been demonstrated between this clinical status and molecular detection of ECoV in faeces ." ]

[ "The objective of this study was to investigate the presence of equine coronavirus ECoV in clinical samples submitted to a diagnostic laboratory in Ireland. A total of 424 clinical samples were examined from equids with enteric disease in 24 Irish counties between 2011 and 2015. A real-time reverse transcription polymerase chain reaction was used to detect ECoV RNA. Nucleocapsid, spike and the region from the p4.7 to p12.7 genes of positive samples were sequenced, and sequence and phylogenetic analyses were conducted. Five samples 1.2% collected in 2011 and 2013 tested positive for ECoV. Positive samples were collected from adult horses, Thoroughbred foals and a donkey foal.", "Five samples 1.2% collected in 2011 and 2013 tested positive for ECoV. Positive samples were collected from adult horses, Thoroughbred foals and a donkey foal. Sequence and/or phylogenetic analysis showed that nucleocapsid, spike and p12.7 genes were highly conserved and were closely related to ECoVs identified in other countries. In contrast, the region from p4.7 and the non-coding region following the p4.7 gene had deletions or insertions. The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the Irish viruses were distinguishable from those circulating in other countries. This is the first report of ECoV detected in both foals and adult horses in Ireland.", "The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the Irish viruses were distinguishable from those circulating in other countries. This is the first report of ECoV detected in both foals and adult horses in Ireland. Text: Equine coronavirus ECoV is a positive-stranded RNA virus and belongs to the species Betacoronavirus 1 in the genus Betacoronavirus . The clinical signs associated with ECoV infection during outbreaks in the USA and Japan were fever, anorexia, lethargy and diarrhoea. The same clinical signs were also recorded in an experimental challenge study using Japanese draft horses . The main transmission route is considered to be faecal-oral and ECoV is usually detected in faecal samples.", "The same clinical signs were also recorded in an experimental challenge study using Japanese draft horses . The main transmission route is considered to be faecal-oral and ECoV is usually detected in faecal samples. However, the molecular detection of ECoV in faeces from horses with diarrhoea, does not prove causation. Coronaviruses can cause both enteric and respiratory disease in many avian and mammalian species but ECoV is less likely to be found in respiratory secretions than in faeces . Both molecular and seroepidemiology studies suggest that ECoV may be more prevalent in the USA than in other countries . ECoV was detected in samples collected from equids in 48 states of the USA .", "Both molecular and seroepidemiology studies suggest that ECoV may be more prevalent in the USA than in other countries . ECoV was detected in samples collected from equids in 48 states of the USA . In central Kentucky, approximately 30% of both healthy and diarrheic Thoroughbred foals were infected with ECoV . All of the qPCR positive foals with diarrhoea were co-infected with other pathogens such as rotavirus or Clostridium perfringens, suggesting that there was potential for ECoV to be over-diagnosed as a causative agent in complex diseases. In contrast in Japan, although an outbreak of diarrhoea occurred among ECoV-infected draft horses at one racecourse , there have been no similar outbreaks subsequently, and all rectal swabs collected from diarrheic Thoroughbred foals were negative. Furthermore, only 2.5% of the rectal swabs collected from healthy foals in the largest Thoroughbred horse breeding region in Japan were positive for ECoV .", "In contrast in Japan, although an outbreak of diarrhoea occurred among ECoV-infected draft horses at one racecourse , there have been no similar outbreaks subsequently, and all rectal swabs collected from diarrheic Thoroughbred foals were negative. Furthermore, only 2.5% of the rectal swabs collected from healthy foals in the largest Thoroughbred horse breeding region in Japan were positive for ECoV . In France, 2.8% of 395 faecal samples and 0.5% of 200 respiratory samples collected in 58 counties tested positive for ECoV . Similar to the reports from Japan and France, a low prevalence of ECoV was also observed in the UK , Saudi Arabia and Oman . The objective of this study was to investigate the presence of ECoV in clinical samples submitted to a diagnostic laboratory in Ireland. The samples were tested by real-time reverse transcription polymerase chain reaction rRT-PCR as it has been shown to be the most sensitive diagnostic method for ECoV and is routinely employed as an alternative to virus isolation in diagnostic laboratories worldwide, both for timely diagnosis and in epidemiological studies .", "The objective of this study was to investigate the presence of ECoV in clinical samples submitted to a diagnostic laboratory in Ireland. The samples were tested by real-time reverse transcription polymerase chain reaction rRT-PCR as it has been shown to be the most sensitive diagnostic method for ECoV and is routinely employed as an alternative to virus isolation in diagnostic laboratories worldwide, both for timely diagnosis and in epidemiological studies . Virus isolation and biological characterisation were beyond the capacity of this study, which was similar in scope to that of the studies in horse populations in the USA, Europe and Asia . The rRT-PCR assay was performed as previously described using a primer set targeting the nucleocapsid N gene ECoV-380f, ECoV-522r and ECoV-436p Table 1 and AgPath-ID One-Step RT-PCR Kit Thermo Fisher Scientific, MA, USA according to the manufacturer's instructions. To prove that the extraction was successful and that there was no inhibition during rRT-PCR amplification, an internal positive control primer/probe PrimerDesign, Southampton, UK was added to the master mix. Thermal cycling conditions were; 48 • C for 10 min and 95 • C for 10 min, followed by 40 cycles at 94 • C for 15 s and 60 • C for 45 s. The SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity Thermo Fisher Scientific, MA, USA was used for sequencing analysis of two of the five ECoV samples identified.", "To prove that the extraction was successful and that there was no inhibition during rRT-PCR amplification, an internal positive control primer/probe PrimerDesign, Southampton, UK was added to the master mix. Thermal cycling conditions were; 48 • C for 10 min and 95 • C for 10 min, followed by 40 cycles at 94 • C for 15 s and 60 • C for 45 s. The SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity Thermo Fisher Scientific, MA, USA was used for sequencing analysis of two of the five ECoV samples identified. There was inadequate viral nucleic acid in the other three samples for sequencing. The primer sets used to amplify the nucleocapsid N gene , the partial spike S gene , and the region from the p4.7 to p12.7 genes of non-structural proteins Oue, personal communication are shown in Table 1 . The RT-PCR products were sequenced commercially by GATC Biotech Cologne, Germany . Sequence analysis was performed using the BLAST and CLUSTALW programs, and Vector NTI Advance 11.5 software Thermo Fisher Scientific, MA, USA .", "The RT-PCR products were sequenced commercially by GATC Biotech Cologne, Germany . Sequence analysis was performed using the BLAST and CLUSTALW programs, and Vector NTI Advance 11.5 software Thermo Fisher Scientific, MA, USA . Phylogenetic analysis of nucleotide sequences was conducted with MEGA software Version 5.2 . A phylogenetic tree was constructed based on nucleotide sequences of the K2+G N gene and TN93 S gene using the maximum likelihood method. MEGA software was used to select the optimal substitution models. Statistical analysis of the tree was performed with the bootstrap test 1000 replicates for multiple alignments.", "MEGA software was used to select the optimal substitution models. Statistical analysis of the tree was performed with the bootstrap test 1000 replicates for multiple alignments. The complete genome sequences of NC99 EF446615 , Tokachi09 LC061272 , Obihiro12-1 LC061273 and Obihiro12-2 LC061274 , the N AB671298 and S AB671299 genes of Obihiro2004, the N gene of Hidaka-No.61/2012 LC054263 and Hidaka-No.119/2012 LC054264 , the S gene of ECoV_FRA_2011/1 KC178705 , ECoV_FRA_2011/2 KC178704 , ECoV_FRA_2012/1 KC178703 , ECoV_FRA_2012/2 KC178702 and ECoV_FRA_2012/3 KC178701 were used in sequence and/or phylogenetic analysis. The accession numbers registered in GenBank/EMBL/DDBJ are as follows: the complete sequences of the N gene; 11V11708/IRL LC149485 and 13V08313/IRL LC149486 , the partial sequences of the S gene; 11V11708/IRL LC149487 and13V08313/IRL LC149488 and the complete sequences from the p4.7 to p12.7 genes; 11V11708/IRL LC149489 and13V08313/IRL LC149490 . One six-week-old foal was the only clinical case on a public Thoroughbred stud farm with approximately 30 mares when it presented with diarrhoea. Recovery took over three weeks during which it received fluid therapy, probiotics, antiulcer medication and antibiotics.", "One six-week-old foal was the only clinical case on a public Thoroughbred stud farm with approximately 30 mares when it presented with diarrhoea. Recovery took over three weeks during which it received fluid therapy, probiotics, antiulcer medication and antibiotics. The second foal was a 14-day-old filly, which had been hospitalised with diarrhoea two days prior to sample collection. The foal responded well to supportive treatment and at the time of sample collection, the diarrhoea had resolved. The five ECoV positive samples tested negative for equine rotavirus. The nucleotide sequences of the complete N gene, the partial S gene and the region from the p4.7 to p12.7 genes of two positive samples 11V11708/IRL/2011 and 13V08313/IRL/2013 were determined.", "The five ECoV positive samples tested negative for equine rotavirus. The nucleotide sequences of the complete N gene, the partial S gene and the region from the p4.7 to p12.7 genes of two positive samples 11V11708/IRL/2011 and 13V08313/IRL/2013 were determined. The nucleotide identities of the N and S genes of the two Irish ECoVs were 99.8% 1338/1341 nucleotides and 99.5% 650/653 nucleotides , respectively. The nucleotide identities of the N gene of the two Irish ECoVs and the ECoVs from other continents are summarised in Table 2 . Phylogenetic analysis was performed for the nucleotide sequences of the complete N and partial S genes Figure 1 . The analysis for the N gene showed that Irish ECoVs were independently clustered although they were closely related to Japanese viruses identified after 2009.", "Phylogenetic analysis was performed for the nucleotide sequences of the complete N and partial S genes Figure 1 . The analysis for the N gene showed that Irish ECoVs were independently clustered although they were closely related to Japanese viruses identified after 2009. In the phylogenetic tree of the S gene, Irish ECoVs were closely related to all other ECoVs analysed. The length of the region from the p4.7 to p12.7 genes in the two viruses was 544 base pairs. Compared with NC99, Irish ECoVs, had a total of 37 nucleotide deletions within p4.7 and the non-coding region following the p4.7 gene. Compared with Obihiro 12-1 and 12-2, Irish ECoVs had a three-nucleotide insertion.", "Compared with NC99, Irish ECoVs, had a total of 37 nucleotide deletions within p4.7 and the non-coding region following the p4.7 gene. Compared with Obihiro 12-1 and 12-2, Irish ECoVs had a three-nucleotide insertion. When compared with Tokachi09, the Irish ECoVs had a 148-nucleotide insertion see Figure S1 . The p12.7 gene of the two Irish ECoVs did not have deletions or insertions, and the nucleotide identities were 98.8-99.7% between these viruses and the other ECoVs NC99, Tokachi09, Obihiro12-1 and Obihiro12-2 . This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease. However, detection of ECoV in faeces samples from horses with enteric disease does not prove This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease.", "This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease. However, detection of ECoV in faeces samples from horses with enteric disease does not prove This study provides the first report of ECoV circulating in Ireland, the third European country with a significant horse industry where the virus has been detected in horses with enteric disease. However, detection of ECoV in faeces samples from horses with enteric disease does not prove causation. In this study, 424 samples collected between 2011 and 2015 from equids with enteric disease were tested, and only five samples 1.2% were positive for ECoV. The inclusion of an internal positive control in the rRT-PCR eliminated the possibility of false negative results due to the presence of PCR inhibitors but the high content of nucleases associated with faeces samples may have caused some RNA degradation. However, this low prevalence of ECoV is similar to that identified in France and among Thoroughbred foals in Japan .", "The inclusion of an internal positive control in the rRT-PCR eliminated the possibility of false negative results due to the presence of PCR inhibitors but the high content of nucleases associated with faeces samples may have caused some RNA degradation. However, this low prevalence of ECoV is similar to that identified in France and among Thoroughbred foals in Japan . Although ECoV has been identified on three continents, little is known about the genetic and pathogenic diversity in field viruses. In this study, sequence and phylogenetic analysis Figure 1 demonstrated a high level of homology between viruses detected in a donkey and a horse in two provinces in Ireland in different years. This suggests that Irish ECoVs may have low genetic diversity. Compared with the ECoVs of other countries, the N, S and p12.7 genes of the two Irish viruses were highly conserved.", "This suggests that Irish ECoVs may have low genetic diversity. Compared with the ECoVs of other countries, the N, S and p12.7 genes of the two Irish viruses were highly conserved. In contrast, the region from p4.7 and the non-coding region following the p4.7 gene had deletions or insertions Figure S1 . Because of polymorphism in this region, this region could be useful for epidemiological investigation . The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the viruses in Ireland may be distinguishable from those circulating in other countries. The positive samples were collected in November ., March . and April . in this study.", "The positive samples were collected in November ., March . and April . in this study. Higher case numbers are identified in the USA during the colder months October to April , and our results were consistent with the circulation period in USA. It has been reported that outbreaks mainly occurred among adult riding, racing and show horses in USA . The choice of cases to include in the current study may not have been optimal for detection of ECoV as the majority of samples were from foals. However, two positive samples were collected from adult horses in a combined riding school/show jumping yard in the West of Ireland.", "The choice of cases to include in the current study may not have been optimal for detection of ECoV as the majority of samples were from foals. However, two positive samples were collected from adult horses in a combined riding school/show jumping yard in the West of Ireland. At the time of sample collection in April 2013, the monthly mean temperatures were below long-term average and in parts of the West, were the coldest in 24 years . Cold weather may have been a predisposing factor to the ECoV infection on the farm. Two positive samples were collected from Thoroughbred foals. A faeces sample collected from one foal with severe watery diarrhoea and inappetance was positive for ECoV but a sample collected three days later tested negative.", "Two positive samples were collected from Thoroughbred foals. A faeces sample collected from one foal with severe watery diarrhoea and inappetance was positive for ECoV but a sample collected three days later tested negative. A potential difficulty in detecting ECoV from naturally infected horses has been noted previously as serial samples from seven sick horses in the USA suggested that ECoV only persisted for three to nine days in faeces . In both cases, the diarrhoea may have been caused by other unidentified coinfecting pathogens as has been suggested by investigators in the USA . This is the first report of ECoV detection in faeces samples from both foals and adult horses in Ireland. The viruses identified in Ireland are genetically closely related to the Japanese viruses and the results of this study give no indication of significant genetic or phenotypic diversity.", "This is the first report of ECoV detection in faeces samples from both foals and adult horses in Ireland. The viruses identified in Ireland are genetically closely related to the Japanese viruses and the results of this study give no indication of significant genetic or phenotypic diversity. In recent years, there has been an increase in awareness and testing for ECoV in the USA and elsewhere . Horse breeding and racing activities in Ireland are the most prominent and important of any country on a per capita basis. There are over 50 Thoroughbred horses per 10,000 of population in Ireland, compared to between three and five for Great Britain, France and the USA . Thus, an investigation of ECoV in Ireland is pertinent not only to increase awareness nationally of the epidemiology of the virus and promote discussion on its clinical importance, but also to inform the industry globally of the health status of Irish horses.", "There are over 50 Thoroughbred horses per 10,000 of population in Ireland, compared to between three and five for Great Britain, France and the USA . Thus, an investigation of ECoV in Ireland is pertinent not only to increase awareness nationally of the epidemiology of the virus and promote discussion on its clinical importance, but also to inform the industry globally of the health status of Irish horses. Ireland exports horses all over the world. By illustration, in 2016 the country was the second biggest seller of bloodstock at public auctions second only to the USA . Many questions remain with regard to the clinical significance of ECoV. The outbreak at a draft-horse racetrack in Japan in 2009 affected 132 of approximately 600 horses and resulted in non-starters and the implementation of movement restrictions .", "Many questions remain with regard to the clinical significance of ECoV. The outbreak at a draft-horse racetrack in Japan in 2009 affected 132 of approximately 600 horses and resulted in non-starters and the implementation of movement restrictions . However, draft horses appear to have a higher infection rate than other breeds and an outbreak of similar severity has not been reported in Thoroughbred racehorses . The much higher incidence of ECoV positive Thoroughbred foals identified in Kentucky compared to similar populations internationally suggests an increased susceptibility to ECoV infection in that population. In the past, specific environmental factors were associated with extensive reproductive loss in the Kentucky area and to a lesser extent in other states , but predisposing regional factors such as differences in management, environment or husbandry have not been identified for ECoV. It has been suggested that ECoV is a coinfecting agent in foals with diarrhoea and clinical infections have predominantly been reported in adult horses with a mono-infection with EcoV .", "In the past, specific environmental factors were associated with extensive reproductive loss in the Kentucky area and to a lesser extent in other states , but predisposing regional factors such as differences in management, environment or husbandry have not been identified for ECoV. It has been suggested that ECoV is a coinfecting agent in foals with diarrhoea and clinical infections have predominantly been reported in adult horses with a mono-infection with EcoV . There was no indication from the results of this study that coronavirus is a major cause of diarrhoea in Irish horses but the introduction of rRT-PCR as a routine diagnostic test will assist in elucidating the significance of this virus to the Irish breeding, racing and sports industries. The primary focus in future will be on testing adult horses that present with anorexia, lethargy, fever and changes in faecal character as a significant association has been demonstrated between this clinical status and molecular detection of ECoV in faeces ." ]

[ "BACKGROUND: Pertussis is estimated to cause 2 percent of childhood deaths globally and is a growing public health problem in developed countries despite high vaccination coverage. Infants are at greatest risk of morbidity and mortality. Maternal vaccination during pregnancy may be effective to prevent pertussis in young infants, but population-based estimates of disease burden in infants are lacking, particularly in low-income countries. The objective of this study was to estimate the incidence of pertussis in infants less than 6 months of age in Sarlahi District, Nepal. METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay.", "METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay. The prospective cohort study includes infants observed between May 2011 and August 2014. RESULTS: The incidence of PCR-confirmed Bordetella pertussis was 13.3 cases per 1000 infant-years 95% confidence interval, 7.7–21.3 in a cohort of 3483 infants with at least 1 day of follow-up. CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants.", "CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants. Text: A resurgence of pertussis across age groups has occurred in several countries in recent years . Middle-and high-income countries that use an acellular pertussis vaccine for the primary vaccination series have been particularly affected , and infants and adolescents have experienced the greatest increase . Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves .", "Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves . Recent data from maternal vaccination trials demonstrate the ability of antibodies to be transferred from mothers to their infants in pregnancy and their persistence in infants . Global estimates of pertussis show the highest childhood burden in Southeast Asia . In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking .", "In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking . The World Health Organization WHO recently recommended that countries using whole cell pertussis vaccines continue to do so in light of recent data indicating that acellular pertussis vaccines are less effective than whole cell pertussis vaccines . Population-based data are needed, especially in low-income settings, to provide a more accurate estimate of the burden of pertussis in infants to inform childhood and maternal immunization policies . We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal.", "We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal. The study was nested within 2 consecutive randomized controlled trials of maternal influenza vaccination during pregnancy set in the Sarlahi District, located in the central Terai low-lying plains region of Nepal . At the start of the trial, prevalent pregnancies were identified through a census of all households in the catchment area. For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled.", "For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled. From April 25, 2011 through September 9, 2013, women between 17 and 34 weeks gestation were randomized and vaccinated with either an influenza vaccine or placebo. The study was a population-based prospective cohort of infants followed from birth through 6 months postpartum. Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 .", "Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 . At baseline, information was collected on household structure, socioeconomic status, and demographics. At enrollment, date of last menstrual period and pregnancy history data were collected. As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days.", "As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days. If an infant had any of the following symptoms, a mid-nasal nylon flocked swab was collected: fever, cough, wheeze, difficulty breathing, or ear infection. Starting on August 17, 2012, new symptoms, more specific for pertussis, were added to the weekly morbidity visit: apnea, cyanosis, cough with vomit, or whoop/whooping cough. The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination .", "The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination . Mid-nasal swabs were also collected on a weekly basis from mothers from enrollment through 6 months postpartum who reported fever plus one additional morbidity cough, sore throat, nasal congestion, or myalgia . All nasal swabs collected from infants were tested for B pertussis, Bordetella parapertussis, and Bordetella bronchispetica. Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods .", "Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods . Two-target PCR was used to assess the presence of 3 Bordetella species: B pertussis, B parapertussis, and B bronchiseptica. The amplified targets were chromosomal repeated insertion sequence IS481 IS and the polymorphic pertussis toxin ptxA promoter region PT . After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42.", "After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42. A sample was negative if none of the targets tested positive or a single positive target was not reproducible. Maternal nasal swabs were tested for those mothers whose infants tested positive for any Bordetella species Polymerase chain reaction was also performed for several viral infections influenza, rhinovirus RV , respiratory syncytial virus RSV , bocavirus BoV , human metapneumovirus, coronavirus, adenovirus, and parainfluenza as previously described . Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 .", "Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 . The final dataset consists of 3483 infants, contributing 1280 infant-years of observation, with at least 1 follow-up visit during the first 6 months. This includes infants from the entire trial period, both before and after more pertussis-specific additions to the weekly symptom questionnaire. At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history.", "At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history. The field workers identified their ethnicity into 2 broad groups Pahadi, a group originating from the hills; or Madeshi, a group originating from north India from names and observation. Women were categorized as nulliparous or multiparous. Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household.", "Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household. Gestational age was measured using a woman's report of date of last menstrual period during pregnancy surveillance. Birth weight was collected as soon as possible after birth using a digital scale Tanita model BD-585, precision to nearest 10 grams . Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards .", "Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards . Women were asked within how many hours of birth breastfeeding was initiated and binary breastfeeding categories were created ≤1 hour versus >1 hour postdelivery . Incidence was calculated as the number of pertussis cases per 1000 infant-years at risk. Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression.", "Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression. Characteristics of all pertussis respiratory episodes were compared with nonpertussis respiratory episodes; t tests were used for continuous predictors and Fisher's exact tests were used for categorical associations due to the low number of pertussis episodes. All statistical analyses were conducted in Stata/SE 14.1. A total of 3483 infants had 4283 episodes of respiratory illness between May 18, 2011 and April 30, 2014. Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 .", "Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 . Mean episode duration was 4.7 days 95% CI, 4.6-4.9 . A total of 3930 92% episodes were matched to 1 or more pertussis-tested nasal swabs from 2026 infants Supplementary Figure 1 . Seventeen cases of B pertussis were identified from 19 nasal swabs nasal swabs were positive on 2 consecutive weeks for 2 infants . The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 .", "The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 . No cases of B bronchiseptica were identified. The average pertussis episode duration was 8 days range, 2-33 Table 1 . Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit.", "Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit. None of the additional symptoms related to pertussis that were added in year 2 cyanosis, apnea, cough with vomit, and whoop resulted in collection of nasal swabs based solely on these additional symptoms. Pertussis episodes were statistically significantly more likely to include difficulty breathing, cough with vomit, and whoop compared with other respiratory illness. Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days.", "Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days. Three fourths of pertussis episodes were coinfected with at least 1 virus, with RV and BoV the most common. Cases of pertussis were more likely to be infected with BoV than respiratory cases due to causes other than pertussis. The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented.", "The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented. Given the low number of pertussis cases, the lack of a statistical association is not evidence of nonassociation. No deaths occurred in infants who had pertussis. Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 .", "Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 . No mothers of infants who had B parapertussis had a nasal swab collected during follow-up. The average B parapertussis episode duration was 4 days Supplementary Table 2 . Mean age of onset of symptoms was 58 days with a range of 7-95 days. The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed.", "The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed. All B parapertussis cases occurred between September 2011 and February 2012 Figure 1 . A low incidence of pertussis and generally mild clinical presentation were found in infants <6 months in Nepal. To our knowledge, this represents one of the first population-based active surveillance of PCR-confirmed pertussis among young infants in Asia. Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range.", "Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range. In the United States in 2014, the estimated pertussis incidence in infants less than 6 months was 2 cases per 1000 infant-years , much lower than observed in our study; however, this passive surveillance system likely vastly underestimates pertussis incidence. Thus, there is a need for active surveillance data such as ours. Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further.", "Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further. The low incidence was found in a population where pentavalent vaccine Pentavac: Diphtheria, Tetanus, Pertussis Whole Cell , Hepatitis-B and Haemophilus Type b Conjugate Vaccine; Serum Institute of India Pvt. Ltd , scheduled for administration at 6, 10, and 14 weeks, is received with significant delays 7% of infants received all 3 recommended pertussis vaccines by 6 months . These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine .", "These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine . Another contributing factor to the low pertussis incidence observed could be that surveillance was conducted during a period of low pertussis transmission. Pertussis is a cyclical disease, thought to peak every 2 to 4 years, and we may have captured the burden at a low circulation period . We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden.", "We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden. Previous research on pertussis seasonality has in different places and time periods demonstrated various periods of peak transmission or no discernable patterns . Although our data do not support a seasonal pattern, the numbers observed are too low to be conclusive. Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting .", "Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting . Studies on pertussis in infants have generally been clinic-based, hospital-based, or in an outbreak, which therefore required a certain severity of illness for parents to recognize a need for medical attention . These study designs and passive surveillance efforts therefore may have missed milder pertussis cases . Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal.", "Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal. Although cough, difficulty breathing, and cough with vomit were the most common symptoms, no symptom was present in all B pertussis cases. During an epidemic period in Washington state, among infants <1 year, who had a minimum of 14 days cough plus an additional symptom, 82% had posttussive emesis, 29% had apnea, 26% had whoop, and 42% had cyanosis . A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever.", "A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever. Fever prevalence was higher in our study, similar to that found in Peru . Although not statistically significant, infants with pertussis were more likely to have been born preterm, low birth weight, and SGA, and their mothers were more likely to be primiparous. These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies .", "These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies . Codetection of B pertussis and B parapertussis with respiratory viruses may be due to asymptomatic pertussis carriage. The incidence of B parapertussis of 4 cases per 1000 person-years was comparable to that of 2 per 1000 person-years found in the Italian acellular pertussis vaccine trial in 1992-1993 . The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection .", "The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection . Bordetella parapertussis cases occurred only during a 5-month period. There were several study design limitations. We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected.", "We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected. Infant respiratory symptoms were reported by parents, who may have missed signs that might have been observed by a healthcare worker. However, the criteria for collection of the nasal swab were broad and did not require sophisticated clinical skills. However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms.", "However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms. These data support our assumption that we were unlikely to have missed pertussis cases in year 1 with our less sensitive respiratory symptom criteria. Nasal swabs were collected in the mid-nasal region for influenza virus detection, which may have lowered the sensitivity of pertussis detection. In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population.", "In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population. Although nasopharyngeal swabs or nasopharyngeal aspirates are the recommended specimen collection method , the nasopharyngeal region was established as the collection area of choice when the diagnostic measure was culture, which has low sensitivity. Recent data demonstrated the comparability of using mid-nasal versus nasopharyngeal swabs in PCR pertussis detection . Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings.", "Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings. The community-based design allows generalizability of these results to the entire population and not just those seeking care at a health facility or in an outbreak situation. The Sarlahi District is located in the Terai region where the majority of Nepalese reside, and it has similar demographics to the entire population of Nepal . Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance.", "Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance. The multitarget PCR method allowed highly sensitive and specific detection of 2 additional Bordetella species beyond the primary B pertussis target. We observed a low incidence of pertussis in infants in a whole cell vaccine environment. Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms.", "Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms. Policymakers in Nepal will need to weigh the benefit of an additional prenatal pertussis vaccine or a switch to acellular primary pertussis vaccine with the low burden of pertussis in infants less than 6 months. Our study demonstrated that mid-nasal swabs were able to detect pertussis using a sensitive multitarget PCR. The less invasive mid-nasal nasal swab is an attractive alternative for pertussis nasal swab collection, and further research is needed to compare this collection site with nasopharyngeal swabs. In the future, this method may enhance population-based surveillance efforts." ]

[ "BACKGROUND: Pertussis is estimated to cause 2 percent of childhood deaths globally and is a growing public health problem in developed countries despite high vaccination coverage. Infants are at greatest risk of morbidity and mortality. Maternal vaccination during pregnancy may be effective to prevent pertussis in young infants, but population-based estimates of disease burden in infants are lacking, particularly in low-income countries. The objective of this study was to estimate the incidence of pertussis in infants less than 6 months of age in Sarlahi District, Nepal. METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay.", "METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay. The prospective cohort study includes infants observed between May 2011 and August 2014. RESULTS: The incidence of PCR-confirmed Bordetella pertussis was 13.3 cases per 1000 infant-years 95% confidence interval, 7.7–21.3 in a cohort of 3483 infants with at least 1 day of follow-up. CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants.", "CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants. Text: A resurgence of pertussis across age groups has occurred in several countries in recent years . Middle-and high-income countries that use an acellular pertussis vaccine for the primary vaccination series have been particularly affected , and infants and adolescents have experienced the greatest increase . Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves .", "Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves . Recent data from maternal vaccination trials demonstrate the ability of antibodies to be transferred from mothers to their infants in pregnancy and their persistence in infants . Global estimates of pertussis show the highest childhood burden in Southeast Asia . In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking .", "In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking . The World Health Organization WHO recently recommended that countries using whole cell pertussis vaccines continue to do so in light of recent data indicating that acellular pertussis vaccines are less effective than whole cell pertussis vaccines . Population-based data are needed, especially in low-income settings, to provide a more accurate estimate of the burden of pertussis in infants to inform childhood and maternal immunization policies . We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal.", "We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal. The study was nested within 2 consecutive randomized controlled trials of maternal influenza vaccination during pregnancy set in the Sarlahi District, located in the central Terai low-lying plains region of Nepal . At the start of the trial, prevalent pregnancies were identified through a census of all households in the catchment area. For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled.", "For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled. From April 25, 2011 through September 9, 2013, women between 17 and 34 weeks gestation were randomized and vaccinated with either an influenza vaccine or placebo. The study was a population-based prospective cohort of infants followed from birth through 6 months postpartum. Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 .", "Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 . At baseline, information was collected on household structure, socioeconomic status, and demographics. At enrollment, date of last menstrual period and pregnancy history data were collected. As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days.", "As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days. If an infant had any of the following symptoms, a mid-nasal nylon flocked swab was collected: fever, cough, wheeze, difficulty breathing, or ear infection. Starting on August 17, 2012, new symptoms, more specific for pertussis, were added to the weekly morbidity visit: apnea, cyanosis, cough with vomit, or whoop/whooping cough. The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination .", "The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination . Mid-nasal swabs were also collected on a weekly basis from mothers from enrollment through 6 months postpartum who reported fever plus one additional morbidity cough, sore throat, nasal congestion, or myalgia . All nasal swabs collected from infants were tested for B pertussis, Bordetella parapertussis, and Bordetella bronchispetica. Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods .", "Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods . Two-target PCR was used to assess the presence of 3 Bordetella species: B pertussis, B parapertussis, and B bronchiseptica. The amplified targets were chromosomal repeated insertion sequence IS481 IS and the polymorphic pertussis toxin ptxA promoter region PT . After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42.", "After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42. A sample was negative if none of the targets tested positive or a single positive target was not reproducible. Maternal nasal swabs were tested for those mothers whose infants tested positive for any Bordetella species Polymerase chain reaction was also performed for several viral infections influenza, rhinovirus RV , respiratory syncytial virus RSV , bocavirus BoV , human metapneumovirus, coronavirus, adenovirus, and parainfluenza as previously described . Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 .", "Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 . The final dataset consists of 3483 infants, contributing 1280 infant-years of observation, with at least 1 follow-up visit during the first 6 months. This includes infants from the entire trial period, both before and after more pertussis-specific additions to the weekly symptom questionnaire. At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history.", "At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history. The field workers identified their ethnicity into 2 broad groups Pahadi, a group originating from the hills; or Madeshi, a group originating from north India from names and observation. Women were categorized as nulliparous or multiparous. Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household.", "Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household. Gestational age was measured using a woman's report of date of last menstrual period during pregnancy surveillance. Birth weight was collected as soon as possible after birth using a digital scale Tanita model BD-585, precision to nearest 10 grams . Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards .", "Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards . Women were asked within how many hours of birth breastfeeding was initiated and binary breastfeeding categories were created ≤1 hour versus >1 hour postdelivery . Incidence was calculated as the number of pertussis cases per 1000 infant-years at risk. Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression.", "Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression. Characteristics of all pertussis respiratory episodes were compared with nonpertussis respiratory episodes; t tests were used for continuous predictors and Fisher's exact tests were used for categorical associations due to the low number of pertussis episodes. All statistical analyses were conducted in Stata/SE 14.1. A total of 3483 infants had 4283 episodes of respiratory illness between May 18, 2011 and April 30, 2014. Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 .", "Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 . Mean episode duration was 4.7 days 95% CI, 4.6-4.9 . A total of 3930 92% episodes were matched to 1 or more pertussis-tested nasal swabs from 2026 infants Supplementary Figure 1 . Seventeen cases of B pertussis were identified from 19 nasal swabs nasal swabs were positive on 2 consecutive weeks for 2 infants . The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 .", "The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 . No cases of B bronchiseptica were identified. The average pertussis episode duration was 8 days range, 2-33 Table 1 . Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit.", "Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit. None of the additional symptoms related to pertussis that were added in year 2 cyanosis, apnea, cough with vomit, and whoop resulted in collection of nasal swabs based solely on these additional symptoms. Pertussis episodes were statistically significantly more likely to include difficulty breathing, cough with vomit, and whoop compared with other respiratory illness. Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days.", "Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days. Three fourths of pertussis episodes were coinfected with at least 1 virus, with RV and BoV the most common. Cases of pertussis were more likely to be infected with BoV than respiratory cases due to causes other than pertussis. The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented.", "The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented. Given the low number of pertussis cases, the lack of a statistical association is not evidence of nonassociation. No deaths occurred in infants who had pertussis. Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 .", "Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 . No mothers of infants who had B parapertussis had a nasal swab collected during follow-up. The average B parapertussis episode duration was 4 days Supplementary Table 2 . Mean age of onset of symptoms was 58 days with a range of 7-95 days. The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed.", "The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed. All B parapertussis cases occurred between September 2011 and February 2012 Figure 1 . A low incidence of pertussis and generally mild clinical presentation were found in infants <6 months in Nepal. To our knowledge, this represents one of the first population-based active surveillance of PCR-confirmed pertussis among young infants in Asia. Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range.", "Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range. In the United States in 2014, the estimated pertussis incidence in infants less than 6 months was 2 cases per 1000 infant-years , much lower than observed in our study; however, this passive surveillance system likely vastly underestimates pertussis incidence. Thus, there is a need for active surveillance data such as ours. Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further.", "Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further. The low incidence was found in a population where pentavalent vaccine Pentavac: Diphtheria, Tetanus, Pertussis Whole Cell , Hepatitis-B and Haemophilus Type b Conjugate Vaccine; Serum Institute of India Pvt. Ltd , scheduled for administration at 6, 10, and 14 weeks, is received with significant delays 7% of infants received all 3 recommended pertussis vaccines by 6 months . These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine .", "These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine . Another contributing factor to the low pertussis incidence observed could be that surveillance was conducted during a period of low pertussis transmission. Pertussis is a cyclical disease, thought to peak every 2 to 4 years, and we may have captured the burden at a low circulation period . We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden.", "We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden. Previous research on pertussis seasonality has in different places and time periods demonstrated various periods of peak transmission or no discernable patterns . Although our data do not support a seasonal pattern, the numbers observed are too low to be conclusive. Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting .", "Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting . Studies on pertussis in infants have generally been clinic-based, hospital-based, or in an outbreak, which therefore required a certain severity of illness for parents to recognize a need for medical attention . These study designs and passive surveillance efforts therefore may have missed milder pertussis cases . Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal.", "Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal. Although cough, difficulty breathing, and cough with vomit were the most common symptoms, no symptom was present in all B pertussis cases. During an epidemic period in Washington state, among infants <1 year, who had a minimum of 14 days cough plus an additional symptom, 82% had posttussive emesis, 29% had apnea, 26% had whoop, and 42% had cyanosis . A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever.", "A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever. Fever prevalence was higher in our study, similar to that found in Peru . Although not statistically significant, infants with pertussis were more likely to have been born preterm, low birth weight, and SGA, and their mothers were more likely to be primiparous. These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies .", "These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies . Codetection of B pertussis and B parapertussis with respiratory viruses may be due to asymptomatic pertussis carriage. The incidence of B parapertussis of 4 cases per 1000 person-years was comparable to that of 2 per 1000 person-years found in the Italian acellular pertussis vaccine trial in 1992-1993 . The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection .", "The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection . Bordetella parapertussis cases occurred only during a 5-month period. There were several study design limitations. We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected.", "We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected. Infant respiratory symptoms were reported by parents, who may have missed signs that might have been observed by a healthcare worker. However, the criteria for collection of the nasal swab were broad and did not require sophisticated clinical skills. However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms.", "However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms. These data support our assumption that we were unlikely to have missed pertussis cases in year 1 with our less sensitive respiratory symptom criteria. Nasal swabs were collected in the mid-nasal region for influenza virus detection, which may have lowered the sensitivity of pertussis detection. In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population.", "In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population. Although nasopharyngeal swabs or nasopharyngeal aspirates are the recommended specimen collection method , the nasopharyngeal region was established as the collection area of choice when the diagnostic measure was culture, which has low sensitivity. Recent data demonstrated the comparability of using mid-nasal versus nasopharyngeal swabs in PCR pertussis detection . Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings.", "Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings. The community-based design allows generalizability of these results to the entire population and not just those seeking care at a health facility or in an outbreak situation. The Sarlahi District is located in the Terai region where the majority of Nepalese reside, and it has similar demographics to the entire population of Nepal . Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance.", "Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance. The multitarget PCR method allowed highly sensitive and specific detection of 2 additional Bordetella species beyond the primary B pertussis target. We observed a low incidence of pertussis in infants in a whole cell vaccine environment. Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms.", "Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms. Policymakers in Nepal will need to weigh the benefit of an additional prenatal pertussis vaccine or a switch to acellular primary pertussis vaccine with the low burden of pertussis in infants less than 6 months. Our study demonstrated that mid-nasal swabs were able to detect pertussis using a sensitive multitarget PCR. The less invasive mid-nasal nasal swab is an attractive alternative for pertussis nasal swab collection, and further research is needed to compare this collection site with nasopharyngeal swabs. In the future, this method may enhance population-based surveillance efforts." ]

[ "BACKGROUND: Pertussis is estimated to cause 2 percent of childhood deaths globally and is a growing public health problem in developed countries despite high vaccination coverage. Infants are at greatest risk of morbidity and mortality. Maternal vaccination during pregnancy may be effective to prevent pertussis in young infants, but population-based estimates of disease burden in infants are lacking, particularly in low-income countries. The objective of this study was to estimate the incidence of pertussis in infants less than 6 months of age in Sarlahi District, Nepal. METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay.", "METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay. The prospective cohort study includes infants observed between May 2011 and August 2014. RESULTS: The incidence of PCR-confirmed Bordetella pertussis was 13.3 cases per 1000 infant-years 95% confidence interval, 7.7–21.3 in a cohort of 3483 infants with at least 1 day of follow-up. CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants.", "CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants. Text: A resurgence of pertussis across age groups has occurred in several countries in recent years . Middle-and high-income countries that use an acellular pertussis vaccine for the primary vaccination series have been particularly affected , and infants and adolescents have experienced the greatest increase . Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves .", "Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves . Recent data from maternal vaccination trials demonstrate the ability of antibodies to be transferred from mothers to their infants in pregnancy and their persistence in infants . Global estimates of pertussis show the highest childhood burden in Southeast Asia . In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking .", "In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking . The World Health Organization WHO recently recommended that countries using whole cell pertussis vaccines continue to do so in light of recent data indicating that acellular pertussis vaccines are less effective than whole cell pertussis vaccines . Population-based data are needed, especially in low-income settings, to provide a more accurate estimate of the burden of pertussis in infants to inform childhood and maternal immunization policies . We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal.", "We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal. The study was nested within 2 consecutive randomized controlled trials of maternal influenza vaccination during pregnancy set in the Sarlahi District, located in the central Terai low-lying plains region of Nepal . At the start of the trial, prevalent pregnancies were identified through a census of all households in the catchment area. For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled.", "For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled. From April 25, 2011 through September 9, 2013, women between 17 and 34 weeks gestation were randomized and vaccinated with either an influenza vaccine or placebo. The study was a population-based prospective cohort of infants followed from birth through 6 months postpartum. Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 .", "Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 . At baseline, information was collected on household structure, socioeconomic status, and demographics. At enrollment, date of last menstrual period and pregnancy history data were collected. As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days.", "As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days. If an infant had any of the following symptoms, a mid-nasal nylon flocked swab was collected: fever, cough, wheeze, difficulty breathing, or ear infection. Starting on August 17, 2012, new symptoms, more specific for pertussis, were added to the weekly morbidity visit: apnea, cyanosis, cough with vomit, or whoop/whooping cough. The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination .", "The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination . Mid-nasal swabs were also collected on a weekly basis from mothers from enrollment through 6 months postpartum who reported fever plus one additional morbidity cough, sore throat, nasal congestion, or myalgia . All nasal swabs collected from infants were tested for B pertussis, Bordetella parapertussis, and Bordetella bronchispetica. Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods .", "Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods . Two-target PCR was used to assess the presence of 3 Bordetella species: B pertussis, B parapertussis, and B bronchiseptica. The amplified targets were chromosomal repeated insertion sequence IS481 IS and the polymorphic pertussis toxin ptxA promoter region PT . After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42.", "After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42. A sample was negative if none of the targets tested positive or a single positive target was not reproducible. Maternal nasal swabs were tested for those mothers whose infants tested positive for any Bordetella species Polymerase chain reaction was also performed for several viral infections influenza, rhinovirus RV , respiratory syncytial virus RSV , bocavirus BoV , human metapneumovirus, coronavirus, adenovirus, and parainfluenza as previously described . Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 .", "Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 . The final dataset consists of 3483 infants, contributing 1280 infant-years of observation, with at least 1 follow-up visit during the first 6 months. This includes infants from the entire trial period, both before and after more pertussis-specific additions to the weekly symptom questionnaire. At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history.", "At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history. The field workers identified their ethnicity into 2 broad groups Pahadi, a group originating from the hills; or Madeshi, a group originating from north India from names and observation. Women were categorized as nulliparous or multiparous. Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household.", "Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household. Gestational age was measured using a woman's report of date of last menstrual period during pregnancy surveillance. Birth weight was collected as soon as possible after birth using a digital scale Tanita model BD-585, precision to nearest 10 grams . Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards .", "Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards . Women were asked within how many hours of birth breastfeeding was initiated and binary breastfeeding categories were created ≤1 hour versus >1 hour postdelivery . Incidence was calculated as the number of pertussis cases per 1000 infant-years at risk. Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression.", "Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression. Characteristics of all pertussis respiratory episodes were compared with nonpertussis respiratory episodes; t tests were used for continuous predictors and Fisher's exact tests were used for categorical associations due to the low number of pertussis episodes. All statistical analyses were conducted in Stata/SE 14.1. A total of 3483 infants had 4283 episodes of respiratory illness between May 18, 2011 and April 30, 2014. Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 .", "Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 . Mean episode duration was 4.7 days 95% CI, 4.6-4.9 . A total of 3930 92% episodes were matched to 1 or more pertussis-tested nasal swabs from 2026 infants Supplementary Figure 1 . Seventeen cases of B pertussis were identified from 19 nasal swabs nasal swabs were positive on 2 consecutive weeks for 2 infants . The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 .", "The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 . No cases of B bronchiseptica were identified. The average pertussis episode duration was 8 days range, 2-33 Table 1 . Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit.", "Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit. None of the additional symptoms related to pertussis that were added in year 2 cyanosis, apnea, cough with vomit, and whoop resulted in collection of nasal swabs based solely on these additional symptoms. Pertussis episodes were statistically significantly more likely to include difficulty breathing, cough with vomit, and whoop compared with other respiratory illness. Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days.", "Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days. Three fourths of pertussis episodes were coinfected with at least 1 virus, with RV and BoV the most common. Cases of pertussis were more likely to be infected with BoV than respiratory cases due to causes other than pertussis. The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented.", "The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented. Given the low number of pertussis cases, the lack of a statistical association is not evidence of nonassociation. No deaths occurred in infants who had pertussis. Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 .", "Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 . No mothers of infants who had B parapertussis had a nasal swab collected during follow-up. The average B parapertussis episode duration was 4 days Supplementary Table 2 . Mean age of onset of symptoms was 58 days with a range of 7-95 days. The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed.", "The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed. All B parapertussis cases occurred between September 2011 and February 2012 Figure 1 . A low incidence of pertussis and generally mild clinical presentation were found in infants <6 months in Nepal. To our knowledge, this represents one of the first population-based active surveillance of PCR-confirmed pertussis among young infants in Asia. Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range.", "Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range. In the United States in 2014, the estimated pertussis incidence in infants less than 6 months was 2 cases per 1000 infant-years , much lower than observed in our study; however, this passive surveillance system likely vastly underestimates pertussis incidence. Thus, there is a need for active surveillance data such as ours. Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further.", "Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further. The low incidence was found in a population where pentavalent vaccine Pentavac: Diphtheria, Tetanus, Pertussis Whole Cell , Hepatitis-B and Haemophilus Type b Conjugate Vaccine; Serum Institute of India Pvt. Ltd , scheduled for administration at 6, 10, and 14 weeks, is received with significant delays 7% of infants received all 3 recommended pertussis vaccines by 6 months . These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine .", "These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine . Another contributing factor to the low pertussis incidence observed could be that surveillance was conducted during a period of low pertussis transmission. Pertussis is a cyclical disease, thought to peak every 2 to 4 years, and we may have captured the burden at a low circulation period . We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden.", "We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden. Previous research on pertussis seasonality has in different places and time periods demonstrated various periods of peak transmission or no discernable patterns . Although our data do not support a seasonal pattern, the numbers observed are too low to be conclusive. Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting .", "Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting . Studies on pertussis in infants have generally been clinic-based, hospital-based, or in an outbreak, which therefore required a certain severity of illness for parents to recognize a need for medical attention . These study designs and passive surveillance efforts therefore may have missed milder pertussis cases . Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal.", "Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal. Although cough, difficulty breathing, and cough with vomit were the most common symptoms, no symptom was present in all B pertussis cases. During an epidemic period in Washington state, among infants <1 year, who had a minimum of 14 days cough plus an additional symptom, 82% had posttussive emesis, 29% had apnea, 26% had whoop, and 42% had cyanosis . A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever.", "A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever. Fever prevalence was higher in our study, similar to that found in Peru . Although not statistically significant, infants with pertussis were more likely to have been born preterm, low birth weight, and SGA, and their mothers were more likely to be primiparous. These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies .", "These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies . Codetection of B pertussis and B parapertussis with respiratory viruses may be due to asymptomatic pertussis carriage. The incidence of B parapertussis of 4 cases per 1000 person-years was comparable to that of 2 per 1000 person-years found in the Italian acellular pertussis vaccine trial in 1992-1993 . The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection .", "The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection . Bordetella parapertussis cases occurred only during a 5-month period. There were several study design limitations. We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected.", "We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected. Infant respiratory symptoms were reported by parents, who may have missed signs that might have been observed by a healthcare worker. However, the criteria for collection of the nasal swab were broad and did not require sophisticated clinical skills. However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms.", "However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms. These data support our assumption that we were unlikely to have missed pertussis cases in year 1 with our less sensitive respiratory symptom criteria. Nasal swabs were collected in the mid-nasal region for influenza virus detection, which may have lowered the sensitivity of pertussis detection. In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population.", "In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population. Although nasopharyngeal swabs or nasopharyngeal aspirates are the recommended specimen collection method , the nasopharyngeal region was established as the collection area of choice when the diagnostic measure was culture, which has low sensitivity. Recent data demonstrated the comparability of using mid-nasal versus nasopharyngeal swabs in PCR pertussis detection . Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings.", "Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings. The community-based design allows generalizability of these results to the entire population and not just those seeking care at a health facility or in an outbreak situation. The Sarlahi District is located in the Terai region where the majority of Nepalese reside, and it has similar demographics to the entire population of Nepal . Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance.", "Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance. The multitarget PCR method allowed highly sensitive and specific detection of 2 additional Bordetella species beyond the primary B pertussis target. We observed a low incidence of pertussis in infants in a whole cell vaccine environment. Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms.", "Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms. Policymakers in Nepal will need to weigh the benefit of an additional prenatal pertussis vaccine or a switch to acellular primary pertussis vaccine with the low burden of pertussis in infants less than 6 months. Our study demonstrated that mid-nasal swabs were able to detect pertussis using a sensitive multitarget PCR. The less invasive mid-nasal nasal swab is an attractive alternative for pertussis nasal swab collection, and further research is needed to compare this collection site with nasopharyngeal swabs. In the future, this method may enhance population-based surveillance efforts." ]

[ "BACKGROUND: Pertussis is estimated to cause 2 percent of childhood deaths globally and is a growing public health problem in developed countries despite high vaccination coverage. Infants are at greatest risk of morbidity and mortality. Maternal vaccination during pregnancy may be effective to prevent pertussis in young infants, but population-based estimates of disease burden in infants are lacking, particularly in low-income countries. The objective of this study was to estimate the incidence of pertussis in infants less than 6 months of age in Sarlahi District, Nepal. METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay.", "METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay. The prospective cohort study includes infants observed between May 2011 and August 2014. RESULTS: The incidence of PCR-confirmed Bordetella pertussis was 13.3 cases per 1000 infant-years 95% confidence interval, 7.7–21.3 in a cohort of 3483 infants with at least 1 day of follow-up. CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants.", "CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants. Text: A resurgence of pertussis across age groups has occurred in several countries in recent years . Middle-and high-income countries that use an acellular pertussis vaccine for the primary vaccination series have been particularly affected , and infants and adolescents have experienced the greatest increase . Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves .", "Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves . Recent data from maternal vaccination trials demonstrate the ability of antibodies to be transferred from mothers to their infants in pregnancy and their persistence in infants . Global estimates of pertussis show the highest childhood burden in Southeast Asia . In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking .", "In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking . The World Health Organization WHO recently recommended that countries using whole cell pertussis vaccines continue to do so in light of recent data indicating that acellular pertussis vaccines are less effective than whole cell pertussis vaccines . Population-based data are needed, especially in low-income settings, to provide a more accurate estimate of the burden of pertussis in infants to inform childhood and maternal immunization policies . We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal.", "We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal. The study was nested within 2 consecutive randomized controlled trials of maternal influenza vaccination during pregnancy set in the Sarlahi District, located in the central Terai low-lying plains region of Nepal . At the start of the trial, prevalent pregnancies were identified through a census of all households in the catchment area. For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled.", "For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled. From April 25, 2011 through September 9, 2013, women between 17 and 34 weeks gestation were randomized and vaccinated with either an influenza vaccine or placebo. The study was a population-based prospective cohort of infants followed from birth through 6 months postpartum. Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 .", "Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 . At baseline, information was collected on household structure, socioeconomic status, and demographics. At enrollment, date of last menstrual period and pregnancy history data were collected. As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days.", "As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days. If an infant had any of the following symptoms, a mid-nasal nylon flocked swab was collected: fever, cough, wheeze, difficulty breathing, or ear infection. Starting on August 17, 2012, new symptoms, more specific for pertussis, were added to the weekly morbidity visit: apnea, cyanosis, cough with vomit, or whoop/whooping cough. The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination .", "The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination . Mid-nasal swabs were also collected on a weekly basis from mothers from enrollment through 6 months postpartum who reported fever plus one additional morbidity cough, sore throat, nasal congestion, or myalgia . All nasal swabs collected from infants were tested for B pertussis, Bordetella parapertussis, and Bordetella bronchispetica. Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods .", "Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods . Two-target PCR was used to assess the presence of 3 Bordetella species: B pertussis, B parapertussis, and B bronchiseptica. The amplified targets were chromosomal repeated insertion sequence IS481 IS and the polymorphic pertussis toxin ptxA promoter region PT . After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42.", "After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42. A sample was negative if none of the targets tested positive or a single positive target was not reproducible. Maternal nasal swabs were tested for those mothers whose infants tested positive for any Bordetella species Polymerase chain reaction was also performed for several viral infections influenza, rhinovirus RV , respiratory syncytial virus RSV , bocavirus BoV , human metapneumovirus, coronavirus, adenovirus, and parainfluenza as previously described . Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 .", "Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 . The final dataset consists of 3483 infants, contributing 1280 infant-years of observation, with at least 1 follow-up visit during the first 6 months. This includes infants from the entire trial period, both before and after more pertussis-specific additions to the weekly symptom questionnaire. At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history.", "At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history. The field workers identified their ethnicity into 2 broad groups Pahadi, a group originating from the hills; or Madeshi, a group originating from north India from names and observation. Women were categorized as nulliparous or multiparous. Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household.", "Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household. Gestational age was measured using a woman's report of date of last menstrual period during pregnancy surveillance. Birth weight was collected as soon as possible after birth using a digital scale Tanita model BD-585, precision to nearest 10 grams . Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards .", "Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards . Women were asked within how many hours of birth breastfeeding was initiated and binary breastfeeding categories were created ≤1 hour versus >1 hour postdelivery . Incidence was calculated as the number of pertussis cases per 1000 infant-years at risk. Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression.", "Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression. Characteristics of all pertussis respiratory episodes were compared with nonpertussis respiratory episodes; t tests were used for continuous predictors and Fisher's exact tests were used for categorical associations due to the low number of pertussis episodes. All statistical analyses were conducted in Stata/SE 14.1. A total of 3483 infants had 4283 episodes of respiratory illness between May 18, 2011 and April 30, 2014. Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 .", "Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 . Mean episode duration was 4.7 days 95% CI, 4.6-4.9 . A total of 3930 92% episodes were matched to 1 or more pertussis-tested nasal swabs from 2026 infants Supplementary Figure 1 . Seventeen cases of B pertussis were identified from 19 nasal swabs nasal swabs were positive on 2 consecutive weeks for 2 infants . The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 .", "The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 . No cases of B bronchiseptica were identified. The average pertussis episode duration was 8 days range, 2-33 Table 1 . Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit.", "Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit. None of the additional symptoms related to pertussis that were added in year 2 cyanosis, apnea, cough with vomit, and whoop resulted in collection of nasal swabs based solely on these additional symptoms. Pertussis episodes were statistically significantly more likely to include difficulty breathing, cough with vomit, and whoop compared with other respiratory illness. Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days.", "Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days. Three fourths of pertussis episodes were coinfected with at least 1 virus, with RV and BoV the most common. Cases of pertussis were more likely to be infected with BoV than respiratory cases due to causes other than pertussis. The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented.", "The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented. Given the low number of pertussis cases, the lack of a statistical association is not evidence of nonassociation. No deaths occurred in infants who had pertussis. Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 .", "Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 . No mothers of infants who had B parapertussis had a nasal swab collected during follow-up. The average B parapertussis episode duration was 4 days Supplementary Table 2 . Mean age of onset of symptoms was 58 days with a range of 7-95 days. The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed.", "The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed. All B parapertussis cases occurred between September 2011 and February 2012 Figure 1 . A low incidence of pertussis and generally mild clinical presentation were found in infants <6 months in Nepal. To our knowledge, this represents one of the first population-based active surveillance of PCR-confirmed pertussis among young infants in Asia. Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range.", "Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range. In the United States in 2014, the estimated pertussis incidence in infants less than 6 months was 2 cases per 1000 infant-years , much lower than observed in our study; however, this passive surveillance system likely vastly underestimates pertussis incidence. Thus, there is a need for active surveillance data such as ours. Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further.", "Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further. The low incidence was found in a population where pentavalent vaccine Pentavac: Diphtheria, Tetanus, Pertussis Whole Cell , Hepatitis-B and Haemophilus Type b Conjugate Vaccine; Serum Institute of India Pvt. Ltd , scheduled for administration at 6, 10, and 14 weeks, is received with significant delays 7% of infants received all 3 recommended pertussis vaccines by 6 months . These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine .", "These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine . Another contributing factor to the low pertussis incidence observed could be that surveillance was conducted during a period of low pertussis transmission. Pertussis is a cyclical disease, thought to peak every 2 to 4 years, and we may have captured the burden at a low circulation period . We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden.", "We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden. Previous research on pertussis seasonality has in different places and time periods demonstrated various periods of peak transmission or no discernable patterns . Although our data do not support a seasonal pattern, the numbers observed are too low to be conclusive. Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting .", "Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting . Studies on pertussis in infants have generally been clinic-based, hospital-based, or in an outbreak, which therefore required a certain severity of illness for parents to recognize a need for medical attention . These study designs and passive surveillance efforts therefore may have missed milder pertussis cases . Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal.", "Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal. Although cough, difficulty breathing, and cough with vomit were the most common symptoms, no symptom was present in all B pertussis cases. During an epidemic period in Washington state, among infants <1 year, who had a minimum of 14 days cough plus an additional symptom, 82% had posttussive emesis, 29% had apnea, 26% had whoop, and 42% had cyanosis . A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever.", "A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever. Fever prevalence was higher in our study, similar to that found in Peru . Although not statistically significant, infants with pertussis were more likely to have been born preterm, low birth weight, and SGA, and their mothers were more likely to be primiparous. These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies .", "These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies . Codetection of B pertussis and B parapertussis with respiratory viruses may be due to asymptomatic pertussis carriage. The incidence of B parapertussis of 4 cases per 1000 person-years was comparable to that of 2 per 1000 person-years found in the Italian acellular pertussis vaccine trial in 1992-1993 . The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection .", "The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection . Bordetella parapertussis cases occurred only during a 5-month period. There were several study design limitations. We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected.", "We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected. Infant respiratory symptoms were reported by parents, who may have missed signs that might have been observed by a healthcare worker. However, the criteria for collection of the nasal swab were broad and did not require sophisticated clinical skills. However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms.", "However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms. These data support our assumption that we were unlikely to have missed pertussis cases in year 1 with our less sensitive respiratory symptom criteria. Nasal swabs were collected in the mid-nasal region for influenza virus detection, which may have lowered the sensitivity of pertussis detection. In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population.", "In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population. Although nasopharyngeal swabs or nasopharyngeal aspirates are the recommended specimen collection method , the nasopharyngeal region was established as the collection area of choice when the diagnostic measure was culture, which has low sensitivity. Recent data demonstrated the comparability of using mid-nasal versus nasopharyngeal swabs in PCR pertussis detection . Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings.", "Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings. The community-based design allows generalizability of these results to the entire population and not just those seeking care at a health facility or in an outbreak situation. The Sarlahi District is located in the Terai region where the majority of Nepalese reside, and it has similar demographics to the entire population of Nepal . Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance.", "Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance. The multitarget PCR method allowed highly sensitive and specific detection of 2 additional Bordetella species beyond the primary B pertussis target. We observed a low incidence of pertussis in infants in a whole cell vaccine environment. Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms.", "Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms. Policymakers in Nepal will need to weigh the benefit of an additional prenatal pertussis vaccine or a switch to acellular primary pertussis vaccine with the low burden of pertussis in infants less than 6 months. Our study demonstrated that mid-nasal swabs were able to detect pertussis using a sensitive multitarget PCR. The less invasive mid-nasal nasal swab is an attractive alternative for pertussis nasal swab collection, and further research is needed to compare this collection site with nasopharyngeal swabs. In the future, this method may enhance population-based surveillance efforts." ]

[ "BACKGROUND: Pertussis is estimated to cause 2 percent of childhood deaths globally and is a growing public health problem in developed countries despite high vaccination coverage. Infants are at greatest risk of morbidity and mortality. Maternal vaccination during pregnancy may be effective to prevent pertussis in young infants, but population-based estimates of disease burden in infants are lacking, particularly in low-income countries. The objective of this study was to estimate the incidence of pertussis in infants less than 6 months of age in Sarlahi District, Nepal. METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay.", "METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay. The prospective cohort study includes infants observed between May 2011 and August 2014. RESULTS: The incidence of PCR-confirmed Bordetella pertussis was 13.3 cases per 1000 infant-years 95% confidence interval, 7.7–21.3 in a cohort of 3483 infants with at least 1 day of follow-up. CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants.", "CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants. Text: A resurgence of pertussis across age groups has occurred in several countries in recent years . Middle-and high-income countries that use an acellular pertussis vaccine for the primary vaccination series have been particularly affected , and infants and adolescents have experienced the greatest increase . Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves .", "Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves . Recent data from maternal vaccination trials demonstrate the ability of antibodies to be transferred from mothers to their infants in pregnancy and their persistence in infants . Global estimates of pertussis show the highest childhood burden in Southeast Asia . In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking .", "In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking . The World Health Organization WHO recently recommended that countries using whole cell pertussis vaccines continue to do so in light of recent data indicating that acellular pertussis vaccines are less effective than whole cell pertussis vaccines . Population-based data are needed, especially in low-income settings, to provide a more accurate estimate of the burden of pertussis in infants to inform childhood and maternal immunization policies . We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal.", "We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal. The study was nested within 2 consecutive randomized controlled trials of maternal influenza vaccination during pregnancy set in the Sarlahi District, located in the central Terai low-lying plains region of Nepal . At the start of the trial, prevalent pregnancies were identified through a census of all households in the catchment area. For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled.", "For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled. From April 25, 2011 through September 9, 2013, women between 17 and 34 weeks gestation were randomized and vaccinated with either an influenza vaccine or placebo. The study was a population-based prospective cohort of infants followed from birth through 6 months postpartum. Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 .", "Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 . At baseline, information was collected on household structure, socioeconomic status, and demographics. At enrollment, date of last menstrual period and pregnancy history data were collected. As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days.", "As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days. If an infant had any of the following symptoms, a mid-nasal nylon flocked swab was collected: fever, cough, wheeze, difficulty breathing, or ear infection. Starting on August 17, 2012, new symptoms, more specific for pertussis, were added to the weekly morbidity visit: apnea, cyanosis, cough with vomit, or whoop/whooping cough. The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination .", "The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination . Mid-nasal swabs were also collected on a weekly basis from mothers from enrollment through 6 months postpartum who reported fever plus one additional morbidity cough, sore throat, nasal congestion, or myalgia . All nasal swabs collected from infants were tested for B pertussis, Bordetella parapertussis, and Bordetella bronchispetica. Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods .", "Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods . Two-target PCR was used to assess the presence of 3 Bordetella species: B pertussis, B parapertussis, and B bronchiseptica. The amplified targets were chromosomal repeated insertion sequence IS481 IS and the polymorphic pertussis toxin ptxA promoter region PT . After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42.", "After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42. A sample was negative if none of the targets tested positive or a single positive target was not reproducible. Maternal nasal swabs were tested for those mothers whose infants tested positive for any Bordetella species Polymerase chain reaction was also performed for several viral infections influenza, rhinovirus RV , respiratory syncytial virus RSV , bocavirus BoV , human metapneumovirus, coronavirus, adenovirus, and parainfluenza as previously described . Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 .", "Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 . The final dataset consists of 3483 infants, contributing 1280 infant-years of observation, with at least 1 follow-up visit during the first 6 months. This includes infants from the entire trial period, both before and after more pertussis-specific additions to the weekly symptom questionnaire. At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history.", "At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history. The field workers identified their ethnicity into 2 broad groups Pahadi, a group originating from the hills; or Madeshi, a group originating from north India from names and observation. Women were categorized as nulliparous or multiparous. Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household.", "Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household. Gestational age was measured using a woman's report of date of last menstrual period during pregnancy surveillance. Birth weight was collected as soon as possible after birth using a digital scale Tanita model BD-585, precision to nearest 10 grams . Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards .", "Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards . Women were asked within how many hours of birth breastfeeding was initiated and binary breastfeeding categories were created ≤1 hour versus >1 hour postdelivery . Incidence was calculated as the number of pertussis cases per 1000 infant-years at risk. Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression.", "Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression. Characteristics of all pertussis respiratory episodes were compared with nonpertussis respiratory episodes; t tests were used for continuous predictors and Fisher's exact tests were used for categorical associations due to the low number of pertussis episodes. All statistical analyses were conducted in Stata/SE 14.1. A total of 3483 infants had 4283 episodes of respiratory illness between May 18, 2011 and April 30, 2014. Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 .", "Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 . Mean episode duration was 4.7 days 95% CI, 4.6-4.9 . A total of 3930 92% episodes were matched to 1 or more pertussis-tested nasal swabs from 2026 infants Supplementary Figure 1 . Seventeen cases of B pertussis were identified from 19 nasal swabs nasal swabs were positive on 2 consecutive weeks for 2 infants . The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 .", "The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 . No cases of B bronchiseptica were identified. The average pertussis episode duration was 8 days range, 2-33 Table 1 . Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit.", "Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit. None of the additional symptoms related to pertussis that were added in year 2 cyanosis, apnea, cough with vomit, and whoop resulted in collection of nasal swabs based solely on these additional symptoms. Pertussis episodes were statistically significantly more likely to include difficulty breathing, cough with vomit, and whoop compared with other respiratory illness. Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days.", "Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days. Three fourths of pertussis episodes were coinfected with at least 1 virus, with RV and BoV the most common. Cases of pertussis were more likely to be infected with BoV than respiratory cases due to causes other than pertussis. The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented.", "The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented. Given the low number of pertussis cases, the lack of a statistical association is not evidence of nonassociation. No deaths occurred in infants who had pertussis. Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 .", "Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 . No mothers of infants who had B parapertussis had a nasal swab collected during follow-up. The average B parapertussis episode duration was 4 days Supplementary Table 2 . Mean age of onset of symptoms was 58 days with a range of 7-95 days. The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed.", "The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed. All B parapertussis cases occurred between September 2011 and February 2012 Figure 1 . A low incidence of pertussis and generally mild clinical presentation were found in infants <6 months in Nepal. To our knowledge, this represents one of the first population-based active surveillance of PCR-confirmed pertussis among young infants in Asia. Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range.", "Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range. In the United States in 2014, the estimated pertussis incidence in infants less than 6 months was 2 cases per 1000 infant-years , much lower than observed in our study; however, this passive surveillance system likely vastly underestimates pertussis incidence. Thus, there is a need for active surveillance data such as ours. Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further.", "Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further. The low incidence was found in a population where pentavalent vaccine Pentavac: Diphtheria, Tetanus, Pertussis Whole Cell , Hepatitis-B and Haemophilus Type b Conjugate Vaccine; Serum Institute of India Pvt. Ltd , scheduled for administration at 6, 10, and 14 weeks, is received with significant delays 7% of infants received all 3 recommended pertussis vaccines by 6 months . These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine .", "These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine . Another contributing factor to the low pertussis incidence observed could be that surveillance was conducted during a period of low pertussis transmission. Pertussis is a cyclical disease, thought to peak every 2 to 4 years, and we may have captured the burden at a low circulation period . We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden.", "We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden. Previous research on pertussis seasonality has in different places and time periods demonstrated various periods of peak transmission or no discernable patterns . Although our data do not support a seasonal pattern, the numbers observed are too low to be conclusive. Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting .", "Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting . Studies on pertussis in infants have generally been clinic-based, hospital-based, or in an outbreak, which therefore required a certain severity of illness for parents to recognize a need for medical attention . These study designs and passive surveillance efforts therefore may have missed milder pertussis cases . Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal.", "Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal. Although cough, difficulty breathing, and cough with vomit were the most common symptoms, no symptom was present in all B pertussis cases. During an epidemic period in Washington state, among infants <1 year, who had a minimum of 14 days cough plus an additional symptom, 82% had posttussive emesis, 29% had apnea, 26% had whoop, and 42% had cyanosis . A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever.", "A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever. Fever prevalence was higher in our study, similar to that found in Peru . Although not statistically significant, infants with pertussis were more likely to have been born preterm, low birth weight, and SGA, and their mothers were more likely to be primiparous. These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies .", "These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies . Codetection of B pertussis and B parapertussis with respiratory viruses may be due to asymptomatic pertussis carriage. The incidence of B parapertussis of 4 cases per 1000 person-years was comparable to that of 2 per 1000 person-years found in the Italian acellular pertussis vaccine trial in 1992-1993 . The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection .", "The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection . Bordetella parapertussis cases occurred only during a 5-month period. There were several study design limitations. We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected.", "We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected. Infant respiratory symptoms were reported by parents, who may have missed signs that might have been observed by a healthcare worker. However, the criteria for collection of the nasal swab were broad and did not require sophisticated clinical skills. However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms.", "However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms. These data support our assumption that we were unlikely to have missed pertussis cases in year 1 with our less sensitive respiratory symptom criteria. Nasal swabs were collected in the mid-nasal region for influenza virus detection, which may have lowered the sensitivity of pertussis detection. In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population.", "In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population. Although nasopharyngeal swabs or nasopharyngeal aspirates are the recommended specimen collection method , the nasopharyngeal region was established as the collection area of choice when the diagnostic measure was culture, which has low sensitivity. Recent data demonstrated the comparability of using mid-nasal versus nasopharyngeal swabs in PCR pertussis detection . Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings.", "Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings. The community-based design allows generalizability of these results to the entire population and not just those seeking care at a health facility or in an outbreak situation. The Sarlahi District is located in the Terai region where the majority of Nepalese reside, and it has similar demographics to the entire population of Nepal . Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance.", "Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance. The multitarget PCR method allowed highly sensitive and specific detection of 2 additional Bordetella species beyond the primary B pertussis target. We observed a low incidence of pertussis in infants in a whole cell vaccine environment. Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms.", "Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms. Policymakers in Nepal will need to weigh the benefit of an additional prenatal pertussis vaccine or a switch to acellular primary pertussis vaccine with the low burden of pertussis in infants less than 6 months. Our study demonstrated that mid-nasal swabs were able to detect pertussis using a sensitive multitarget PCR. The less invasive mid-nasal nasal swab is an attractive alternative for pertussis nasal swab collection, and further research is needed to compare this collection site with nasopharyngeal swabs. In the future, this method may enhance population-based surveillance efforts." ]

[ "BACKGROUND: Pertussis is estimated to cause 2 percent of childhood deaths globally and is a growing public health problem in developed countries despite high vaccination coverage. Infants are at greatest risk of morbidity and mortality. Maternal vaccination during pregnancy may be effective to prevent pertussis in young infants, but population-based estimates of disease burden in infants are lacking, particularly in low-income countries. The objective of this study was to estimate the incidence of pertussis in infants less than 6 months of age in Sarlahi District, Nepal. METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay.", "METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay. The prospective cohort study includes infants observed between May 2011 and August 2014. RESULTS: The incidence of PCR-confirmed Bordetella pertussis was 13.3 cases per 1000 infant-years 95% confidence interval, 7.7–21.3 in a cohort of 3483 infants with at least 1 day of follow-up. CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants.", "CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants. Text: A resurgence of pertussis across age groups has occurred in several countries in recent years . Middle-and high-income countries that use an acellular pertussis vaccine for the primary vaccination series have been particularly affected , and infants and adolescents have experienced the greatest increase . Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves .", "Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves . Recent data from maternal vaccination trials demonstrate the ability of antibodies to be transferred from mothers to their infants in pregnancy and their persistence in infants . Global estimates of pertussis show the highest childhood burden in Southeast Asia . In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking .", "In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking . The World Health Organization WHO recently recommended that countries using whole cell pertussis vaccines continue to do so in light of recent data indicating that acellular pertussis vaccines are less effective than whole cell pertussis vaccines . Population-based data are needed, especially in low-income settings, to provide a more accurate estimate of the burden of pertussis in infants to inform childhood and maternal immunization policies . We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal.", "We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal. The study was nested within 2 consecutive randomized controlled trials of maternal influenza vaccination during pregnancy set in the Sarlahi District, located in the central Terai low-lying plains region of Nepal . At the start of the trial, prevalent pregnancies were identified through a census of all households in the catchment area. For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled.", "For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled. From April 25, 2011 through September 9, 2013, women between 17 and 34 weeks gestation were randomized and vaccinated with either an influenza vaccine or placebo. The study was a population-based prospective cohort of infants followed from birth through 6 months postpartum. Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 .", "Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 . At baseline, information was collected on household structure, socioeconomic status, and demographics. At enrollment, date of last menstrual period and pregnancy history data were collected. As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days.", "As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days. If an infant had any of the following symptoms, a mid-nasal nylon flocked swab was collected: fever, cough, wheeze, difficulty breathing, or ear infection. Starting on August 17, 2012, new symptoms, more specific for pertussis, were added to the weekly morbidity visit: apnea, cyanosis, cough with vomit, or whoop/whooping cough. The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination .", "The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination . Mid-nasal swabs were also collected on a weekly basis from mothers from enrollment through 6 months postpartum who reported fever plus one additional morbidity cough, sore throat, nasal congestion, or myalgia . All nasal swabs collected from infants were tested for B pertussis, Bordetella parapertussis, and Bordetella bronchispetica. Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods .", "Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods . Two-target PCR was used to assess the presence of 3 Bordetella species: B pertussis, B parapertussis, and B bronchiseptica. The amplified targets were chromosomal repeated insertion sequence IS481 IS and the polymorphic pertussis toxin ptxA promoter region PT . After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42.", "After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42. A sample was negative if none of the targets tested positive or a single positive target was not reproducible. Maternal nasal swabs were tested for those mothers whose infants tested positive for any Bordetella species Polymerase chain reaction was also performed for several viral infections influenza, rhinovirus RV , respiratory syncytial virus RSV , bocavirus BoV , human metapneumovirus, coronavirus, adenovirus, and parainfluenza as previously described . Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 .", "Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 . The final dataset consists of 3483 infants, contributing 1280 infant-years of observation, with at least 1 follow-up visit during the first 6 months. This includes infants from the entire trial period, both before and after more pertussis-specific additions to the weekly symptom questionnaire. At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history.", "At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history. The field workers identified their ethnicity into 2 broad groups Pahadi, a group originating from the hills; or Madeshi, a group originating from north India from names and observation. Women were categorized as nulliparous or multiparous. Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household.", "Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household. Gestational age was measured using a woman's report of date of last menstrual period during pregnancy surveillance. Birth weight was collected as soon as possible after birth using a digital scale Tanita model BD-585, precision to nearest 10 grams . Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards .", "Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards . Women were asked within how many hours of birth breastfeeding was initiated and binary breastfeeding categories were created ≤1 hour versus >1 hour postdelivery . Incidence was calculated as the number of pertussis cases per 1000 infant-years at risk. Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression.", "Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression. Characteristics of all pertussis respiratory episodes were compared with nonpertussis respiratory episodes; t tests were used for continuous predictors and Fisher's exact tests were used for categorical associations due to the low number of pertussis episodes. All statistical analyses were conducted in Stata/SE 14.1. A total of 3483 infants had 4283 episodes of respiratory illness between May 18, 2011 and April 30, 2014. Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 .", "Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 . Mean episode duration was 4.7 days 95% CI, 4.6-4.9 . A total of 3930 92% episodes were matched to 1 or more pertussis-tested nasal swabs from 2026 infants Supplementary Figure 1 . Seventeen cases of B pertussis were identified from 19 nasal swabs nasal swabs were positive on 2 consecutive weeks for 2 infants . The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 .", "The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 . No cases of B bronchiseptica were identified. The average pertussis episode duration was 8 days range, 2-33 Table 1 . Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit.", "Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit. None of the additional symptoms related to pertussis that were added in year 2 cyanosis, apnea, cough with vomit, and whoop resulted in collection of nasal swabs based solely on these additional symptoms. Pertussis episodes were statistically significantly more likely to include difficulty breathing, cough with vomit, and whoop compared with other respiratory illness. Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days.", "Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days. Three fourths of pertussis episodes were coinfected with at least 1 virus, with RV and BoV the most common. Cases of pertussis were more likely to be infected with BoV than respiratory cases due to causes other than pertussis. The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented.", "The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented. Given the low number of pertussis cases, the lack of a statistical association is not evidence of nonassociation. No deaths occurred in infants who had pertussis. Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 .", "Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 . No mothers of infants who had B parapertussis had a nasal swab collected during follow-up. The average B parapertussis episode duration was 4 days Supplementary Table 2 . Mean age of onset of symptoms was 58 days with a range of 7-95 days. The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed.", "The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed. All B parapertussis cases occurred between September 2011 and February 2012 Figure 1 . A low incidence of pertussis and generally mild clinical presentation were found in infants <6 months in Nepal. To our knowledge, this represents one of the first population-based active surveillance of PCR-confirmed pertussis among young infants in Asia. Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range.", "Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range. In the United States in 2014, the estimated pertussis incidence in infants less than 6 months was 2 cases per 1000 infant-years , much lower than observed in our study; however, this passive surveillance system likely vastly underestimates pertussis incidence. Thus, there is a need for active surveillance data such as ours. Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further.", "Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further. The low incidence was found in a population where pentavalent vaccine Pentavac: Diphtheria, Tetanus, Pertussis Whole Cell , Hepatitis-B and Haemophilus Type b Conjugate Vaccine; Serum Institute of India Pvt. Ltd , scheduled for administration at 6, 10, and 14 weeks, is received with significant delays 7% of infants received all 3 recommended pertussis vaccines by 6 months . These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine .", "These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine . Another contributing factor to the low pertussis incidence observed could be that surveillance was conducted during a period of low pertussis transmission. Pertussis is a cyclical disease, thought to peak every 2 to 4 years, and we may have captured the burden at a low circulation period . We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden.", "We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden. Previous research on pertussis seasonality has in different places and time periods demonstrated various periods of peak transmission or no discernable patterns . Although our data do not support a seasonal pattern, the numbers observed are too low to be conclusive. Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting .", "Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting . Studies on pertussis in infants have generally been clinic-based, hospital-based, or in an outbreak, which therefore required a certain severity of illness for parents to recognize a need for medical attention . These study designs and passive surveillance efforts therefore may have missed milder pertussis cases . Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal.", "Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal. Although cough, difficulty breathing, and cough with vomit were the most common symptoms, no symptom was present in all B pertussis cases. During an epidemic period in Washington state, among infants <1 year, who had a minimum of 14 days cough plus an additional symptom, 82% had posttussive emesis, 29% had apnea, 26% had whoop, and 42% had cyanosis . A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever.", "A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever. Fever prevalence was higher in our study, similar to that found in Peru . Although not statistically significant, infants with pertussis were more likely to have been born preterm, low birth weight, and SGA, and their mothers were more likely to be primiparous. These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies .", "These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies . Codetection of B pertussis and B parapertussis with respiratory viruses may be due to asymptomatic pertussis carriage. The incidence of B parapertussis of 4 cases per 1000 person-years was comparable to that of 2 per 1000 person-years found in the Italian acellular pertussis vaccine trial in 1992-1993 . The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection .", "The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection . Bordetella parapertussis cases occurred only during a 5-month period. There were several study design limitations. We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected.", "We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected. Infant respiratory symptoms were reported by parents, who may have missed signs that might have been observed by a healthcare worker. However, the criteria for collection of the nasal swab were broad and did not require sophisticated clinical skills. However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms.", "However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms. These data support our assumption that we were unlikely to have missed pertussis cases in year 1 with our less sensitive respiratory symptom criteria. Nasal swabs were collected in the mid-nasal region for influenza virus detection, which may have lowered the sensitivity of pertussis detection. In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population.", "In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population. Although nasopharyngeal swabs or nasopharyngeal aspirates are the recommended specimen collection method , the nasopharyngeal region was established as the collection area of choice when the diagnostic measure was culture, which has low sensitivity. Recent data demonstrated the comparability of using mid-nasal versus nasopharyngeal swabs in PCR pertussis detection . Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings.", "Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings. The community-based design allows generalizability of these results to the entire population and not just those seeking care at a health facility or in an outbreak situation. The Sarlahi District is located in the Terai region where the majority of Nepalese reside, and it has similar demographics to the entire population of Nepal . Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance.", "Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance. The multitarget PCR method allowed highly sensitive and specific detection of 2 additional Bordetella species beyond the primary B pertussis target. We observed a low incidence of pertussis in infants in a whole cell vaccine environment. Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms.", "Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms. Policymakers in Nepal will need to weigh the benefit of an additional prenatal pertussis vaccine or a switch to acellular primary pertussis vaccine with the low burden of pertussis in infants less than 6 months. Our study demonstrated that mid-nasal swabs were able to detect pertussis using a sensitive multitarget PCR. The less invasive mid-nasal nasal swab is an attractive alternative for pertussis nasal swab collection, and further research is needed to compare this collection site with nasopharyngeal swabs. In the future, this method may enhance population-based surveillance efforts." ]

[ "BACKGROUND: Pertussis is estimated to cause 2 percent of childhood deaths globally and is a growing public health problem in developed countries despite high vaccination coverage. Infants are at greatest risk of morbidity and mortality. Maternal vaccination during pregnancy may be effective to prevent pertussis in young infants, but population-based estimates of disease burden in infants are lacking, particularly in low-income countries. The objective of this study was to estimate the incidence of pertussis in infants less than 6 months of age in Sarlahi District, Nepal. METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay.", "METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction PCR assay. The prospective cohort study includes infants observed between May 2011 and August 2014. RESULTS: The incidence of PCR-confirmed Bordetella pertussis was 13.3 cases per 1000 infant-years 95% confidence interval, 7.7–21.3 in a cohort of 3483 infants with at least 1 day of follow-up. CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants.", "CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants. Text: A resurgence of pertussis across age groups has occurred in several countries in recent years . Middle-and high-income countries that use an acellular pertussis vaccine for the primary vaccination series have been particularly affected , and infants and adolescents have experienced the greatest increase . Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves .", "Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines , asymptomatic transmission from individuals vaccinated with acellular vaccines , genetic adaption of Bordetella pertussis , vaccination delay or refusal , improved surveillance and laboratory capabilities , and overall increased awareness of the continuing circulation of B pertussis . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts to protect the youngest infants from pertussis before they can be vaccinated themselves . Recent data from maternal vaccination trials demonstrate the ability of antibodies to be transferred from mothers to their infants in pregnancy and their persistence in infants . Global estimates of pertussis show the highest childhood burden in Southeast Asia . In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking .", "In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted Senegal , and surveillance and laboratory capabilities in Asia are lacking . The World Health Organization WHO recently recommended that countries using whole cell pertussis vaccines continue to do so in light of recent data indicating that acellular pertussis vaccines are less effective than whole cell pertussis vaccines . Population-based data are needed, especially in low-income settings, to provide a more accurate estimate of the burden of pertussis in infants to inform childhood and maternal immunization policies . We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal.", "We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal. The study was nested within 2 consecutive randomized controlled trials of maternal influenza vaccination during pregnancy set in the Sarlahi District, located in the central Terai low-lying plains region of Nepal . At the start of the trial, prevalent pregnancies were identified through a census of all households in the catchment area. For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled.", "For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women 15-40 years resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled. From April 25, 2011 through September 9, 2013, women between 17 and 34 weeks gestation were randomized and vaccinated with either an influenza vaccine or placebo. The study was a population-based prospective cohort of infants followed from birth through 6 months postpartum. Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 .", "Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov NCT01034254 . At baseline, information was collected on household structure, socioeconomic status, and demographics. At enrollment, date of last menstrual period and pregnancy history data were collected. As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days.", "As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days. If an infant had any of the following symptoms, a mid-nasal nylon flocked swab was collected: fever, cough, wheeze, difficulty breathing, or ear infection. Starting on August 17, 2012, new symptoms, more specific for pertussis, were added to the weekly morbidity visit: apnea, cyanosis, cough with vomit, or whoop/whooping cough. The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination .", "The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium Longhorn Diagnostics LLC, Bethesda, MD . In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination . Mid-nasal swabs were also collected on a weekly basis from mothers from enrollment through 6 months postpartum who reported fever plus one additional morbidity cough, sore throat, nasal congestion, or myalgia . All nasal swabs collected from infants were tested for B pertussis, Bordetella parapertussis, and Bordetella bronchispetica. Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods .", "Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction PCR testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods . Two-target PCR was used to assess the presence of 3 Bordetella species: B pertussis, B parapertussis, and B bronchiseptica. The amplified targets were chromosomal repeated insertion sequence IS481 IS and the polymorphic pertussis toxin ptxA promoter region PT . After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42.", "After amplification, the melting points of the amplicons were measured in an iCycler Bio-Rad . A sample was interpreted as positive when the target s had a melting temperature within the species-specific acceptable range and a computed tomography ≤42. A sample was negative if none of the targets tested positive or a single positive target was not reproducible. Maternal nasal swabs were tested for those mothers whose infants tested positive for any Bordetella species Polymerase chain reaction was also performed for several viral infections influenza, rhinovirus RV , respiratory syncytial virus RSV , bocavirus BoV , human metapneumovirus, coronavirus, adenovirus, and parainfluenza as previously described . Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 .", "Of 3693 women enrolled, 3646 infants were live born to 3621 women Supplementary Figure 1 . Infants were included in this analysis if they were followed for any length of the follow-up period 0 to 180 days ; median total follow-up was 146 days per infant Supplementary Figure 2 . The final dataset consists of 3483 infants, contributing 1280 infant-years of observation, with at least 1 follow-up visit during the first 6 months. This includes infants from the entire trial period, both before and after more pertussis-specific additions to the weekly symptom questionnaire. At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history.", "At baseline, data on household structure were gathered. At enrollment, women reported their literacy status binary and pregnancy history. The field workers identified their ethnicity into 2 broad groups Pahadi, a group originating from the hills; or Madeshi, a group originating from north India from names and observation. Women were categorized as nulliparous or multiparous. Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household.", "Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household. Gestational age was measured using a woman's report of date of last menstrual period during pregnancy surveillance. Birth weight was collected as soon as possible after birth using a digital scale Tanita model BD-585, precision to nearest 10 grams . Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards .", "Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age SGA was calculated using the sex-specific 10th percentile cutoff described by Alexander et al and the INTERGROWTH-21 standards . Women were asked within how many hours of birth breastfeeding was initiated and binary breastfeeding categories were created ≤1 hour versus >1 hour postdelivery . Incidence was calculated as the number of pertussis cases per 1000 infant-years at risk. Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression.", "Poisson exact 95% confidence intervals CIs were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression. Characteristics of all pertussis respiratory episodes were compared with nonpertussis respiratory episodes; t tests were used for continuous predictors and Fisher's exact tests were used for categorical associations due to the low number of pertussis episodes. All statistical analyses were conducted in Stata/SE 14.1. A total of 3483 infants had 4283 episodes of respiratory illness between May 18, 2011 and April 30, 2014. Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 .", "Thirty-nine percent n = 1350 of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year 95% CI, 3.5-3.7 . Mean episode duration was 4.7 days 95% CI, 4.6-4.9 . A total of 3930 92% episodes were matched to 1 or more pertussis-tested nasal swabs from 2026 infants Supplementary Figure 1 . Seventeen cases of B pertussis were identified from 19 nasal swabs nasal swabs were positive on 2 consecutive weeks for 2 infants . The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 .", "The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years 95% CI, 7.7-21.3 . Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years 95% CI, 1.3-9.1 . No cases of B bronchiseptica were identified. The average pertussis episode duration was 8 days range, 2-33 Table 1 . Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit.", "Mean age of onset of symptoms was 83 days range, 19-137 median, 80; interquartile range, 63-109 . The most common symptoms were cough, difficulty breathing, and cough with vomit. None of the additional symptoms related to pertussis that were added in year 2 cyanosis, apnea, cough with vomit, and whoop resulted in collection of nasal swabs based solely on these additional symptoms. Pertussis episodes were statistically significantly more likely to include difficulty breathing, cough with vomit, and whoop compared with other respiratory illness. Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days.", "Six infants had at least 1 pertussis vaccination before pertussis disease onset three <2 weeks and three >2 weeks before pertussis illness with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes P = .03 . Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days. Three fourths of pertussis episodes were coinfected with at least 1 virus, with RV and BoV the most common. Cases of pertussis were more likely to be infected with BoV than respiratory cases due to causes other than pertussis. The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented.", "The majority of cases occurred between February 2013 and January 2014 Figure 1 . No statistically significant differences between risk factors for pertussis and nonpertussis cases Table 2 were documented. Given the low number of pertussis cases, the lack of a statistical association is not evidence of nonassociation. No deaths occurred in infants who had pertussis. Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 .", "Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected 14 nasal swabs total during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity Supplementary Table 1 . No mothers of infants who had B parapertussis had a nasal swab collected during follow-up. The average B parapertussis episode duration was 4 days Supplementary Table 2 . Mean age of onset of symptoms was 58 days with a range of 7-95 days. The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed.", "The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed. All B parapertussis cases occurred between September 2011 and February 2012 Figure 1 . A low incidence of pertussis and generally mild clinical presentation were found in infants <6 months in Nepal. To our knowledge, this represents one of the first population-based active surveillance of PCR-confirmed pertussis among young infants in Asia. Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range.", "Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range. In the United States in 2014, the estimated pertussis incidence in infants less than 6 months was 2 cases per 1000 infant-years , much lower than observed in our study; however, this passive surveillance system likely vastly underestimates pertussis incidence. Thus, there is a need for active surveillance data such as ours. Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further.", "Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further. The low incidence was found in a population where pentavalent vaccine Pentavac: Diphtheria, Tetanus, Pertussis Whole Cell , Hepatitis-B and Haemophilus Type b Conjugate Vaccine; Serum Institute of India Pvt. Ltd , scheduled for administration at 6, 10, and 14 weeks, is received with significant delays 7% of infants received all 3 recommended pertussis vaccines by 6 months . These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine .", "These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine . Another contributing factor to the low pertussis incidence observed could be that surveillance was conducted during a period of low pertussis transmission. Pertussis is a cyclical disease, thought to peak every 2 to 4 years, and we may have captured the burden at a low circulation period . We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden.", "We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden. Previous research on pertussis seasonality has in different places and time periods demonstrated various periods of peak transmission or no discernable patterns . Although our data do not support a seasonal pattern, the numbers observed are too low to be conclusive. Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting .", "Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting . Studies on pertussis in infants have generally been clinic-based, hospital-based, or in an outbreak, which therefore required a certain severity of illness for parents to recognize a need for medical attention . These study designs and passive surveillance efforts therefore may have missed milder pertussis cases . Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal.", "Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal. Although cough, difficulty breathing, and cough with vomit were the most common symptoms, no symptom was present in all B pertussis cases. During an epidemic period in Washington state, among infants <1 year, who had a minimum of 14 days cough plus an additional symptom, 82% had posttussive emesis, 29% had apnea, 26% had whoop, and 42% had cyanosis . A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever.", "A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever . Our study found lower or equal symptom prevalence with the exception of fever. Fever prevalence was higher in our study, similar to that found in Peru . Although not statistically significant, infants with pertussis were more likely to have been born preterm, low birth weight, and SGA, and their mothers were more likely to be primiparous. These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies .", "These findings are similar to previous studies showing no difference in pertussis cases by sex or crowding but showing differences by birth weight . Coinfections were common, consistent with findings from other hospital-based studies . Codetection of B pertussis and B parapertussis with respiratory viruses may be due to asymptomatic pertussis carriage. The incidence of B parapertussis of 4 cases per 1000 person-years was comparable to that of 2 per 1000 person-years found in the Italian acellular pertussis vaccine trial in 1992-1993 . The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection .", "The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection . Bordetella parapertussis cases occurred only during a 5-month period. There were several study design limitations. We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected.", "We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected. Infant respiratory symptoms were reported by parents, who may have missed signs that might have been observed by a healthcare worker. However, the criteria for collection of the nasal swab were broad and did not require sophisticated clinical skills. However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms.", "However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms. These data support our assumption that we were unlikely to have missed pertussis cases in year 1 with our less sensitive respiratory symptom criteria. Nasal swabs were collected in the mid-nasal region for influenza virus detection, which may have lowered the sensitivity of pertussis detection. In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population.", "In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population. Although nasopharyngeal swabs or nasopharyngeal aspirates are the recommended specimen collection method , the nasopharyngeal region was established as the collection area of choice when the diagnostic measure was culture, which has low sensitivity. Recent data demonstrated the comparability of using mid-nasal versus nasopharyngeal swabs in PCR pertussis detection . Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings.", "Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings. The community-based design allows generalizability of these results to the entire population and not just those seeking care at a health facility or in an outbreak situation. The Sarlahi District is located in the Terai region where the majority of Nepalese reside, and it has similar demographics to the entire population of Nepal . Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance.", "Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance. The multitarget PCR method allowed highly sensitive and specific detection of 2 additional Bordetella species beyond the primary B pertussis target. We observed a low incidence of pertussis in infants in a whole cell vaccine environment. Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms.", "Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms. Policymakers in Nepal will need to weigh the benefit of an additional prenatal pertussis vaccine or a switch to acellular primary pertussis vaccine with the low burden of pertussis in infants less than 6 months. Our study demonstrated that mid-nasal swabs were able to detect pertussis using a sensitive multitarget PCR. The less invasive mid-nasal nasal swab is an attractive alternative for pertussis nasal swab collection, and further research is needed to compare this collection site with nasopharyngeal swabs. In the future, this method may enhance population-based surveillance efforts." ]

[ "Nuclear receptors NRs are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. Therefore, NRs have become a frequent target for drug development. During the process of developing drugs against these diseases by targeting NRs, we are often facing a problem: Given a NR and chemical compound, can we identify whether they are really in interaction with each other in a cell? To address this problem, a predictor called “iNR-Drug” was developed. In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner.", "In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. It is anticipated that the iNR-Drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Text: With the ability to directly bind to DNA Figure 1 and regulate the expression of adjacent genes, nuclear receptors NRs are a class of ligand-inducible transcription factors. They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like .", "They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like . Since they are involved in almost all aspects of human physiology and are implicated in many major diseases such as cancer, diabetes and osteoporosis, nuclear receptors have become major drug targets , along with G protein-coupled receptors GPCRs , ion channels , and kinase proteins . Identification of drug-target interactions is one of the most important steps for the new medicine development . The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition.", "The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition. Although X-ray crystallography is a powerful tool in determining protein 3D structures, it is time-consuming and expensive. Particularly, not all proteins can be successfully crystallized. For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined.", "For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined. Although NMR Nuclear Magnetic Resonance is indeed a very powerful tool in determining the 3D structures of membrane proteins as indicated by a series of recent publications see, e.g., and a review article , it is also time-consuming and costly. To acquire the 3D structural information in a timely manner, one has to resort to various structural bioinformatics tools see, e.g., , particularly the homologous modeling approach as utilized for a series of protein receptors urgently needed during the process of drug development 19, . Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter.", "Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter. The results thus obtained can be used to pre-exclude the compounds identified not in interaction with the nuclear receptors, so as to timely stop wasting time and money on those unpromising compounds . Actually, based on the functional groups and biological features, a powerful method was developed recently for this purpose. However, further development in this regard is definitely needed due to the following reasons. a He et al.", "However, further development in this regard is definitely needed due to the following reasons. a He et al. did not provide a publicly accessible web-server for their method, and hence its practical application value is quite limited, particularly for the broad experimental scientists; b The prediction quality can be further enhanced by incorporating some key features into the formulation of NR-drug nuclear receptor and drug samples via the general form of pseudo amino acid composition . The present study was initiated with an attempt to develop a new method for predicting the interaction of drugs with nuclear receptors by addressing the two points. As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps.", "As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps. The data used in the current study were collected from KEGG Kyoto Encyclopedia of Genes and Genomes at KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. Here, the benchmark dataset can be formulated as where is the positive subset that consists of the interactive drug-NR pairs only, while the negative subset that contains of the non-interactive drug-NR pairs only, and the symbol represents the union in the set theory. The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al.", "The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al. . The negative dataset contains 172 non-interactive drug-NR pairs, which were derived according to the following procedures: a separating each of the pairs in into single drug and NR; b re-coupling each of the single drugs with each of the single NRs into pairs in a way that none of them occurred in ; c randomly picking the pairs thus formed until reaching the number two times as many as the pairs in . The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample.", "The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample. First, for the drug part in the current benchmark dataset, we can use a 256-D vector to formulate it as given by where D represents the vector for a drug compound, and d i its i-th i = 1,2, ,256 component that can be derived by following the \"2D molecular fingerprint procedure\" as elaborated in . The 53 molecular fingerprint vectors thus obtained for the 53 drugs in are, respectively, given in Supplementary Information S2. The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth.", "The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth. Now the problem is how to effectively represent the sequence of Equation . with a non-sequential or discrete model . This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction.", "This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction. Facing such a dilemma, can we find an approach to partially incorporate the sequence-order effects? Actually, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or a vector, yet still keep considerable sequence order information. To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of .", "To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of . Moreover, the concept of PseAAC was further extended to represent the feature vectors of nucleotides , as well as other biological samples see, e.g., . Because it has been widely and increasingly used, recently two powerful soft-wares, called \"PseAAC-Builder\" and \"propy\" , were established for generating various special Chou's pseudo-amino acid compositions, in addition to the web-server \"PseAAC\" built in 2008. According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation .", "According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation . . Below, let us describe how to extract useful information to define the components of PseAAC for the NR samples concerned. First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned.", "First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned. Thus, such 20 AAC components were used here to define the first 20 elements in Equation . ; i.e., . 1, 2, , 20 ii fi   . where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned.", "where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned. Since AAC did not contain any sequence order information, the following steps were taken to make up this shortcoming. To avoid completely losing the local or short-range sequence order information, we considered the approach of dipeptide composition. It contained 20 × 20 = 400 components . Such 400 components were used to define the next 400 elements in Equation . ; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned.", "; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned. To incorporate the global or long-range sequence order information, let us consider the following approach. According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion.", "According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion. With the accumulation of these changes over a long period of time, many original similarities between initial and resultant amino acid sequences are gradually faded out, but the corresponding proteins may still share many common attributes , such as having basically the same biological function and residing at a same subcellular location . To extract the sequential evolution information and use it to define the components of Equation ., the PSSM Position Specific Scoring Matrix was used as described below. According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned.", "According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned. In order to make every element in Equation . be scaled from their original score ranges into the region of , we performed a conversion through the standard sigmoid function to make it become Now we extract the useful information from Equation . Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j .", "Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j . has the same meaning as in Equation . where   and Combining Equations ., ., . and ., we found that the total number of the components obtained via the current approach for the PseAAC of Equation . and each of the 500 components is given by . Since the elements in Equations . and .", "and each of the 500 components is given by . Since the elements in Equations . and . are well defined, we can now formulate the drug-NR pair by combining the two equations as given by   . where G represents the drug-NR pair, Å the orthogonal sum, and the 256 + 500 = 756 components are defined by Equations . and . . For the sake of convenience, let us use x i i = 1, 2, , 756 to represent the 756 components in Equation . ; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation .", "; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation . to where the symbol means taking the average of the quantity therein, and SD means the corresponding standard derivation. In this study, the SVM support vector machine was used as the operation engine. SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function.", "SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function. For a brief formulation of SVM and how it works, see the papers ; for more details about SVM, see a monograph . In this study, the LIBSVM package was used as an implementation of SVM, which can be downloaded from the popular radial basis function RBF was taken as the kernel function. For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later.", "For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later. The predictor obtained via the aforementioned procedure is called iNR-Drug, where \"i\" means identify, and \"NR-Drug\" means the interaction between nuclear receptor and drug compound. To provide an intuitive overall picture, a flowchart is provided in Figure 2 to show the process of how the predictor works in identifying the interactions between nuclear receptors and drug compounds. To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs.", "To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs. According to Equation . we can easily see the following. When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation.", "When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation. As we can see from the above discussion, it is much more intuitive and easier to understand when using Equation . to examine a predictor for its four metrics, particularly for its Mathew's correlation coefficient. It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in .", "It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in . How to properly test a predictor for its anticipated success rates is very important for its development as well as its potential application value. Generally speaking, the following three cross-validation methods are often used to examine the quality of a predictor and its effectiveness in practical application: independent dataset test, subsampling or K-fold such as five-fold, seven-fold, or 10-fold crossover test and jackknife test . However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either.", "However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either. Only the jackknife test is the least arbitrary that can always yield a unique result for a given benchmark dataset 73, 74, 156, . Therefore, the jackknife test has been widely recognized and increasingly utilized by investigators to examine the quality of various predictors see, e.g., . Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  .", "Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  . To find their optimal values, a 2-D grid search was conducted by the jackknife test on the benchmark dataset . The results thus obtained are shown in Figure 3 , from which it can be seen that the iNR-Drug predictor reaches its optimal status when C = 2 3 and 9 2    . The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al.", "The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al. on the same benchmark dataset is also given although no results were reported by them for Sn, Sp and MCC. It can be observed from the table that the overall accuracy obtained by iNR-Drug is remarkably higher that of He et al. , and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates.", ", and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates. As mentioned above Section 3.2 , the jackknife test is the most objective method for examining the quality of a predictor. However, as a demonstration to show how to practically use the current predictor, we took 41 NR-drug pairs from the study by Yamanishi et al. that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 .", "that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 . It is anticipated that the iNR-Drug predictor developed in this paper may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful predictors , a publicly accessible web-server for iNR-Drug was established. For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1.", "For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1. Open the web server at the site and you will see the top page of the predictor on your computer screen, as shown in Figure 4 . Click on the Read Me button to see a brief introduction about iNR-Drug predictor and the caveat when using it. Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 .", "Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 . Each query pair consists of two parts: one is for the nuclear receptor sequence, and the other for the drug. The NR sequence should be in FASTA format, while the drug in the KEGG code beginning with the symbol #. Examples for the query pairs input and the corresponding output can be seen by clicking on the Example button right above the input box. Step 3. Click on the Submit button to see the predicted result.", "Step 3. Click on the Submit button to see the predicted result. For example, if you use the three query pairs in the Example window as the input, after clicking the Submit button, you will see on your screen that the \"hsa:2099\" NR and the \"D00066\" drug are an interactive pair, and that the \"hsa:2908\" NR and the \"D00088\" drug are also an interactive pair, but that the \"hsa:5468\" NR and the \"D00279\" drug are not an interactive pair. All these results are fully consistent with the experimental observations. It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4.", "It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4. Click on the Citation button to find the relevant paper that documents the detailed development and algorithm of iNR-Durg. Step 5. Click on the Data button to download the benchmark dataset used to train and test the iNR-Durg predictor. Step 6. The program code is also available by clicking the button download on the lower panel of Figure 4 ." ]

[ "Nuclear receptors NRs are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. Therefore, NRs have become a frequent target for drug development. During the process of developing drugs against these diseases by targeting NRs, we are often facing a problem: Given a NR and chemical compound, can we identify whether they are really in interaction with each other in a cell? To address this problem, a predictor called “iNR-Drug” was developed. In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner.", "In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. It is anticipated that the iNR-Drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Text: With the ability to directly bind to DNA Figure 1 and regulate the expression of adjacent genes, nuclear receptors NRs are a class of ligand-inducible transcription factors. They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like .", "They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like . Since they are involved in almost all aspects of human physiology and are implicated in many major diseases such as cancer, diabetes and osteoporosis, nuclear receptors have become major drug targets , along with G protein-coupled receptors GPCRs , ion channels , and kinase proteins . Identification of drug-target interactions is one of the most important steps for the new medicine development . The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition.", "The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition. Although X-ray crystallography is a powerful tool in determining protein 3D structures, it is time-consuming and expensive. Particularly, not all proteins can be successfully crystallized. For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined.", "For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined. Although NMR Nuclear Magnetic Resonance is indeed a very powerful tool in determining the 3D structures of membrane proteins as indicated by a series of recent publications see, e.g., and a review article , it is also time-consuming and costly. To acquire the 3D structural information in a timely manner, one has to resort to various structural bioinformatics tools see, e.g., , particularly the homologous modeling approach as utilized for a series of protein receptors urgently needed during the process of drug development 19, . Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter.", "Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter. The results thus obtained can be used to pre-exclude the compounds identified not in interaction with the nuclear receptors, so as to timely stop wasting time and money on those unpromising compounds . Actually, based on the functional groups and biological features, a powerful method was developed recently for this purpose. However, further development in this regard is definitely needed due to the following reasons. a He et al.", "However, further development in this regard is definitely needed due to the following reasons. a He et al. did not provide a publicly accessible web-server for their method, and hence its practical application value is quite limited, particularly for the broad experimental scientists; b The prediction quality can be further enhanced by incorporating some key features into the formulation of NR-drug nuclear receptor and drug samples via the general form of pseudo amino acid composition . The present study was initiated with an attempt to develop a new method for predicting the interaction of drugs with nuclear receptors by addressing the two points. As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps.", "As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps. The data used in the current study were collected from KEGG Kyoto Encyclopedia of Genes and Genomes at KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. Here, the benchmark dataset can be formulated as where is the positive subset that consists of the interactive drug-NR pairs only, while the negative subset that contains of the non-interactive drug-NR pairs only, and the symbol represents the union in the set theory. The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al.", "The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al. . The negative dataset contains 172 non-interactive drug-NR pairs, which were derived according to the following procedures: a separating each of the pairs in into single drug and NR; b re-coupling each of the single drugs with each of the single NRs into pairs in a way that none of them occurred in ; c randomly picking the pairs thus formed until reaching the number two times as many as the pairs in . The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample.", "The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample. First, for the drug part in the current benchmark dataset, we can use a 256-D vector to formulate it as given by where D represents the vector for a drug compound, and d i its i-th i = 1,2, ,256 component that can be derived by following the \"2D molecular fingerprint procedure\" as elaborated in . The 53 molecular fingerprint vectors thus obtained for the 53 drugs in are, respectively, given in Supplementary Information S2. The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth.", "The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth. Now the problem is how to effectively represent the sequence of Equation . with a non-sequential or discrete model . This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction.", "This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction. Facing such a dilemma, can we find an approach to partially incorporate the sequence-order effects? Actually, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or a vector, yet still keep considerable sequence order information. To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of .", "To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of . Moreover, the concept of PseAAC was further extended to represent the feature vectors of nucleotides , as well as other biological samples see, e.g., . Because it has been widely and increasingly used, recently two powerful soft-wares, called \"PseAAC-Builder\" and \"propy\" , were established for generating various special Chou's pseudo-amino acid compositions, in addition to the web-server \"PseAAC\" built in 2008. According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation .", "According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation . . Below, let us describe how to extract useful information to define the components of PseAAC for the NR samples concerned. First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned.", "First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned. Thus, such 20 AAC components were used here to define the first 20 elements in Equation . ; i.e., . 1, 2, , 20 ii fi   . where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned.", "where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned. Since AAC did not contain any sequence order information, the following steps were taken to make up this shortcoming. To avoid completely losing the local or short-range sequence order information, we considered the approach of dipeptide composition. It contained 20 × 20 = 400 components . Such 400 components were used to define the next 400 elements in Equation . ; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned.", "; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned. To incorporate the global or long-range sequence order information, let us consider the following approach. According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion.", "According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion. With the accumulation of these changes over a long period of time, many original similarities between initial and resultant amino acid sequences are gradually faded out, but the corresponding proteins may still share many common attributes , such as having basically the same biological function and residing at a same subcellular location . To extract the sequential evolution information and use it to define the components of Equation ., the PSSM Position Specific Scoring Matrix was used as described below. According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned.", "According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned. In order to make every element in Equation . be scaled from their original score ranges into the region of , we performed a conversion through the standard sigmoid function to make it become Now we extract the useful information from Equation . Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j .", "Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j . has the same meaning as in Equation . where   and Combining Equations ., ., . and ., we found that the total number of the components obtained via the current approach for the PseAAC of Equation . and each of the 500 components is given by . Since the elements in Equations . and .", "and each of the 500 components is given by . Since the elements in Equations . and . are well defined, we can now formulate the drug-NR pair by combining the two equations as given by   . where G represents the drug-NR pair, Å the orthogonal sum, and the 256 + 500 = 756 components are defined by Equations . and . . For the sake of convenience, let us use x i i = 1, 2, , 756 to represent the 756 components in Equation . ; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation .", "; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation . to where the symbol means taking the average of the quantity therein, and SD means the corresponding standard derivation. In this study, the SVM support vector machine was used as the operation engine. SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function.", "SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function. For a brief formulation of SVM and how it works, see the papers ; for more details about SVM, see a monograph . In this study, the LIBSVM package was used as an implementation of SVM, which can be downloaded from the popular radial basis function RBF was taken as the kernel function. For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later.", "For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later. The predictor obtained via the aforementioned procedure is called iNR-Drug, where \"i\" means identify, and \"NR-Drug\" means the interaction between nuclear receptor and drug compound. To provide an intuitive overall picture, a flowchart is provided in Figure 2 to show the process of how the predictor works in identifying the interactions between nuclear receptors and drug compounds. To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs.", "To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs. According to Equation . we can easily see the following. When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation.", "When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation. As we can see from the above discussion, it is much more intuitive and easier to understand when using Equation . to examine a predictor for its four metrics, particularly for its Mathew's correlation coefficient. It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in .", "It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in . How to properly test a predictor for its anticipated success rates is very important for its development as well as its potential application value. Generally speaking, the following three cross-validation methods are often used to examine the quality of a predictor and its effectiveness in practical application: independent dataset test, subsampling or K-fold such as five-fold, seven-fold, or 10-fold crossover test and jackknife test . However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either.", "However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either. Only the jackknife test is the least arbitrary that can always yield a unique result for a given benchmark dataset 73, 74, 156, . Therefore, the jackknife test has been widely recognized and increasingly utilized by investigators to examine the quality of various predictors see, e.g., . Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  .", "Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  . To find their optimal values, a 2-D grid search was conducted by the jackknife test on the benchmark dataset . The results thus obtained are shown in Figure 3 , from which it can be seen that the iNR-Drug predictor reaches its optimal status when C = 2 3 and 9 2    . The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al.", "The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al. on the same benchmark dataset is also given although no results were reported by them for Sn, Sp and MCC. It can be observed from the table that the overall accuracy obtained by iNR-Drug is remarkably higher that of He et al. , and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates.", ", and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates. As mentioned above Section 3.2 , the jackknife test is the most objective method for examining the quality of a predictor. However, as a demonstration to show how to practically use the current predictor, we took 41 NR-drug pairs from the study by Yamanishi et al. that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 .", "that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 . It is anticipated that the iNR-Drug predictor developed in this paper may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful predictors , a publicly accessible web-server for iNR-Drug was established. For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1.", "For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1. Open the web server at the site and you will see the top page of the predictor on your computer screen, as shown in Figure 4 . Click on the Read Me button to see a brief introduction about iNR-Drug predictor and the caveat when using it. Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 .", "Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 . Each query pair consists of two parts: one is for the nuclear receptor sequence, and the other for the drug. The NR sequence should be in FASTA format, while the drug in the KEGG code beginning with the symbol #. Examples for the query pairs input and the corresponding output can be seen by clicking on the Example button right above the input box. Step 3. Click on the Submit button to see the predicted result.", "Step 3. Click on the Submit button to see the predicted result. For example, if you use the three query pairs in the Example window as the input, after clicking the Submit button, you will see on your screen that the \"hsa:2099\" NR and the \"D00066\" drug are an interactive pair, and that the \"hsa:2908\" NR and the \"D00088\" drug are also an interactive pair, but that the \"hsa:5468\" NR and the \"D00279\" drug are not an interactive pair. All these results are fully consistent with the experimental observations. It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4.", "It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4. Click on the Citation button to find the relevant paper that documents the detailed development and algorithm of iNR-Durg. Step 5. Click on the Data button to download the benchmark dataset used to train and test the iNR-Durg predictor. Step 6. The program code is also available by clicking the button download on the lower panel of Figure 4 ." ]

[ "Nuclear receptors NRs are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. Therefore, NRs have become a frequent target for drug development. During the process of developing drugs against these diseases by targeting NRs, we are often facing a problem: Given a NR and chemical compound, can we identify whether they are really in interaction with each other in a cell? To address this problem, a predictor called “iNR-Drug” was developed. In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner.", "In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. It is anticipated that the iNR-Drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Text: With the ability to directly bind to DNA Figure 1 and regulate the expression of adjacent genes, nuclear receptors NRs are a class of ligand-inducible transcription factors. They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like .", "They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like . Since they are involved in almost all aspects of human physiology and are implicated in many major diseases such as cancer, diabetes and osteoporosis, nuclear receptors have become major drug targets , along with G protein-coupled receptors GPCRs , ion channels , and kinase proteins . Identification of drug-target interactions is one of the most important steps for the new medicine development . The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition.", "The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition. Although X-ray crystallography is a powerful tool in determining protein 3D structures, it is time-consuming and expensive. Particularly, not all proteins can be successfully crystallized. For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined.", "For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined. Although NMR Nuclear Magnetic Resonance is indeed a very powerful tool in determining the 3D structures of membrane proteins as indicated by a series of recent publications see, e.g., and a review article , it is also time-consuming and costly. To acquire the 3D structural information in a timely manner, one has to resort to various structural bioinformatics tools see, e.g., , particularly the homologous modeling approach as utilized for a series of protein receptors urgently needed during the process of drug development 19, . Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter.", "Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter. The results thus obtained can be used to pre-exclude the compounds identified not in interaction with the nuclear receptors, so as to timely stop wasting time and money on those unpromising compounds . Actually, based on the functional groups and biological features, a powerful method was developed recently for this purpose. However, further development in this regard is definitely needed due to the following reasons. a He et al.", "However, further development in this regard is definitely needed due to the following reasons. a He et al. did not provide a publicly accessible web-server for their method, and hence its practical application value is quite limited, particularly for the broad experimental scientists; b The prediction quality can be further enhanced by incorporating some key features into the formulation of NR-drug nuclear receptor and drug samples via the general form of pseudo amino acid composition . The present study was initiated with an attempt to develop a new method for predicting the interaction of drugs with nuclear receptors by addressing the two points. As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps.", "As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps. The data used in the current study were collected from KEGG Kyoto Encyclopedia of Genes and Genomes at KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. Here, the benchmark dataset can be formulated as where is the positive subset that consists of the interactive drug-NR pairs only, while the negative subset that contains of the non-interactive drug-NR pairs only, and the symbol represents the union in the set theory. The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al.", "The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al. . The negative dataset contains 172 non-interactive drug-NR pairs, which were derived according to the following procedures: a separating each of the pairs in into single drug and NR; b re-coupling each of the single drugs with each of the single NRs into pairs in a way that none of them occurred in ; c randomly picking the pairs thus formed until reaching the number two times as many as the pairs in . The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample.", "The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample. First, for the drug part in the current benchmark dataset, we can use a 256-D vector to formulate it as given by where D represents the vector for a drug compound, and d i its i-th i = 1,2, ,256 component that can be derived by following the \"2D molecular fingerprint procedure\" as elaborated in . The 53 molecular fingerprint vectors thus obtained for the 53 drugs in are, respectively, given in Supplementary Information S2. The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth.", "The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth. Now the problem is how to effectively represent the sequence of Equation . with a non-sequential or discrete model . This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction.", "This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction. Facing such a dilemma, can we find an approach to partially incorporate the sequence-order effects? Actually, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or a vector, yet still keep considerable sequence order information. To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of .", "To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of . Moreover, the concept of PseAAC was further extended to represent the feature vectors of nucleotides , as well as other biological samples see, e.g., . Because it has been widely and increasingly used, recently two powerful soft-wares, called \"PseAAC-Builder\" and \"propy\" , were established for generating various special Chou's pseudo-amino acid compositions, in addition to the web-server \"PseAAC\" built in 2008. According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation .", "According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation . . Below, let us describe how to extract useful information to define the components of PseAAC for the NR samples concerned. First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned.", "First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned. Thus, such 20 AAC components were used here to define the first 20 elements in Equation . ; i.e., . 1, 2, , 20 ii fi   . where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned.", "where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned. Since AAC did not contain any sequence order information, the following steps were taken to make up this shortcoming. To avoid completely losing the local or short-range sequence order information, we considered the approach of dipeptide composition. It contained 20 × 20 = 400 components . Such 400 components were used to define the next 400 elements in Equation . ; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned.", "; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned. To incorporate the global or long-range sequence order information, let us consider the following approach. According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion.", "According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion. With the accumulation of these changes over a long period of time, many original similarities between initial and resultant amino acid sequences are gradually faded out, but the corresponding proteins may still share many common attributes , such as having basically the same biological function and residing at a same subcellular location . To extract the sequential evolution information and use it to define the components of Equation ., the PSSM Position Specific Scoring Matrix was used as described below. According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned.", "According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned. In order to make every element in Equation . be scaled from their original score ranges into the region of , we performed a conversion through the standard sigmoid function to make it become Now we extract the useful information from Equation . Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j .", "Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j . has the same meaning as in Equation . where   and Combining Equations ., ., . and ., we found that the total number of the components obtained via the current approach for the PseAAC of Equation . and each of the 500 components is given by . Since the elements in Equations . and .", "and each of the 500 components is given by . Since the elements in Equations . and . are well defined, we can now formulate the drug-NR pair by combining the two equations as given by   . where G represents the drug-NR pair, Å the orthogonal sum, and the 256 + 500 = 756 components are defined by Equations . and . . For the sake of convenience, let us use x i i = 1, 2, , 756 to represent the 756 components in Equation . ; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation .", "; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation . to where the symbol means taking the average of the quantity therein, and SD means the corresponding standard derivation. In this study, the SVM support vector machine was used as the operation engine. SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function.", "SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function. For a brief formulation of SVM and how it works, see the papers ; for more details about SVM, see a monograph . In this study, the LIBSVM package was used as an implementation of SVM, which can be downloaded from the popular radial basis function RBF was taken as the kernel function. For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later.", "For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later. The predictor obtained via the aforementioned procedure is called iNR-Drug, where \"i\" means identify, and \"NR-Drug\" means the interaction between nuclear receptor and drug compound. To provide an intuitive overall picture, a flowchart is provided in Figure 2 to show the process of how the predictor works in identifying the interactions between nuclear receptors and drug compounds. To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs.", "To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs. According to Equation . we can easily see the following. When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation.", "When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation. As we can see from the above discussion, it is much more intuitive and easier to understand when using Equation . to examine a predictor for its four metrics, particularly for its Mathew's correlation coefficient. It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in .", "It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in . How to properly test a predictor for its anticipated success rates is very important for its development as well as its potential application value. Generally speaking, the following three cross-validation methods are often used to examine the quality of a predictor and its effectiveness in practical application: independent dataset test, subsampling or K-fold such as five-fold, seven-fold, or 10-fold crossover test and jackknife test . However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either.", "However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either. Only the jackknife test is the least arbitrary that can always yield a unique result for a given benchmark dataset 73, 74, 156, . Therefore, the jackknife test has been widely recognized and increasingly utilized by investigators to examine the quality of various predictors see, e.g., . Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  .", "Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  . To find their optimal values, a 2-D grid search was conducted by the jackknife test on the benchmark dataset . The results thus obtained are shown in Figure 3 , from which it can be seen that the iNR-Drug predictor reaches its optimal status when C = 2 3 and 9 2    . The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al.", "The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al. on the same benchmark dataset is also given although no results were reported by them for Sn, Sp and MCC. It can be observed from the table that the overall accuracy obtained by iNR-Drug is remarkably higher that of He et al. , and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates.", ", and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates. As mentioned above Section 3.2 , the jackknife test is the most objective method for examining the quality of a predictor. However, as a demonstration to show how to practically use the current predictor, we took 41 NR-drug pairs from the study by Yamanishi et al. that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 .", "that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 . It is anticipated that the iNR-Drug predictor developed in this paper may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful predictors , a publicly accessible web-server for iNR-Drug was established. For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1.", "For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1. Open the web server at the site and you will see the top page of the predictor on your computer screen, as shown in Figure 4 . Click on the Read Me button to see a brief introduction about iNR-Drug predictor and the caveat when using it. Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 .", "Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 . Each query pair consists of two parts: one is for the nuclear receptor sequence, and the other for the drug. The NR sequence should be in FASTA format, while the drug in the KEGG code beginning with the symbol #. Examples for the query pairs input and the corresponding output can be seen by clicking on the Example button right above the input box. Step 3. Click on the Submit button to see the predicted result.", "Step 3. Click on the Submit button to see the predicted result. For example, if you use the three query pairs in the Example window as the input, after clicking the Submit button, you will see on your screen that the \"hsa:2099\" NR and the \"D00066\" drug are an interactive pair, and that the \"hsa:2908\" NR and the \"D00088\" drug are also an interactive pair, but that the \"hsa:5468\" NR and the \"D00279\" drug are not an interactive pair. All these results are fully consistent with the experimental observations. It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4.", "It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4. Click on the Citation button to find the relevant paper that documents the detailed development and algorithm of iNR-Durg. Step 5. Click on the Data button to download the benchmark dataset used to train and test the iNR-Durg predictor. Step 6. The program code is also available by clicking the button download on the lower panel of Figure 4 ." ]

[ "Nuclear receptors NRs are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. Therefore, NRs have become a frequent target for drug development. During the process of developing drugs against these diseases by targeting NRs, we are often facing a problem: Given a NR and chemical compound, can we identify whether they are really in interaction with each other in a cell? To address this problem, a predictor called “iNR-Drug” was developed. In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner.", "In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. It is anticipated that the iNR-Drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Text: With the ability to directly bind to DNA Figure 1 and regulate the expression of adjacent genes, nuclear receptors NRs are a class of ligand-inducible transcription factors. They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like .", "They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like . Since they are involved in almost all aspects of human physiology and are implicated in many major diseases such as cancer, diabetes and osteoporosis, nuclear receptors have become major drug targets , along with G protein-coupled receptors GPCRs , ion channels , and kinase proteins . Identification of drug-target interactions is one of the most important steps for the new medicine development . The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition.", "The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition. Although X-ray crystallography is a powerful tool in determining protein 3D structures, it is time-consuming and expensive. Particularly, not all proteins can be successfully crystallized. For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined.", "For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined. Although NMR Nuclear Magnetic Resonance is indeed a very powerful tool in determining the 3D structures of membrane proteins as indicated by a series of recent publications see, e.g., and a review article , it is also time-consuming and costly. To acquire the 3D structural information in a timely manner, one has to resort to various structural bioinformatics tools see, e.g., , particularly the homologous modeling approach as utilized for a series of protein receptors urgently needed during the process of drug development 19, . Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter.", "Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter. The results thus obtained can be used to pre-exclude the compounds identified not in interaction with the nuclear receptors, so as to timely stop wasting time and money on those unpromising compounds . Actually, based on the functional groups and biological features, a powerful method was developed recently for this purpose. However, further development in this regard is definitely needed due to the following reasons. a He et al.", "However, further development in this regard is definitely needed due to the following reasons. a He et al. did not provide a publicly accessible web-server for their method, and hence its practical application value is quite limited, particularly for the broad experimental scientists; b The prediction quality can be further enhanced by incorporating some key features into the formulation of NR-drug nuclear receptor and drug samples via the general form of pseudo amino acid composition . The present study was initiated with an attempt to develop a new method for predicting the interaction of drugs with nuclear receptors by addressing the two points. As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps.", "As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps. The data used in the current study were collected from KEGG Kyoto Encyclopedia of Genes and Genomes at KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. Here, the benchmark dataset can be formulated as where is the positive subset that consists of the interactive drug-NR pairs only, while the negative subset that contains of the non-interactive drug-NR pairs only, and the symbol represents the union in the set theory. The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al.", "The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al. . The negative dataset contains 172 non-interactive drug-NR pairs, which were derived according to the following procedures: a separating each of the pairs in into single drug and NR; b re-coupling each of the single drugs with each of the single NRs into pairs in a way that none of them occurred in ; c randomly picking the pairs thus formed until reaching the number two times as many as the pairs in . The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample.", "The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample. First, for the drug part in the current benchmark dataset, we can use a 256-D vector to formulate it as given by where D represents the vector for a drug compound, and d i its i-th i = 1,2, ,256 component that can be derived by following the \"2D molecular fingerprint procedure\" as elaborated in . The 53 molecular fingerprint vectors thus obtained for the 53 drugs in are, respectively, given in Supplementary Information S2. The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth.", "The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth. Now the problem is how to effectively represent the sequence of Equation . with a non-sequential or discrete model . This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction.", "This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction. Facing such a dilemma, can we find an approach to partially incorporate the sequence-order effects? Actually, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or a vector, yet still keep considerable sequence order information. To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of .", "To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of . Moreover, the concept of PseAAC was further extended to represent the feature vectors of nucleotides , as well as other biological samples see, e.g., . Because it has been widely and increasingly used, recently two powerful soft-wares, called \"PseAAC-Builder\" and \"propy\" , were established for generating various special Chou's pseudo-amino acid compositions, in addition to the web-server \"PseAAC\" built in 2008. According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation .", "According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation . . Below, let us describe how to extract useful information to define the components of PseAAC for the NR samples concerned. First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned.", "First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned. Thus, such 20 AAC components were used here to define the first 20 elements in Equation . ; i.e., . 1, 2, , 20 ii fi   . where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned.", "where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned. Since AAC did not contain any sequence order information, the following steps were taken to make up this shortcoming. To avoid completely losing the local or short-range sequence order information, we considered the approach of dipeptide composition. It contained 20 × 20 = 400 components . Such 400 components were used to define the next 400 elements in Equation . ; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned.", "; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned. To incorporate the global or long-range sequence order information, let us consider the following approach. According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion.", "According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion. With the accumulation of these changes over a long period of time, many original similarities between initial and resultant amino acid sequences are gradually faded out, but the corresponding proteins may still share many common attributes , such as having basically the same biological function and residing at a same subcellular location . To extract the sequential evolution information and use it to define the components of Equation ., the PSSM Position Specific Scoring Matrix was used as described below. According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned.", "According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned. In order to make every element in Equation . be scaled from their original score ranges into the region of , we performed a conversion through the standard sigmoid function to make it become Now we extract the useful information from Equation . Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j .", "Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j . has the same meaning as in Equation . where   and Combining Equations ., ., . and ., we found that the total number of the components obtained via the current approach for the PseAAC of Equation . and each of the 500 components is given by . Since the elements in Equations . and .", "and each of the 500 components is given by . Since the elements in Equations . and . are well defined, we can now formulate the drug-NR pair by combining the two equations as given by   . where G represents the drug-NR pair, Å the orthogonal sum, and the 256 + 500 = 756 components are defined by Equations . and . . For the sake of convenience, let us use x i i = 1, 2, , 756 to represent the 756 components in Equation . ; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation .", "; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation . to where the symbol means taking the average of the quantity therein, and SD means the corresponding standard derivation. In this study, the SVM support vector machine was used as the operation engine. SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function.", "SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function. For a brief formulation of SVM and how it works, see the papers ; for more details about SVM, see a monograph . In this study, the LIBSVM package was used as an implementation of SVM, which can be downloaded from the popular radial basis function RBF was taken as the kernel function. For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later.", "For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later. The predictor obtained via the aforementioned procedure is called iNR-Drug, where \"i\" means identify, and \"NR-Drug\" means the interaction between nuclear receptor and drug compound. To provide an intuitive overall picture, a flowchart is provided in Figure 2 to show the process of how the predictor works in identifying the interactions between nuclear receptors and drug compounds. To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs.", "To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs. According to Equation . we can easily see the following. When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation.", "When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation. As we can see from the above discussion, it is much more intuitive and easier to understand when using Equation . to examine a predictor for its four metrics, particularly for its Mathew's correlation coefficient. It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in .", "It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in . How to properly test a predictor for its anticipated success rates is very important for its development as well as its potential application value. Generally speaking, the following three cross-validation methods are often used to examine the quality of a predictor and its effectiveness in practical application: independent dataset test, subsampling or K-fold such as five-fold, seven-fold, or 10-fold crossover test and jackknife test . However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either.", "However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either. Only the jackknife test is the least arbitrary that can always yield a unique result for a given benchmark dataset 73, 74, 156, . Therefore, the jackknife test has been widely recognized and increasingly utilized by investigators to examine the quality of various predictors see, e.g., . Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  .", "Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  . To find their optimal values, a 2-D grid search was conducted by the jackknife test on the benchmark dataset . The results thus obtained are shown in Figure 3 , from which it can be seen that the iNR-Drug predictor reaches its optimal status when C = 2 3 and 9 2    . The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al.", "The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al. on the same benchmark dataset is also given although no results were reported by them for Sn, Sp and MCC. It can be observed from the table that the overall accuracy obtained by iNR-Drug is remarkably higher that of He et al. , and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates.", ", and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates. As mentioned above Section 3.2 , the jackknife test is the most objective method for examining the quality of a predictor. However, as a demonstration to show how to practically use the current predictor, we took 41 NR-drug pairs from the study by Yamanishi et al. that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 .", "that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 . It is anticipated that the iNR-Drug predictor developed in this paper may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful predictors , a publicly accessible web-server for iNR-Drug was established. For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1.", "For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1. Open the web server at the site and you will see the top page of the predictor on your computer screen, as shown in Figure 4 . Click on the Read Me button to see a brief introduction about iNR-Drug predictor and the caveat when using it. Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 .", "Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 . Each query pair consists of two parts: one is for the nuclear receptor sequence, and the other for the drug. The NR sequence should be in FASTA format, while the drug in the KEGG code beginning with the symbol #. Examples for the query pairs input and the corresponding output can be seen by clicking on the Example button right above the input box. Step 3. Click on the Submit button to see the predicted result.", "Step 3. Click on the Submit button to see the predicted result. For example, if you use the three query pairs in the Example window as the input, after clicking the Submit button, you will see on your screen that the \"hsa:2099\" NR and the \"D00066\" drug are an interactive pair, and that the \"hsa:2908\" NR and the \"D00088\" drug are also an interactive pair, but that the \"hsa:5468\" NR and the \"D00279\" drug are not an interactive pair. All these results are fully consistent with the experimental observations. It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4.", "It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4. Click on the Citation button to find the relevant paper that documents the detailed development and algorithm of iNR-Durg. Step 5. Click on the Data button to download the benchmark dataset used to train and test the iNR-Durg predictor. Step 6. The program code is also available by clicking the button download on the lower panel of Figure 4 ." ]

[ "Nuclear receptors NRs are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. Therefore, NRs have become a frequent target for drug development. During the process of developing drugs against these diseases by targeting NRs, we are often facing a problem: Given a NR and chemical compound, can we identify whether they are really in interaction with each other in a cell? To address this problem, a predictor called “iNR-Drug” was developed. In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner.", "In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. It is anticipated that the iNR-Drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Text: With the ability to directly bind to DNA Figure 1 and regulate the expression of adjacent genes, nuclear receptors NRs are a class of ligand-inducible transcription factors. They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like .", "They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like . Since they are involved in almost all aspects of human physiology and are implicated in many major diseases such as cancer, diabetes and osteoporosis, nuclear receptors have become major drug targets , along with G protein-coupled receptors GPCRs , ion channels , and kinase proteins . Identification of drug-target interactions is one of the most important steps for the new medicine development . The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition.", "The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition. Although X-ray crystallography is a powerful tool in determining protein 3D structures, it is time-consuming and expensive. Particularly, not all proteins can be successfully crystallized. For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined.", "For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined. Although NMR Nuclear Magnetic Resonance is indeed a very powerful tool in determining the 3D structures of membrane proteins as indicated by a series of recent publications see, e.g., and a review article , it is also time-consuming and costly. To acquire the 3D structural information in a timely manner, one has to resort to various structural bioinformatics tools see, e.g., , particularly the homologous modeling approach as utilized for a series of protein receptors urgently needed during the process of drug development 19, . Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter.", "Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter. The results thus obtained can be used to pre-exclude the compounds identified not in interaction with the nuclear receptors, so as to timely stop wasting time and money on those unpromising compounds . Actually, based on the functional groups and biological features, a powerful method was developed recently for this purpose. However, further development in this regard is definitely needed due to the following reasons. a He et al.", "However, further development in this regard is definitely needed due to the following reasons. a He et al. did not provide a publicly accessible web-server for their method, and hence its practical application value is quite limited, particularly for the broad experimental scientists; b The prediction quality can be further enhanced by incorporating some key features into the formulation of NR-drug nuclear receptor and drug samples via the general form of pseudo amino acid composition . The present study was initiated with an attempt to develop a new method for predicting the interaction of drugs with nuclear receptors by addressing the two points. As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps.", "As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps. The data used in the current study were collected from KEGG Kyoto Encyclopedia of Genes and Genomes at KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. Here, the benchmark dataset can be formulated as where is the positive subset that consists of the interactive drug-NR pairs only, while the negative subset that contains of the non-interactive drug-NR pairs only, and the symbol represents the union in the set theory. The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al.", "The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al. . The negative dataset contains 172 non-interactive drug-NR pairs, which were derived according to the following procedures: a separating each of the pairs in into single drug and NR; b re-coupling each of the single drugs with each of the single NRs into pairs in a way that none of them occurred in ; c randomly picking the pairs thus formed until reaching the number two times as many as the pairs in . The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample.", "The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample. First, for the drug part in the current benchmark dataset, we can use a 256-D vector to formulate it as given by where D represents the vector for a drug compound, and d i its i-th i = 1,2, ,256 component that can be derived by following the \"2D molecular fingerprint procedure\" as elaborated in . The 53 molecular fingerprint vectors thus obtained for the 53 drugs in are, respectively, given in Supplementary Information S2. The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth.", "The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth. Now the problem is how to effectively represent the sequence of Equation . with a non-sequential or discrete model . This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction.", "This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction. Facing such a dilemma, can we find an approach to partially incorporate the sequence-order effects? Actually, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or a vector, yet still keep considerable sequence order information. To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of .", "To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of . Moreover, the concept of PseAAC was further extended to represent the feature vectors of nucleotides , as well as other biological samples see, e.g., . Because it has been widely and increasingly used, recently two powerful soft-wares, called \"PseAAC-Builder\" and \"propy\" , were established for generating various special Chou's pseudo-amino acid compositions, in addition to the web-server \"PseAAC\" built in 2008. According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation .", "According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation . . Below, let us describe how to extract useful information to define the components of PseAAC for the NR samples concerned. First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned.", "First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned. Thus, such 20 AAC components were used here to define the first 20 elements in Equation . ; i.e., . 1, 2, , 20 ii fi   . where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned.", "where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned. Since AAC did not contain any sequence order information, the following steps were taken to make up this shortcoming. To avoid completely losing the local or short-range sequence order information, we considered the approach of dipeptide composition. It contained 20 × 20 = 400 components . Such 400 components were used to define the next 400 elements in Equation . ; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned.", "; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned. To incorporate the global or long-range sequence order information, let us consider the following approach. According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion.", "According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion. With the accumulation of these changes over a long period of time, many original similarities between initial and resultant amino acid sequences are gradually faded out, but the corresponding proteins may still share many common attributes , such as having basically the same biological function and residing at a same subcellular location . To extract the sequential evolution information and use it to define the components of Equation ., the PSSM Position Specific Scoring Matrix was used as described below. According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned.", "According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned. In order to make every element in Equation . be scaled from their original score ranges into the region of , we performed a conversion through the standard sigmoid function to make it become Now we extract the useful information from Equation . Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j .", "Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j . has the same meaning as in Equation . where   and Combining Equations ., ., . and ., we found that the total number of the components obtained via the current approach for the PseAAC of Equation . and each of the 500 components is given by . Since the elements in Equations . and .", "and each of the 500 components is given by . Since the elements in Equations . and . are well defined, we can now formulate the drug-NR pair by combining the two equations as given by   . where G represents the drug-NR pair, Å the orthogonal sum, and the 256 + 500 = 756 components are defined by Equations . and . . For the sake of convenience, let us use x i i = 1, 2, , 756 to represent the 756 components in Equation . ; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation .", "; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation . to where the symbol means taking the average of the quantity therein, and SD means the corresponding standard derivation. In this study, the SVM support vector machine was used as the operation engine. SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function.", "SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function. For a brief formulation of SVM and how it works, see the papers ; for more details about SVM, see a monograph . In this study, the LIBSVM package was used as an implementation of SVM, which can be downloaded from the popular radial basis function RBF was taken as the kernel function. For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later.", "For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later. The predictor obtained via the aforementioned procedure is called iNR-Drug, where \"i\" means identify, and \"NR-Drug\" means the interaction between nuclear receptor and drug compound. To provide an intuitive overall picture, a flowchart is provided in Figure 2 to show the process of how the predictor works in identifying the interactions between nuclear receptors and drug compounds. To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs.", "To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs. According to Equation . we can easily see the following. When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation.", "When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation. As we can see from the above discussion, it is much more intuitive and easier to understand when using Equation . to examine a predictor for its four metrics, particularly for its Mathew's correlation coefficient. It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in .", "It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in . How to properly test a predictor for its anticipated success rates is very important for its development as well as its potential application value. Generally speaking, the following three cross-validation methods are often used to examine the quality of a predictor and its effectiveness in practical application: independent dataset test, subsampling or K-fold such as five-fold, seven-fold, or 10-fold crossover test and jackknife test . However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either.", "However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either. Only the jackknife test is the least arbitrary that can always yield a unique result for a given benchmark dataset 73, 74, 156, . Therefore, the jackknife test has been widely recognized and increasingly utilized by investigators to examine the quality of various predictors see, e.g., . Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  .", "Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  . To find their optimal values, a 2-D grid search was conducted by the jackknife test on the benchmark dataset . The results thus obtained are shown in Figure 3 , from which it can be seen that the iNR-Drug predictor reaches its optimal status when C = 2 3 and 9 2    . The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al.", "The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al. on the same benchmark dataset is also given although no results were reported by them for Sn, Sp and MCC. It can be observed from the table that the overall accuracy obtained by iNR-Drug is remarkably higher that of He et al. , and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates.", ", and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates. As mentioned above Section 3.2 , the jackknife test is the most objective method for examining the quality of a predictor. However, as a demonstration to show how to practically use the current predictor, we took 41 NR-drug pairs from the study by Yamanishi et al. that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 .", "that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 . It is anticipated that the iNR-Drug predictor developed in this paper may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful predictors , a publicly accessible web-server for iNR-Drug was established. For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1.", "For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1. Open the web server at the site and you will see the top page of the predictor on your computer screen, as shown in Figure 4 . Click on the Read Me button to see a brief introduction about iNR-Drug predictor and the caveat when using it. Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 .", "Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 . Each query pair consists of two parts: one is for the nuclear receptor sequence, and the other for the drug. The NR sequence should be in FASTA format, while the drug in the KEGG code beginning with the symbol #. Examples for the query pairs input and the corresponding output can be seen by clicking on the Example button right above the input box. Step 3. Click on the Submit button to see the predicted result.", "Step 3. Click on the Submit button to see the predicted result. For example, if you use the three query pairs in the Example window as the input, after clicking the Submit button, you will see on your screen that the \"hsa:2099\" NR and the \"D00066\" drug are an interactive pair, and that the \"hsa:2908\" NR and the \"D00088\" drug are also an interactive pair, but that the \"hsa:5468\" NR and the \"D00279\" drug are not an interactive pair. All these results are fully consistent with the experimental observations. It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4.", "It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4. Click on the Citation button to find the relevant paper that documents the detailed development and algorithm of iNR-Durg. Step 5. Click on the Data button to download the benchmark dataset used to train and test the iNR-Durg predictor. Step 6. The program code is also available by clicking the button download on the lower panel of Figure 4 ." ]

[ "Nuclear receptors NRs are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. Therefore, NRs have become a frequent target for drug development. During the process of developing drugs against these diseases by targeting NRs, we are often facing a problem: Given a NR and chemical compound, can we identify whether they are really in interaction with each other in a cell? To address this problem, a predictor called “iNR-Drug” was developed. In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner.", "In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. It is anticipated that the iNR-Drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Text: With the ability to directly bind to DNA Figure 1 and regulate the expression of adjacent genes, nuclear receptors NRs are a class of ligand-inducible transcription factors. They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like .", "They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like . Since they are involved in almost all aspects of human physiology and are implicated in many major diseases such as cancer, diabetes and osteoporosis, nuclear receptors have become major drug targets , along with G protein-coupled receptors GPCRs , ion channels , and kinase proteins . Identification of drug-target interactions is one of the most important steps for the new medicine development . The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition.", "The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition. Although X-ray crystallography is a powerful tool in determining protein 3D structures, it is time-consuming and expensive. Particularly, not all proteins can be successfully crystallized. For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined.", "For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined. Although NMR Nuclear Magnetic Resonance is indeed a very powerful tool in determining the 3D structures of membrane proteins as indicated by a series of recent publications see, e.g., and a review article , it is also time-consuming and costly. To acquire the 3D structural information in a timely manner, one has to resort to various structural bioinformatics tools see, e.g., , particularly the homologous modeling approach as utilized for a series of protein receptors urgently needed during the process of drug development 19, . Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter.", "Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter. The results thus obtained can be used to pre-exclude the compounds identified not in interaction with the nuclear receptors, so as to timely stop wasting time and money on those unpromising compounds . Actually, based on the functional groups and biological features, a powerful method was developed recently for this purpose. However, further development in this regard is definitely needed due to the following reasons. a He et al.", "However, further development in this regard is definitely needed due to the following reasons. a He et al. did not provide a publicly accessible web-server for their method, and hence its practical application value is quite limited, particularly for the broad experimental scientists; b The prediction quality can be further enhanced by incorporating some key features into the formulation of NR-drug nuclear receptor and drug samples via the general form of pseudo amino acid composition . The present study was initiated with an attempt to develop a new method for predicting the interaction of drugs with nuclear receptors by addressing the two points. As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps.", "As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps. The data used in the current study were collected from KEGG Kyoto Encyclopedia of Genes and Genomes at KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. Here, the benchmark dataset can be formulated as where is the positive subset that consists of the interactive drug-NR pairs only, while the negative subset that contains of the non-interactive drug-NR pairs only, and the symbol represents the union in the set theory. The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al.", "The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al. . The negative dataset contains 172 non-interactive drug-NR pairs, which were derived according to the following procedures: a separating each of the pairs in into single drug and NR; b re-coupling each of the single drugs with each of the single NRs into pairs in a way that none of them occurred in ; c randomly picking the pairs thus formed until reaching the number two times as many as the pairs in . The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample.", "The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample. First, for the drug part in the current benchmark dataset, we can use a 256-D vector to formulate it as given by where D represents the vector for a drug compound, and d i its i-th i = 1,2, ,256 component that can be derived by following the \"2D molecular fingerprint procedure\" as elaborated in . The 53 molecular fingerprint vectors thus obtained for the 53 drugs in are, respectively, given in Supplementary Information S2. The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth.", "The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth. Now the problem is how to effectively represent the sequence of Equation . with a non-sequential or discrete model . This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction.", "This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction. Facing such a dilemma, can we find an approach to partially incorporate the sequence-order effects? Actually, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or a vector, yet still keep considerable sequence order information. To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of .", "To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of . Moreover, the concept of PseAAC was further extended to represent the feature vectors of nucleotides , as well as other biological samples see, e.g., . Because it has been widely and increasingly used, recently two powerful soft-wares, called \"PseAAC-Builder\" and \"propy\" , were established for generating various special Chou's pseudo-amino acid compositions, in addition to the web-server \"PseAAC\" built in 2008. According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation .", "According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation . . Below, let us describe how to extract useful information to define the components of PseAAC for the NR samples concerned. First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned.", "First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned. Thus, such 20 AAC components were used here to define the first 20 elements in Equation . ; i.e., . 1, 2, , 20 ii fi   . where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned.", "where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned. Since AAC did not contain any sequence order information, the following steps were taken to make up this shortcoming. To avoid completely losing the local or short-range sequence order information, we considered the approach of dipeptide composition. It contained 20 × 20 = 400 components . Such 400 components were used to define the next 400 elements in Equation . ; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned.", "; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned. To incorporate the global or long-range sequence order information, let us consider the following approach. According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion.", "According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion. With the accumulation of these changes over a long period of time, many original similarities between initial and resultant amino acid sequences are gradually faded out, but the corresponding proteins may still share many common attributes , such as having basically the same biological function and residing at a same subcellular location . To extract the sequential evolution information and use it to define the components of Equation ., the PSSM Position Specific Scoring Matrix was used as described below. According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned.", "According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned. In order to make every element in Equation . be scaled from their original score ranges into the region of , we performed a conversion through the standard sigmoid function to make it become Now we extract the useful information from Equation . Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j .", "Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j . has the same meaning as in Equation . where   and Combining Equations ., ., . and ., we found that the total number of the components obtained via the current approach for the PseAAC of Equation . and each of the 500 components is given by . Since the elements in Equations . and .", "and each of the 500 components is given by . Since the elements in Equations . and . are well defined, we can now formulate the drug-NR pair by combining the two equations as given by   . where G represents the drug-NR pair, Å the orthogonal sum, and the 256 + 500 = 756 components are defined by Equations . and . . For the sake of convenience, let us use x i i = 1, 2, , 756 to represent the 756 components in Equation . ; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation .", "; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation . to where the symbol means taking the average of the quantity therein, and SD means the corresponding standard derivation. In this study, the SVM support vector machine was used as the operation engine. SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function.", "SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function. For a brief formulation of SVM and how it works, see the papers ; for more details about SVM, see a monograph . In this study, the LIBSVM package was used as an implementation of SVM, which can be downloaded from the popular radial basis function RBF was taken as the kernel function. For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later.", "For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later. The predictor obtained via the aforementioned procedure is called iNR-Drug, where \"i\" means identify, and \"NR-Drug\" means the interaction between nuclear receptor and drug compound. To provide an intuitive overall picture, a flowchart is provided in Figure 2 to show the process of how the predictor works in identifying the interactions between nuclear receptors and drug compounds. To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs.", "To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs. According to Equation . we can easily see the following. When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation.", "When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation. As we can see from the above discussion, it is much more intuitive and easier to understand when using Equation . to examine a predictor for its four metrics, particularly for its Mathew's correlation coefficient. It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in .", "It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in . How to properly test a predictor for its anticipated success rates is very important for its development as well as its potential application value. Generally speaking, the following three cross-validation methods are often used to examine the quality of a predictor and its effectiveness in practical application: independent dataset test, subsampling or K-fold such as five-fold, seven-fold, or 10-fold crossover test and jackknife test . However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either.", "However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either. Only the jackknife test is the least arbitrary that can always yield a unique result for a given benchmark dataset 73, 74, 156, . Therefore, the jackknife test has been widely recognized and increasingly utilized by investigators to examine the quality of various predictors see, e.g., . Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  .", "Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  . To find their optimal values, a 2-D grid search was conducted by the jackknife test on the benchmark dataset . The results thus obtained are shown in Figure 3 , from which it can be seen that the iNR-Drug predictor reaches its optimal status when C = 2 3 and 9 2    . The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al.", "The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al. on the same benchmark dataset is also given although no results were reported by them for Sn, Sp and MCC. It can be observed from the table that the overall accuracy obtained by iNR-Drug is remarkably higher that of He et al. , and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates.", ", and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates. As mentioned above Section 3.2 , the jackknife test is the most objective method for examining the quality of a predictor. However, as a demonstration to show how to practically use the current predictor, we took 41 NR-drug pairs from the study by Yamanishi et al. that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 .", "that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 . It is anticipated that the iNR-Drug predictor developed in this paper may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful predictors , a publicly accessible web-server for iNR-Drug was established. For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1.", "For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1. Open the web server at the site and you will see the top page of the predictor on your computer screen, as shown in Figure 4 . Click on the Read Me button to see a brief introduction about iNR-Drug predictor and the caveat when using it. Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 .", "Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 . Each query pair consists of two parts: one is for the nuclear receptor sequence, and the other for the drug. The NR sequence should be in FASTA format, while the drug in the KEGG code beginning with the symbol #. Examples for the query pairs input and the corresponding output can be seen by clicking on the Example button right above the input box. Step 3. Click on the Submit button to see the predicted result.", "Step 3. Click on the Submit button to see the predicted result. For example, if you use the three query pairs in the Example window as the input, after clicking the Submit button, you will see on your screen that the \"hsa:2099\" NR and the \"D00066\" drug are an interactive pair, and that the \"hsa:2908\" NR and the \"D00088\" drug are also an interactive pair, but that the \"hsa:5468\" NR and the \"D00279\" drug are not an interactive pair. All these results are fully consistent with the experimental observations. It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4.", "It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4. Click on the Citation button to find the relevant paper that documents the detailed development and algorithm of iNR-Durg. Step 5. Click on the Data button to download the benchmark dataset used to train and test the iNR-Durg predictor. Step 6. The program code is also available by clicking the button download on the lower panel of Figure 4 ." ]

[ "Nuclear receptors NRs are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. Therefore, NRs have become a frequent target for drug development. During the process of developing drugs against these diseases by targeting NRs, we are often facing a problem: Given a NR and chemical compound, can we identify whether they are really in interaction with each other in a cell? To address this problem, a predictor called “iNR-Drug” was developed. In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner.", "In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. It is anticipated that the iNR-Drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Text: With the ability to directly bind to DNA Figure 1 and regulate the expression of adjacent genes, nuclear receptors NRs are a class of ligand-inducible transcription factors. They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like .", "They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like . Since they are involved in almost all aspects of human physiology and are implicated in many major diseases such as cancer, diabetes and osteoporosis, nuclear receptors have become major drug targets , along with G protein-coupled receptors GPCRs , ion channels , and kinase proteins . Identification of drug-target interactions is one of the most important steps for the new medicine development . The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition.", "The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition. Although X-ray crystallography is a powerful tool in determining protein 3D structures, it is time-consuming and expensive. Particularly, not all proteins can be successfully crystallized. For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined.", "For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined. Although NMR Nuclear Magnetic Resonance is indeed a very powerful tool in determining the 3D structures of membrane proteins as indicated by a series of recent publications see, e.g., and a review article , it is also time-consuming and costly. To acquire the 3D structural information in a timely manner, one has to resort to various structural bioinformatics tools see, e.g., , particularly the homologous modeling approach as utilized for a series of protein receptors urgently needed during the process of drug development 19, . Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter.", "Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter. The results thus obtained can be used to pre-exclude the compounds identified not in interaction with the nuclear receptors, so as to timely stop wasting time and money on those unpromising compounds . Actually, based on the functional groups and biological features, a powerful method was developed recently for this purpose. However, further development in this regard is definitely needed due to the following reasons. a He et al.", "However, further development in this regard is definitely needed due to the following reasons. a He et al. did not provide a publicly accessible web-server for their method, and hence its practical application value is quite limited, particularly for the broad experimental scientists; b The prediction quality can be further enhanced by incorporating some key features into the formulation of NR-drug nuclear receptor and drug samples via the general form of pseudo amino acid composition . The present study was initiated with an attempt to develop a new method for predicting the interaction of drugs with nuclear receptors by addressing the two points. As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps.", "As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps. The data used in the current study were collected from KEGG Kyoto Encyclopedia of Genes and Genomes at KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. Here, the benchmark dataset can be formulated as where is the positive subset that consists of the interactive drug-NR pairs only, while the negative subset that contains of the non-interactive drug-NR pairs only, and the symbol represents the union in the set theory. The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al.", "The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al. . The negative dataset contains 172 non-interactive drug-NR pairs, which were derived according to the following procedures: a separating each of the pairs in into single drug and NR; b re-coupling each of the single drugs with each of the single NRs into pairs in a way that none of them occurred in ; c randomly picking the pairs thus formed until reaching the number two times as many as the pairs in . The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample.", "The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample. First, for the drug part in the current benchmark dataset, we can use a 256-D vector to formulate it as given by where D represents the vector for a drug compound, and d i its i-th i = 1,2, ,256 component that can be derived by following the \"2D molecular fingerprint procedure\" as elaborated in . The 53 molecular fingerprint vectors thus obtained for the 53 drugs in are, respectively, given in Supplementary Information S2. The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth.", "The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth. Now the problem is how to effectively represent the sequence of Equation . with a non-sequential or discrete model . This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction.", "This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction. Facing such a dilemma, can we find an approach to partially incorporate the sequence-order effects? Actually, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or a vector, yet still keep considerable sequence order information. To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of .", "To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of . Moreover, the concept of PseAAC was further extended to represent the feature vectors of nucleotides , as well as other biological samples see, e.g., . Because it has been widely and increasingly used, recently two powerful soft-wares, called \"PseAAC-Builder\" and \"propy\" , were established for generating various special Chou's pseudo-amino acid compositions, in addition to the web-server \"PseAAC\" built in 2008. According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation .", "According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation . . Below, let us describe how to extract useful information to define the components of PseAAC for the NR samples concerned. First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned.", "First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned. Thus, such 20 AAC components were used here to define the first 20 elements in Equation . ; i.e., . 1, 2, , 20 ii fi   . where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned.", "where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned. Since AAC did not contain any sequence order information, the following steps were taken to make up this shortcoming. To avoid completely losing the local or short-range sequence order information, we considered the approach of dipeptide composition. It contained 20 × 20 = 400 components . Such 400 components were used to define the next 400 elements in Equation . ; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned.", "; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned. To incorporate the global or long-range sequence order information, let us consider the following approach. According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion.", "According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion. With the accumulation of these changes over a long period of time, many original similarities between initial and resultant amino acid sequences are gradually faded out, but the corresponding proteins may still share many common attributes , such as having basically the same biological function and residing at a same subcellular location . To extract the sequential evolution information and use it to define the components of Equation ., the PSSM Position Specific Scoring Matrix was used as described below. According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned.", "According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned. In order to make every element in Equation . be scaled from their original score ranges into the region of , we performed a conversion through the standard sigmoid function to make it become Now we extract the useful information from Equation . Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j .", "Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j . has the same meaning as in Equation . where   and Combining Equations ., ., . and ., we found that the total number of the components obtained via the current approach for the PseAAC of Equation . and each of the 500 components is given by . Since the elements in Equations . and .", "and each of the 500 components is given by . Since the elements in Equations . and . are well defined, we can now formulate the drug-NR pair by combining the two equations as given by   . where G represents the drug-NR pair, Å the orthogonal sum, and the 256 + 500 = 756 components are defined by Equations . and . . For the sake of convenience, let us use x i i = 1, 2, , 756 to represent the 756 components in Equation . ; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation .", "; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation . to where the symbol means taking the average of the quantity therein, and SD means the corresponding standard derivation. In this study, the SVM support vector machine was used as the operation engine. SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function.", "SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function. For a brief formulation of SVM and how it works, see the papers ; for more details about SVM, see a monograph . In this study, the LIBSVM package was used as an implementation of SVM, which can be downloaded from the popular radial basis function RBF was taken as the kernel function. For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later.", "For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later. The predictor obtained via the aforementioned procedure is called iNR-Drug, where \"i\" means identify, and \"NR-Drug\" means the interaction between nuclear receptor and drug compound. To provide an intuitive overall picture, a flowchart is provided in Figure 2 to show the process of how the predictor works in identifying the interactions between nuclear receptors and drug compounds. To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs.", "To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs. According to Equation . we can easily see the following. When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation.", "When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation. As we can see from the above discussion, it is much more intuitive and easier to understand when using Equation . to examine a predictor for its four metrics, particularly for its Mathew's correlation coefficient. It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in .", "It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in . How to properly test a predictor for its anticipated success rates is very important for its development as well as its potential application value. Generally speaking, the following three cross-validation methods are often used to examine the quality of a predictor and its effectiveness in practical application: independent dataset test, subsampling or K-fold such as five-fold, seven-fold, or 10-fold crossover test and jackknife test . However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either.", "However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either. Only the jackknife test is the least arbitrary that can always yield a unique result for a given benchmark dataset 73, 74, 156, . Therefore, the jackknife test has been widely recognized and increasingly utilized by investigators to examine the quality of various predictors see, e.g., . Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  .", "Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  . To find their optimal values, a 2-D grid search was conducted by the jackknife test on the benchmark dataset . The results thus obtained are shown in Figure 3 , from which it can be seen that the iNR-Drug predictor reaches its optimal status when C = 2 3 and 9 2    . The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al.", "The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al. on the same benchmark dataset is also given although no results were reported by them for Sn, Sp and MCC. It can be observed from the table that the overall accuracy obtained by iNR-Drug is remarkably higher that of He et al. , and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates.", ", and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates. As mentioned above Section 3.2 , the jackknife test is the most objective method for examining the quality of a predictor. However, as a demonstration to show how to practically use the current predictor, we took 41 NR-drug pairs from the study by Yamanishi et al. that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 .", "that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 . It is anticipated that the iNR-Drug predictor developed in this paper may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful predictors , a publicly accessible web-server for iNR-Drug was established. For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1.", "For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1. Open the web server at the site and you will see the top page of the predictor on your computer screen, as shown in Figure 4 . Click on the Read Me button to see a brief introduction about iNR-Drug predictor and the caveat when using it. Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 .", "Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 . Each query pair consists of two parts: one is for the nuclear receptor sequence, and the other for the drug. The NR sequence should be in FASTA format, while the drug in the KEGG code beginning with the symbol #. Examples for the query pairs input and the corresponding output can be seen by clicking on the Example button right above the input box. Step 3. Click on the Submit button to see the predicted result.", "Step 3. Click on the Submit button to see the predicted result. For example, if you use the three query pairs in the Example window as the input, after clicking the Submit button, you will see on your screen that the \"hsa:2099\" NR and the \"D00066\" drug are an interactive pair, and that the \"hsa:2908\" NR and the \"D00088\" drug are also an interactive pair, but that the \"hsa:5468\" NR and the \"D00279\" drug are not an interactive pair. All these results are fully consistent with the experimental observations. It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4.", "It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4. Click on the Citation button to find the relevant paper that documents the detailed development and algorithm of iNR-Durg. Step 5. Click on the Data button to download the benchmark dataset used to train and test the iNR-Durg predictor. Step 6. The program code is also available by clicking the button download on the lower panel of Figure 4 ." ]

[ "Nuclear receptors NRs are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. Therefore, NRs have become a frequent target for drug development. During the process of developing drugs against these diseases by targeting NRs, we are often facing a problem: Given a NR and chemical compound, can we identify whether they are really in interaction with each other in a cell? To address this problem, a predictor called “iNR-Drug” was developed. In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner.", "In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. It is anticipated that the iNR-Drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Text: With the ability to directly bind to DNA Figure 1 and regulate the expression of adjacent genes, nuclear receptors NRs are a class of ligand-inducible transcription factors. They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like .", "They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like . Since they are involved in almost all aspects of human physiology and are implicated in many major diseases such as cancer, diabetes and osteoporosis, nuclear receptors have become major drug targets , along with G protein-coupled receptors GPCRs , ion channels , and kinase proteins . Identification of drug-target interactions is one of the most important steps for the new medicine development . The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition.", "The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition. Although X-ray crystallography is a powerful tool in determining protein 3D structures, it is time-consuming and expensive. Particularly, not all proteins can be successfully crystallized. For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined.", "For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined. Although NMR Nuclear Magnetic Resonance is indeed a very powerful tool in determining the 3D structures of membrane proteins as indicated by a series of recent publications see, e.g., and a review article , it is also time-consuming and costly. To acquire the 3D structural information in a timely manner, one has to resort to various structural bioinformatics tools see, e.g., , particularly the homologous modeling approach as utilized for a series of protein receptors urgently needed during the process of drug development 19, . Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter.", "Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter. The results thus obtained can be used to pre-exclude the compounds identified not in interaction with the nuclear receptors, so as to timely stop wasting time and money on those unpromising compounds . Actually, based on the functional groups and biological features, a powerful method was developed recently for this purpose. However, further development in this regard is definitely needed due to the following reasons. a He et al.", "However, further development in this regard is definitely needed due to the following reasons. a He et al. did not provide a publicly accessible web-server for their method, and hence its practical application value is quite limited, particularly for the broad experimental scientists; b The prediction quality can be further enhanced by incorporating some key features into the formulation of NR-drug nuclear receptor and drug samples via the general form of pseudo amino acid composition . The present study was initiated with an attempt to develop a new method for predicting the interaction of drugs with nuclear receptors by addressing the two points. As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps.", "As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps. The data used in the current study were collected from KEGG Kyoto Encyclopedia of Genes and Genomes at KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. Here, the benchmark dataset can be formulated as where is the positive subset that consists of the interactive drug-NR pairs only, while the negative subset that contains of the non-interactive drug-NR pairs only, and the symbol represents the union in the set theory. The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al.", "The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al. . The negative dataset contains 172 non-interactive drug-NR pairs, which were derived according to the following procedures: a separating each of the pairs in into single drug and NR; b re-coupling each of the single drugs with each of the single NRs into pairs in a way that none of them occurred in ; c randomly picking the pairs thus formed until reaching the number two times as many as the pairs in . The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample.", "The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample. First, for the drug part in the current benchmark dataset, we can use a 256-D vector to formulate it as given by where D represents the vector for a drug compound, and d i its i-th i = 1,2, ,256 component that can be derived by following the \"2D molecular fingerprint procedure\" as elaborated in . The 53 molecular fingerprint vectors thus obtained for the 53 drugs in are, respectively, given in Supplementary Information S2. The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth.", "The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth. Now the problem is how to effectively represent the sequence of Equation . with a non-sequential or discrete model . This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction.", "This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction. Facing such a dilemma, can we find an approach to partially incorporate the sequence-order effects? Actually, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or a vector, yet still keep considerable sequence order information. To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of .", "To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of . Moreover, the concept of PseAAC was further extended to represent the feature vectors of nucleotides , as well as other biological samples see, e.g., . Because it has been widely and increasingly used, recently two powerful soft-wares, called \"PseAAC-Builder\" and \"propy\" , were established for generating various special Chou's pseudo-amino acid compositions, in addition to the web-server \"PseAAC\" built in 2008. According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation .", "According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation . . Below, let us describe how to extract useful information to define the components of PseAAC for the NR samples concerned. First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned.", "First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned. Thus, such 20 AAC components were used here to define the first 20 elements in Equation . ; i.e., . 1, 2, , 20 ii fi   . where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned.", "where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned. Since AAC did not contain any sequence order information, the following steps were taken to make up this shortcoming. To avoid completely losing the local or short-range sequence order information, we considered the approach of dipeptide composition. It contained 20 × 20 = 400 components . Such 400 components were used to define the next 400 elements in Equation . ; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned.", "; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned. To incorporate the global or long-range sequence order information, let us consider the following approach. According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion.", "According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion. With the accumulation of these changes over a long period of time, many original similarities between initial and resultant amino acid sequences are gradually faded out, but the corresponding proteins may still share many common attributes , such as having basically the same biological function and residing at a same subcellular location . To extract the sequential evolution information and use it to define the components of Equation ., the PSSM Position Specific Scoring Matrix was used as described below. According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned.", "According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned. In order to make every element in Equation . be scaled from their original score ranges into the region of , we performed a conversion through the standard sigmoid function to make it become Now we extract the useful information from Equation . Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j .", "Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j . has the same meaning as in Equation . where   and Combining Equations ., ., . and ., we found that the total number of the components obtained via the current approach for the PseAAC of Equation . and each of the 500 components is given by . Since the elements in Equations . and .", "and each of the 500 components is given by . Since the elements in Equations . and . are well defined, we can now formulate the drug-NR pair by combining the two equations as given by   . where G represents the drug-NR pair, Å the orthogonal sum, and the 256 + 500 = 756 components are defined by Equations . and . . For the sake of convenience, let us use x i i = 1, 2, , 756 to represent the 756 components in Equation . ; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation .", "; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation . to where the symbol means taking the average of the quantity therein, and SD means the corresponding standard derivation. In this study, the SVM support vector machine was used as the operation engine. SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function.", "SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function. For a brief formulation of SVM and how it works, see the papers ; for more details about SVM, see a monograph . In this study, the LIBSVM package was used as an implementation of SVM, which can be downloaded from the popular radial basis function RBF was taken as the kernel function. For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later.", "For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later. The predictor obtained via the aforementioned procedure is called iNR-Drug, where \"i\" means identify, and \"NR-Drug\" means the interaction between nuclear receptor and drug compound. To provide an intuitive overall picture, a flowchart is provided in Figure 2 to show the process of how the predictor works in identifying the interactions between nuclear receptors and drug compounds. To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs.", "To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs. According to Equation . we can easily see the following. When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation.", "When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation. As we can see from the above discussion, it is much more intuitive and easier to understand when using Equation . to examine a predictor for its four metrics, particularly for its Mathew's correlation coefficient. It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in .", "It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in . How to properly test a predictor for its anticipated success rates is very important for its development as well as its potential application value. Generally speaking, the following three cross-validation methods are often used to examine the quality of a predictor and its effectiveness in practical application: independent dataset test, subsampling or K-fold such as five-fold, seven-fold, or 10-fold crossover test and jackknife test . However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either.", "However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either. Only the jackknife test is the least arbitrary that can always yield a unique result for a given benchmark dataset 73, 74, 156, . Therefore, the jackknife test has been widely recognized and increasingly utilized by investigators to examine the quality of various predictors see, e.g., . Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  .", "Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  . To find their optimal values, a 2-D grid search was conducted by the jackknife test on the benchmark dataset . The results thus obtained are shown in Figure 3 , from which it can be seen that the iNR-Drug predictor reaches its optimal status when C = 2 3 and 9 2    . The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al.", "The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al. on the same benchmark dataset is also given although no results were reported by them for Sn, Sp and MCC. It can be observed from the table that the overall accuracy obtained by iNR-Drug is remarkably higher that of He et al. , and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates.", ", and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates. As mentioned above Section 3.2 , the jackknife test is the most objective method for examining the quality of a predictor. However, as a demonstration to show how to practically use the current predictor, we took 41 NR-drug pairs from the study by Yamanishi et al. that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 .", "that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 . It is anticipated that the iNR-Drug predictor developed in this paper may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful predictors , a publicly accessible web-server for iNR-Drug was established. For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1.", "For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1. Open the web server at the site and you will see the top page of the predictor on your computer screen, as shown in Figure 4 . Click on the Read Me button to see a brief introduction about iNR-Drug predictor and the caveat when using it. Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 .", "Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 . Each query pair consists of two parts: one is for the nuclear receptor sequence, and the other for the drug. The NR sequence should be in FASTA format, while the drug in the KEGG code beginning with the symbol #. Examples for the query pairs input and the corresponding output can be seen by clicking on the Example button right above the input box. Step 3. Click on the Submit button to see the predicted result.", "Step 3. Click on the Submit button to see the predicted result. For example, if you use the three query pairs in the Example window as the input, after clicking the Submit button, you will see on your screen that the \"hsa:2099\" NR and the \"D00066\" drug are an interactive pair, and that the \"hsa:2908\" NR and the \"D00088\" drug are also an interactive pair, but that the \"hsa:5468\" NR and the \"D00279\" drug are not an interactive pair. All these results are fully consistent with the experimental observations. It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4.", "It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4. Click on the Citation button to find the relevant paper that documents the detailed development and algorithm of iNR-Durg. Step 5. Click on the Data button to download the benchmark dataset used to train and test the iNR-Durg predictor. Step 6. The program code is also available by clicking the button download on the lower panel of Figure 4 ." ]

[ "Nuclear receptors NRs are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. Therefore, NRs have become a frequent target for drug development. During the process of developing drugs against these diseases by targeting NRs, we are often facing a problem: Given a NR and chemical compound, can we identify whether they are really in interaction with each other in a cell? To address this problem, a predictor called “iNR-Drug” was developed. In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner.", "In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. It is anticipated that the iNR-Drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Text: With the ability to directly bind to DNA Figure 1 and regulate the expression of adjacent genes, nuclear receptors NRs are a class of ligand-inducible transcription factors. They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like .", "They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like . Since they are involved in almost all aspects of human physiology and are implicated in many major diseases such as cancer, diabetes and osteoporosis, nuclear receptors have become major drug targets , along with G protein-coupled receptors GPCRs , ion channels , and kinase proteins . Identification of drug-target interactions is one of the most important steps for the new medicine development . The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition.", "The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition. Although X-ray crystallography is a powerful tool in determining protein 3D structures, it is time-consuming and expensive. Particularly, not all proteins can be successfully crystallized. For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined.", "For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined. Although NMR Nuclear Magnetic Resonance is indeed a very powerful tool in determining the 3D structures of membrane proteins as indicated by a series of recent publications see, e.g., and a review article , it is also time-consuming and costly. To acquire the 3D structural information in a timely manner, one has to resort to various structural bioinformatics tools see, e.g., , particularly the homologous modeling approach as utilized for a series of protein receptors urgently needed during the process of drug development 19, . Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter.", "Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter. The results thus obtained can be used to pre-exclude the compounds identified not in interaction with the nuclear receptors, so as to timely stop wasting time and money on those unpromising compounds . Actually, based on the functional groups and biological features, a powerful method was developed recently for this purpose. However, further development in this regard is definitely needed due to the following reasons. a He et al.", "However, further development in this regard is definitely needed due to the following reasons. a He et al. did not provide a publicly accessible web-server for their method, and hence its practical application value is quite limited, particularly for the broad experimental scientists; b The prediction quality can be further enhanced by incorporating some key features into the formulation of NR-drug nuclear receptor and drug samples via the general form of pseudo amino acid composition . The present study was initiated with an attempt to develop a new method for predicting the interaction of drugs with nuclear receptors by addressing the two points. As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps.", "As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps. The data used in the current study were collected from KEGG Kyoto Encyclopedia of Genes and Genomes at KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. Here, the benchmark dataset can be formulated as where is the positive subset that consists of the interactive drug-NR pairs only, while the negative subset that contains of the non-interactive drug-NR pairs only, and the symbol represents the union in the set theory. The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al.", "The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al. . The negative dataset contains 172 non-interactive drug-NR pairs, which were derived according to the following procedures: a separating each of the pairs in into single drug and NR; b re-coupling each of the single drugs with each of the single NRs into pairs in a way that none of them occurred in ; c randomly picking the pairs thus formed until reaching the number two times as many as the pairs in . The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample.", "The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample. First, for the drug part in the current benchmark dataset, we can use a 256-D vector to formulate it as given by where D represents the vector for a drug compound, and d i its i-th i = 1,2, ,256 component that can be derived by following the \"2D molecular fingerprint procedure\" as elaborated in . The 53 molecular fingerprint vectors thus obtained for the 53 drugs in are, respectively, given in Supplementary Information S2. The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth.", "The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth. Now the problem is how to effectively represent the sequence of Equation . with a non-sequential or discrete model . This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction.", "This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction. Facing such a dilemma, can we find an approach to partially incorporate the sequence-order effects? Actually, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or a vector, yet still keep considerable sequence order information. To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of .", "To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of . Moreover, the concept of PseAAC was further extended to represent the feature vectors of nucleotides , as well as other biological samples see, e.g., . Because it has been widely and increasingly used, recently two powerful soft-wares, called \"PseAAC-Builder\" and \"propy\" , were established for generating various special Chou's pseudo-amino acid compositions, in addition to the web-server \"PseAAC\" built in 2008. According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation .", "According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation . . Below, let us describe how to extract useful information to define the components of PseAAC for the NR samples concerned. First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned.", "First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned. Thus, such 20 AAC components were used here to define the first 20 elements in Equation . ; i.e., . 1, 2, , 20 ii fi   . where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned.", "where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned. Since AAC did not contain any sequence order information, the following steps were taken to make up this shortcoming. To avoid completely losing the local or short-range sequence order information, we considered the approach of dipeptide composition. It contained 20 × 20 = 400 components . Such 400 components were used to define the next 400 elements in Equation . ; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned.", "; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned. To incorporate the global or long-range sequence order information, let us consider the following approach. According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion.", "According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion. With the accumulation of these changes over a long period of time, many original similarities between initial and resultant amino acid sequences are gradually faded out, but the corresponding proteins may still share many common attributes , such as having basically the same biological function and residing at a same subcellular location . To extract the sequential evolution information and use it to define the components of Equation ., the PSSM Position Specific Scoring Matrix was used as described below. According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned.", "According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned. In order to make every element in Equation . be scaled from their original score ranges into the region of , we performed a conversion through the standard sigmoid function to make it become Now we extract the useful information from Equation . Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j .", "Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j . has the same meaning as in Equation . where   and Combining Equations ., ., . and ., we found that the total number of the components obtained via the current approach for the PseAAC of Equation . and each of the 500 components is given by . Since the elements in Equations . and .", "and each of the 500 components is given by . Since the elements in Equations . and . are well defined, we can now formulate the drug-NR pair by combining the two equations as given by   . where G represents the drug-NR pair, Å the orthogonal sum, and the 256 + 500 = 756 components are defined by Equations . and . . For the sake of convenience, let us use x i i = 1, 2, , 756 to represent the 756 components in Equation . ; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation .", "; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation . to where the symbol means taking the average of the quantity therein, and SD means the corresponding standard derivation. In this study, the SVM support vector machine was used as the operation engine. SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function.", "SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function. For a brief formulation of SVM and how it works, see the papers ; for more details about SVM, see a monograph . In this study, the LIBSVM package was used as an implementation of SVM, which can be downloaded from the popular radial basis function RBF was taken as the kernel function. For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later.", "For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later. The predictor obtained via the aforementioned procedure is called iNR-Drug, where \"i\" means identify, and \"NR-Drug\" means the interaction between nuclear receptor and drug compound. To provide an intuitive overall picture, a flowchart is provided in Figure 2 to show the process of how the predictor works in identifying the interactions between nuclear receptors and drug compounds. To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs.", "To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs. According to Equation . we can easily see the following. When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation.", "When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation. As we can see from the above discussion, it is much more intuitive and easier to understand when using Equation . to examine a predictor for its four metrics, particularly for its Mathew's correlation coefficient. It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in .", "It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in . How to properly test a predictor for its anticipated success rates is very important for its development as well as its potential application value. Generally speaking, the following three cross-validation methods are often used to examine the quality of a predictor and its effectiveness in practical application: independent dataset test, subsampling or K-fold such as five-fold, seven-fold, or 10-fold crossover test and jackknife test . However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either.", "However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either. Only the jackknife test is the least arbitrary that can always yield a unique result for a given benchmark dataset 73, 74, 156, . Therefore, the jackknife test has been widely recognized and increasingly utilized by investigators to examine the quality of various predictors see, e.g., . Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  .", "Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  . To find their optimal values, a 2-D grid search was conducted by the jackknife test on the benchmark dataset . The results thus obtained are shown in Figure 3 , from which it can be seen that the iNR-Drug predictor reaches its optimal status when C = 2 3 and 9 2    . The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al.", "The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al. on the same benchmark dataset is also given although no results were reported by them for Sn, Sp and MCC. It can be observed from the table that the overall accuracy obtained by iNR-Drug is remarkably higher that of He et al. , and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates.", ", and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates. As mentioned above Section 3.2 , the jackknife test is the most objective method for examining the quality of a predictor. However, as a demonstration to show how to practically use the current predictor, we took 41 NR-drug pairs from the study by Yamanishi et al. that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 .", "that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 . It is anticipated that the iNR-Drug predictor developed in this paper may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful predictors , a publicly accessible web-server for iNR-Drug was established. For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1.", "For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1. Open the web server at the site and you will see the top page of the predictor on your computer screen, as shown in Figure 4 . Click on the Read Me button to see a brief introduction about iNR-Drug predictor and the caveat when using it. Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 .", "Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 . Each query pair consists of two parts: one is for the nuclear receptor sequence, and the other for the drug. The NR sequence should be in FASTA format, while the drug in the KEGG code beginning with the symbol #. Examples for the query pairs input and the corresponding output can be seen by clicking on the Example button right above the input box. Step 3. Click on the Submit button to see the predicted result.", "Step 3. Click on the Submit button to see the predicted result. For example, if you use the three query pairs in the Example window as the input, after clicking the Submit button, you will see on your screen that the \"hsa:2099\" NR and the \"D00066\" drug are an interactive pair, and that the \"hsa:2908\" NR and the \"D00088\" drug are also an interactive pair, but that the \"hsa:5468\" NR and the \"D00279\" drug are not an interactive pair. All these results are fully consistent with the experimental observations. It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4.", "It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4. Click on the Citation button to find the relevant paper that documents the detailed development and algorithm of iNR-Durg. Step 5. Click on the Data button to download the benchmark dataset used to train and test the iNR-Durg predictor. Step 6. The program code is also available by clicking the button download on the lower panel of Figure 4 ." ]

[ "Nuclear receptors NRs are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. Therefore, NRs have become a frequent target for drug development. During the process of developing drugs against these diseases by targeting NRs, we are often facing a problem: Given a NR and chemical compound, can we identify whether they are really in interaction with each other in a cell? To address this problem, a predictor called “iNR-Drug” was developed. In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner.", "In the predictor, the drug compound concerned was formulated by a 256-D dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. It is anticipated that the iNR-Drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Text: With the ability to directly bind to DNA Figure 1 and regulate the expression of adjacent genes, nuclear receptors NRs are a class of ligand-inducible transcription factors. They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like .", "They regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology . The NR superfamily has been classified into seven families: NR0 knirps or DAX like ; NR1 thyroid hormone like , NR2 HNF4-like , NR3 estrogen like , NR4 nerve growth factor IB-like , NR5 fushi tarazu-F1 like , and NR6 germ cell nuclear factor like . Since they are involved in almost all aspects of human physiology and are implicated in many major diseases such as cancer, diabetes and osteoporosis, nuclear receptors have become major drug targets , along with G protein-coupled receptors GPCRs , ion channels , and kinase proteins . Identification of drug-target interactions is one of the most important steps for the new medicine development . The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition.", "The method usually adopted in this step is molecular docking simulation . However, to make molecular docking study feasible, a reliable 3D three dimensional structure of the target protein is the prerequisite condition. Although X-ray crystallography is a powerful tool in determining protein 3D structures, it is time-consuming and expensive. Particularly, not all proteins can be successfully crystallized. For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined.", "For example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein 3D structures have been determined. Although NMR Nuclear Magnetic Resonance is indeed a very powerful tool in determining the 3D structures of membrane proteins as indicated by a series of recent publications see, e.g., and a review article , it is also time-consuming and costly. To acquire the 3D structural information in a timely manner, one has to resort to various structural bioinformatics tools see, e.g., , particularly the homologous modeling approach as utilized for a series of protein receptors urgently needed during the process of drug development 19, . Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter.", "Unfortunately, the number of dependable templates for developing high quality 3D structures by means of homology modeling is very limited . To overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter. The results thus obtained can be used to pre-exclude the compounds identified not in interaction with the nuclear receptors, so as to timely stop wasting time and money on those unpromising compounds . Actually, based on the functional groups and biological features, a powerful method was developed recently for this purpose. However, further development in this regard is definitely needed due to the following reasons. a He et al.", "However, further development in this regard is definitely needed due to the following reasons. a He et al. did not provide a publicly accessible web-server for their method, and hence its practical application value is quite limited, particularly for the broad experimental scientists; b The prediction quality can be further enhanced by incorporating some key features into the formulation of NR-drug nuclear receptor and drug samples via the general form of pseudo amino acid composition . The present study was initiated with an attempt to develop a new method for predicting the interaction of drugs with nuclear receptors by addressing the two points. As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps.", "As demonstrated by a series of recent publications 10, 18, and summarized in a comprehensive review , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: a select or construct a valid benchmark dataset to train and test the predictor; b represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; c introduce or develop a powerful algorithm or engine to operate the prediction; d properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; e establish a user-friendly web-server for the predictor that is accessible to the public. Below, let us elaborate how to deal with these steps. The data used in the current study were collected from KEGG Kyoto Encyclopedia of Genes and Genomes at KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. Here, the benchmark dataset can be formulated as where is the positive subset that consists of the interactive drug-NR pairs only, while the negative subset that contains of the non-interactive drug-NR pairs only, and the symbol represents the union in the set theory. The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al.", "The so-called \"interactive\" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the KEGG database ; while the \"non-interactive\" pair means that its two counterparts are not interacting with each other in the drug-target networks. The positive dataset contains 86 drug-NR pairs, which were taken from He et al. . The negative dataset contains 172 non-interactive drug-NR pairs, which were derived according to the following procedures: a separating each of the pairs in into single drug and NR; b re-coupling each of the single drugs with each of the single NRs into pairs in a way that none of them occurred in ; c randomly picking the pairs thus formed until reaching the number two times as many as the pairs in . The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample.", "The 86 interactive drug-NR pairs and 172 non-interactive drug-NR pairs are given in Supplementary Information S1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different NRs and 53 different compounds. Since each of the samples in the current network system contains a drug compound and a NR protein , the following procedures were taken to represent the drug-NR pair sample. First, for the drug part in the current benchmark dataset, we can use a 256-D vector to formulate it as given by where D represents the vector for a drug compound, and d i its i-th i = 1,2, ,256 component that can be derived by following the \"2D molecular fingerprint procedure\" as elaborated in . The 53 molecular fingerprint vectors thus obtained for the 53 drugs in are, respectively, given in Supplementary Information S2. The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth.", "The protein sequences of the 25 different NRs in are listed in Supplementary Information S3. Suppose the sequence of a nuclear receptor protein P with L residues is generally expressed by where 1 R represents the 1st residue of the protein sequence P , 2 R the 2nd residue, and so forth. Now the problem is how to effectively represent the sequence of Equation . with a non-sequential or discrete model . This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction.", "This is because all the existing operation engines, such as covariance discriminant CD 17, 65, , neural network , support vector machine SVM 83 , random forest , conditional random field , nearest neighbor NN ; K-nearest neighbor KNN , OET-KNN , and Fuzzy K-nearest neighbor , can only handle vector but not sequence samples. However, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction. Facing such a dilemma, can we find an approach to partially incorporate the sequence-order effects? Actually, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or a vector, yet still keep considerable sequence order information. To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of .", "To avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition or Chou's PseAAC was proposed. Ever since the concept of PseAAC was proposed in 2001 , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides , predicting protein subcellular location , predicting membrane protein types , predicting protein submitochondria locations , predicting GABA A receptor proteins , predicting enzyme subfamily classes , predicting antibacterial peptides , predicting supersecondary structure , predicting bacterial virulent proteins , predicting protein structural class , predicting the cofactors of oxidoreductases , predicting metalloproteinase family , identifying cysteine S-nitrosylation sites in proteins , identifying bacterial secreted proteins , identifying antibacterial peptides , identifying allergenic proteins , identifying protein quaternary structural attributes , identifying risk type of human papillomaviruses , identifying cyclin proteins , identifying GPCRs and their types , discriminating outer membrane proteins , classifying amino acids , detecting remote homologous proteins , among many others see a long list of papers cited in the References section of . Moreover, the concept of PseAAC was further extended to represent the feature vectors of nucleotides , as well as other biological samples see, e.g., . Because it has been widely and increasingly used, recently two powerful soft-wares, called \"PseAAC-Builder\" and \"propy\" , were established for generating various special Chou's pseudo-amino acid compositions, in addition to the web-server \"PseAAC\" built in 2008. According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation .", "According to a comprehensive review , the general form of PseAAC for a protein sequence P is formulated by where the subscript  is an integer, and its value as well as the components 1, 2, , u u   will depend on how to extract the desired information from the amino acid sequence of P cf. Equation . . Below, let us describe how to extract useful information to define the components of PseAAC for the NR samples concerned. First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned.", "First, many earlier studies see, e.g., have indicated that the amino acid composition AAC of a protein plays an important role in determining its attributes. The AAC contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned. Thus, such 20 AAC components were used here to define the first 20 elements in Equation . ; i.e., . 1, 2, , 20 ii fi   . where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned.", "where f i . is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned. Since AAC did not contain any sequence order information, the following steps were taken to make up this shortcoming. To avoid completely losing the local or short-range sequence order information, we considered the approach of dipeptide composition. It contained 20 × 20 = 400 components . Such 400 components were used to define the next 400 elements in Equation . ; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned.", "; i.e., . 20 1, 2, , 400 jj fj where . j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned. To incorporate the global or long-range sequence order information, let us consider the following approach. According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion.", "According to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. Driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues , gene doubling, and gene fusion. With the accumulation of these changes over a long period of time, many original similarities between initial and resultant amino acid sequences are gradually faded out, but the corresponding proteins may still share many common attributes , such as having basically the same biological function and residing at a same subcellular location . To extract the sequential evolution information and use it to define the components of Equation ., the PSSM Position Specific Scoring Matrix was used as described below. According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned.", "According to Schaffer , the sequence evolution information of a nuclear receptor protein P with L amino acid residues can be expressed by a 20 L matrix, as given by where . were generated by using PSI-BLAST to search the UniProtKB/Swiss-Prot database The Universal Protein Resource UniProt ; through three iterations with 0.001 as the E-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned. In order to make every element in Equation . be scaled from their original score ranges into the region of , we performed a conversion through the standard sigmoid function to make it become Now we extract the useful information from Equation . Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j .", "Moreover, we used the grey system model approach as elaborated in to further define the next 60 components of Equation . 1, 2, , 20 In the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j . has the same meaning as in Equation . where   and Combining Equations ., ., . and ., we found that the total number of the components obtained via the current approach for the PseAAC of Equation . and each of the 500 components is given by . Since the elements in Equations . and .", "and each of the 500 components is given by . Since the elements in Equations . and . are well defined, we can now formulate the drug-NR pair by combining the two equations as given by   . where G represents the drug-NR pair, Å the orthogonal sum, and the 256 + 500 = 756 components are defined by Equations . and . . For the sake of convenience, let us use x i i = 1, 2, , 756 to represent the 756 components in Equation . ; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation .", "; i.e., . To optimize the prediction quality with a time-saving approach, similar to the treatment , let us convert Equation . to where the symbol means taking the average of the quantity therein, and SD means the corresponding standard derivation. In this study, the SVM support vector machine was used as the operation engine. SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function.", "SVM has been widely used in the realm of bioinformatics see, e.g., . The basic idea of SVM is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function. For a brief formulation of SVM and how it works, see the papers ; for more details about SVM, see a monograph . In this study, the LIBSVM package was used as an implementation of SVM, which can be downloaded from the popular radial basis function RBF was taken as the kernel function. For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later.", "For the current SVM classifier, there were two uncertain parameters: penalty parameter C and kernel parameter  . The method of how to determine the two parameters will be given later. The predictor obtained via the aforementioned procedure is called iNR-Drug, where \"i\" means identify, and \"NR-Drug\" means the interaction between nuclear receptor and drug compound. To provide an intuitive overall picture, a flowchart is provided in Figure 2 to show the process of how the predictor works in identifying the interactions between nuclear receptors and drug compounds. To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs.", "To provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by Chou in predicting signal peptides was adopted. According to Chou's formulation, the sensitivity, specificity, overall accuracy, and Matthew's correlation coefficient can be respectively expressed as 62, Sn 1 where N  is the total number of the interactive NR-drug pairs investigated while N   the number of the interactive NR-drug pairs incorrectly predicted as the non-interactive NR-drug pairs; N  the total number of the non-interactive NR-drug pairs investigated while N   the number of the non-interactive NR-drug pairs incorrectly predicted as the interactive NR-drug pairs. According to Equation . we can easily see the following. When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation.", "When 0 N    meaning none of the interactive NR-drug pairs was mispredicted to be a non-interactive NR-drug pair, we have the sensitivity Sn = 1; while NN    meaning that all the interactive NR-drug pairs were mispredicted to be the non-interactive NR-drug pairs, we have the sensitivity Sn = 0 . Likewise, when 0 N    meaning none of the non-interactive NR-drug pairs was mispredicted, we have the specificity Sp we have MCC = 0 meaning total disagreement between prediction and observation. As we can see from the above discussion, it is much more intuitive and easier to understand when using Equation . to examine a predictor for its four metrics, particularly for its Mathew's correlation coefficient. It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in .", "It is instructive to point out that the metrics as defined in Equation . are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in . How to properly test a predictor for its anticipated success rates is very important for its development as well as its potential application value. Generally speaking, the following three cross-validation methods are often used to examine the quality of a predictor and its effectiveness in practical application: independent dataset test, subsampling or K-fold such as five-fold, seven-fold, or 10-fold crossover test and jackknife test . However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either.", "However, as elaborated by a penetrating analysis in , considerable arbitrariness exists in the independent dataset test. Also, as demonstrated in , the subsampling or K-fold crossover validation test cannot avoid arbitrariness either. Only the jackknife test is the least arbitrary that can always yield a unique result for a given benchmark dataset 73, 74, 156, . Therefore, the jackknife test has been widely recognized and increasingly utilized by investigators to examine the quality of various predictors see, e.g., . Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  .", "Accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. As mentioned above, the SVM operation engine contains two uncertain parameters C and  . To find their optimal values, a 2-D grid search was conducted by the jackknife test on the benchmark dataset . The results thus obtained are shown in Figure 3 , from which it can be seen that the iNR-Drug predictor reaches its optimal status when C = 2 3 and 9 2    . The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al.", "The corresponding rates for the four metrics cf. Equation . are given in Table 1 , where for facilitating comparison, the overall accuracy Acc reported by He et al. on the same benchmark dataset is also given although no results were reported by them for Sn, Sp and MCC. It can be observed from the table that the overall accuracy obtained by iNR-Drug is remarkably higher that of He et al. , and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates.", ", and that the rates achieved by iNR-Drug for the other three metrics are also quite higher. These facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates. As mentioned above Section 3.2 , the jackknife test is the most objective method for examining the quality of a predictor. However, as a demonstration to show how to practically use the current predictor, we took 41 NR-drug pairs from the study by Yamanishi et al. that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 .", "that had been confirmed by experiments as interactive pairs. For such an independent dataset, 34 were correctly identified by iNR-Drug as interactive pairs, i.e., Sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in Table 1 . It is anticipated that the iNR-Drug predictor developed in this paper may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. Since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful predictors , a publicly accessible web-server for iNR-Drug was established. For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1.", "For the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using iNR-Drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. Step 1. Open the web server at the site and you will see the top page of the predictor on your computer screen, as shown in Figure 4 . Click on the Read Me button to see a brief introduction about iNR-Drug predictor and the caveat when using it. Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 .", "Step 2. Either type or copy/paste the query NR-drug pairs into the input box at the center of Figure 4 . Each query pair consists of two parts: one is for the nuclear receptor sequence, and the other for the drug. The NR sequence should be in FASTA format, while the drug in the KEGG code beginning with the symbol #. Examples for the query pairs input and the corresponding output can be seen by clicking on the Example button right above the input box. Step 3. Click on the Submit button to see the predicted result.", "Step 3. Click on the Submit button to see the predicted result. For example, if you use the three query pairs in the Example window as the input, after clicking the Submit button, you will see on your screen that the \"hsa:2099\" NR and the \"D00066\" drug are an interactive pair, and that the \"hsa:2908\" NR and the \"D00088\" drug are also an interactive pair, but that the \"hsa:5468\" NR and the \"D00279\" drug are not an interactive pair. All these results are fully consistent with the experimental observations. It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4.", "It takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. Step 4. Click on the Citation button to find the relevant paper that documents the detailed development and algorithm of iNR-Durg. Step 5. Click on the Data button to download the benchmark dataset used to train and test the iNR-Durg predictor. Step 6. The program code is also available by clicking the button download on the lower panel of Figure 4 ." ]

[ "Feline Infectious Peritonitis FIP is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus FIPV , a virulent mutant strain of Feline Enteric Coronavirus FECV . Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide TFO RNAs TFO1 to TFO5 , which target the different regions of virulent feline coronavirus FCoV strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney CRFK cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus FCoV , under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus FECV , a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory.", "The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response .", "In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined .", "Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells.", "serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects CPE development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 TCID 50 was calculated using Reed and Muench's method . Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed Table 1 . The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO.", "The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 ssRNA ligase New England Biolabs Inc., England , was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA .", "Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA . The target regions of the FIPV genome were synthesized by Dharmacon Research USA Table 1 . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter ITC TA instruments, Newcastle, UK . The RNA sample mixtures, consisting of circular TFOs 0.0002 mM , were incubated with their respective synthetic target regions 0.015 mM using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent Qiagen, Germany , as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well .", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well . Finally, the viral inoculum was replaced by fresh maintenance media MEM containing 1% FBS and 1% pen/strep . Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA 25 nM, 50 nM, 100 nM, and 500 nM were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above. Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours ORF1a/1b and 530-541 ORF1a/1b and 7399-7411 ORF1a/1b and 14048-14061 - * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA .", "* * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA . The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit Bioline, UK , which consisted of 12.5 L 2X SensiMix SYBR No-Rox One- Step reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis.", "Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 .", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 . The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps C-C to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay EMSA . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ≤ 0.05 . The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection.", "The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect CPE , the same as virus inoculated cells supplementary Figure 2 . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication.", "Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 .", "The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 . The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ≥ 0.05 supplementary Figure 3 . Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins .", "in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA and viral protease inhibitors Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests mean ± SE . * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization Figure 2 . The EMSA result also concurred with the antiviral study, where all circular TFOs except TFO2 were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells Figure 3 . However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value Table 3 , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours .", "However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells data not shown . The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases .", "Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study Figure 4 . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy . Recently, McDonagh et al. developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells .", "The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers Table 1 , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 i.e., 24 mers , which might be insufficient to a triplex formation upon hybridization Figure 2 , be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "Feline Infectious Peritonitis FIP is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus FIPV , a virulent mutant strain of Feline Enteric Coronavirus FECV . Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide TFO RNAs TFO1 to TFO5 , which target the different regions of virulent feline coronavirus FCoV strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney CRFK cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus FCoV , under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus FECV , a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory.", "The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response .", "In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined .", "Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells.", "serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects CPE development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 TCID 50 was calculated using Reed and Muench's method . Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed Table 1 . The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO.", "The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 ssRNA ligase New England Biolabs Inc., England , was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA .", "Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA . The target regions of the FIPV genome were synthesized by Dharmacon Research USA Table 1 . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter ITC TA instruments, Newcastle, UK . The RNA sample mixtures, consisting of circular TFOs 0.0002 mM , were incubated with their respective synthetic target regions 0.015 mM using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent Qiagen, Germany , as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well .", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well . Finally, the viral inoculum was replaced by fresh maintenance media MEM containing 1% FBS and 1% pen/strep . Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA 25 nM, 50 nM, 100 nM, and 500 nM were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above. Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours ORF1a/1b and 530-541 ORF1a/1b and 7399-7411 ORF1a/1b and 14048-14061 - * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA .", "* * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA . The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit Bioline, UK , which consisted of 12.5 L 2X SensiMix SYBR No-Rox One- Step reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis.", "Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 .", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 . The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps C-C to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay EMSA . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ≤ 0.05 . The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection.", "The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect CPE , the same as virus inoculated cells supplementary Figure 2 . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication.", "Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 .", "The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 . The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ≥ 0.05 supplementary Figure 3 . Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins .", "in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA and viral protease inhibitors Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests mean ± SE . * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization Figure 2 . The EMSA result also concurred with the antiviral study, where all circular TFOs except TFO2 were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells Figure 3 . However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value Table 3 , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours .", "However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells data not shown . The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases .", "Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study Figure 4 . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy . Recently, McDonagh et al. developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells .", "The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers Table 1 , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 i.e., 24 mers , which might be insufficient to a triplex formation upon hybridization Figure 2 , be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "Feline Infectious Peritonitis FIP is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus FIPV , a virulent mutant strain of Feline Enteric Coronavirus FECV . Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide TFO RNAs TFO1 to TFO5 , which target the different regions of virulent feline coronavirus FCoV strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney CRFK cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus FCoV , under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus FECV , a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory.", "The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response .", "In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined .", "Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells.", "serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects CPE development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 TCID 50 was calculated using Reed and Muench's method . Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed Table 1 . The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO.", "The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 ssRNA ligase New England Biolabs Inc., England , was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA .", "Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA . The target regions of the FIPV genome were synthesized by Dharmacon Research USA Table 1 . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter ITC TA instruments, Newcastle, UK . The RNA sample mixtures, consisting of circular TFOs 0.0002 mM , were incubated with their respective synthetic target regions 0.015 mM using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent Qiagen, Germany , as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well .", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well . Finally, the viral inoculum was replaced by fresh maintenance media MEM containing 1% FBS and 1% pen/strep . Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA 25 nM, 50 nM, 100 nM, and 500 nM were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above. Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours ORF1a/1b and 530-541 ORF1a/1b and 7399-7411 ORF1a/1b and 14048-14061 - * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA .", "* * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA . The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit Bioline, UK , which consisted of 12.5 L 2X SensiMix SYBR No-Rox One- Step reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis.", "Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 .", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 . The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps C-C to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay EMSA . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ≤ 0.05 . The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection.", "The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect CPE , the same as virus inoculated cells supplementary Figure 2 . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication.", "Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 .", "The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 . The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ≥ 0.05 supplementary Figure 3 . Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins .", "in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA and viral protease inhibitors Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests mean ± SE . * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization Figure 2 . The EMSA result also concurred with the antiviral study, where all circular TFOs except TFO2 were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells Figure 3 . However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value Table 3 , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours .", "However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells data not shown . The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases .", "Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study Figure 4 . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy . Recently, McDonagh et al. developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells .", "The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers Table 1 , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 i.e., 24 mers , which might be insufficient to a triplex formation upon hybridization Figure 2 , be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "Feline Infectious Peritonitis FIP is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus FIPV , a virulent mutant strain of Feline Enteric Coronavirus FECV . Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide TFO RNAs TFO1 to TFO5 , which target the different regions of virulent feline coronavirus FCoV strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney CRFK cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus FCoV , under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus FECV , a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory.", "The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response .", "In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined .", "Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells.", "serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects CPE development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 TCID 50 was calculated using Reed and Muench's method . Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed Table 1 . The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO.", "The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 ssRNA ligase New England Biolabs Inc., England , was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA .", "Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA . The target regions of the FIPV genome were synthesized by Dharmacon Research USA Table 1 . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter ITC TA instruments, Newcastle, UK . The RNA sample mixtures, consisting of circular TFOs 0.0002 mM , were incubated with their respective synthetic target regions 0.015 mM using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent Qiagen, Germany , as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well .", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well . Finally, the viral inoculum was replaced by fresh maintenance media MEM containing 1% FBS and 1% pen/strep . Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA 25 nM, 50 nM, 100 nM, and 500 nM were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above. Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours ORF1a/1b and 530-541 ORF1a/1b and 7399-7411 ORF1a/1b and 14048-14061 - * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA .", "* * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA . The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit Bioline, UK , which consisted of 12.5 L 2X SensiMix SYBR No-Rox One- Step reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis.", "Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 .", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 . The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps C-C to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay EMSA . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ≤ 0.05 . The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection.", "The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect CPE , the same as virus inoculated cells supplementary Figure 2 . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication.", "Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 .", "The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 . The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ≥ 0.05 supplementary Figure 3 . Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins .", "in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA and viral protease inhibitors Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests mean ± SE . * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization Figure 2 . The EMSA result also concurred with the antiviral study, where all circular TFOs except TFO2 were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells Figure 3 . However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value Table 3 , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours .", "However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells data not shown . The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases .", "Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study Figure 4 . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy . Recently, McDonagh et al. developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells .", "The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers Table 1 , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 i.e., 24 mers , which might be insufficient to a triplex formation upon hybridization Figure 2 , be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "Feline Infectious Peritonitis FIP is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus FIPV , a virulent mutant strain of Feline Enteric Coronavirus FECV . Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide TFO RNAs TFO1 to TFO5 , which target the different regions of virulent feline coronavirus FCoV strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney CRFK cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus FCoV , under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus FECV , a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory.", "The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response .", "In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined .", "Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells.", "serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects CPE development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 TCID 50 was calculated using Reed and Muench's method . Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed Table 1 . The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO.", "The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 ssRNA ligase New England Biolabs Inc., England , was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA .", "Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA . The target regions of the FIPV genome were synthesized by Dharmacon Research USA Table 1 . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter ITC TA instruments, Newcastle, UK . The RNA sample mixtures, consisting of circular TFOs 0.0002 mM , were incubated with their respective synthetic target regions 0.015 mM using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent Qiagen, Germany , as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well .", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well . Finally, the viral inoculum was replaced by fresh maintenance media MEM containing 1% FBS and 1% pen/strep . Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA 25 nM, 50 nM, 100 nM, and 500 nM were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above. Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours ORF1a/1b and 530-541 ORF1a/1b and 7399-7411 ORF1a/1b and 14048-14061 - * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA .", "* * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA . The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit Bioline, UK , which consisted of 12.5 L 2X SensiMix SYBR No-Rox One- Step reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis.", "Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 .", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 . The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps C-C to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay EMSA . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ≤ 0.05 . The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection.", "The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect CPE , the same as virus inoculated cells supplementary Figure 2 . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication.", "Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 .", "The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 . The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ≥ 0.05 supplementary Figure 3 . Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins .", "in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA and viral protease inhibitors Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests mean ± SE . * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization Figure 2 . The EMSA result also concurred with the antiviral study, where all circular TFOs except TFO2 were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells Figure 3 . However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value Table 3 , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours .", "However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells data not shown . The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases .", "Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study Figure 4 . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy . Recently, McDonagh et al. developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells .", "The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers Table 1 , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 i.e., 24 mers , which might be insufficient to a triplex formation upon hybridization Figure 2 , be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "Feline Infectious Peritonitis FIP is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus FIPV , a virulent mutant strain of Feline Enteric Coronavirus FECV . Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide TFO RNAs TFO1 to TFO5 , which target the different regions of virulent feline coronavirus FCoV strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney CRFK cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus FCoV , under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus FECV , a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory.", "The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response .", "In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined .", "Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells.", "serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects CPE development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 TCID 50 was calculated using Reed and Muench's method . Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed Table 1 . The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO.", "The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 ssRNA ligase New England Biolabs Inc., England , was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA .", "Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA . The target regions of the FIPV genome were synthesized by Dharmacon Research USA Table 1 . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter ITC TA instruments, Newcastle, UK . The RNA sample mixtures, consisting of circular TFOs 0.0002 mM , were incubated with their respective synthetic target regions 0.015 mM using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent Qiagen, Germany , as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well .", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well . Finally, the viral inoculum was replaced by fresh maintenance media MEM containing 1% FBS and 1% pen/strep . Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA 25 nM, 50 nM, 100 nM, and 500 nM were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above. Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours ORF1a/1b and 530-541 ORF1a/1b and 7399-7411 ORF1a/1b and 14048-14061 - * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA .", "* * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA . The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit Bioline, UK , which consisted of 12.5 L 2X SensiMix SYBR No-Rox One- Step reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis.", "Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 .", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 . The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps C-C to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay EMSA . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ≤ 0.05 . The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection.", "The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect CPE , the same as virus inoculated cells supplementary Figure 2 . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication.", "Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 .", "The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 . The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ≥ 0.05 supplementary Figure 3 . Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins .", "in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA and viral protease inhibitors Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests mean ± SE . * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization Figure 2 . The EMSA result also concurred with the antiviral study, where all circular TFOs except TFO2 were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells Figure 3 . However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value Table 3 , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours .", "However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells data not shown . The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases .", "Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study Figure 4 . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy . Recently, McDonagh et al. developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells .", "The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers Table 1 , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 i.e., 24 mers , which might be insufficient to a triplex formation upon hybridization Figure 2 , be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "Feline Infectious Peritonitis FIP is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus FIPV , a virulent mutant strain of Feline Enteric Coronavirus FECV . Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide TFO RNAs TFO1 to TFO5 , which target the different regions of virulent feline coronavirus FCoV strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney CRFK cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log. from 10. in the virus-inoculated cells to 10. in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus FIPV is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus FCoV , under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus FECV , a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory.", "The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, i internal mutation theory and ii circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response .", "In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined .", "Triple Helix Forming Oligonucleotide TFO is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells.", "serotype II strain WSU 79-1146 ATCC no. VR-1777 was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects CPE development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 TCID 50 was calculated using Reed and Muench's method . Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides TFOs were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 Accession no: AY994055 . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed Table 1 . The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO.", "The designed linear TFOs were synthesized by Dharmacon Research USA , whereby the 5 and 3 ends of the linear TFOs were modified with phosphate PO 4 group and hydroxide OH group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 ssRNA ligase New England Biolabs Inc., England , was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA .", "Finally, the gel was stained with ethidium bromide Sigma, USA and viewed with a Bio-Rad Gel Doc XR system CA, USA . EMSA . The target regions of the FIPV genome were synthesized by Dharmacon Research USA Table 1 . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter ITC TA instruments, Newcastle, UK . The RNA sample mixtures, consisting of circular TFOs 0.0002 mM , were incubated with their respective synthetic target regions 0.015 mM using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent Qiagen, Germany , as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well .", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C 100 L/well . Finally, the viral inoculum was replaced by fresh maintenance media MEM containing 1% FBS and 1% pen/strep . Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA 25 nM, 50 nM, 100 nM, and 500 nM were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above. Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "Madin-Darby Canine Kidney MDCK cell ATCC no. CCL-34 , at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours ORF1a/1b and 530-541 ORF1a/1b and 7399-7411 ORF1a/1b and 14048-14061 - * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA .", "* * Unrelated circular TFO. , respectively. The reverse transcriptase quantitative real-time PCR RT-qPCR was performed using a Bio-Rad CFX96 real-time system BioRad, USA . The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit Bioline, UK , which consisted of 12.5 L 2X SensiMix SYBR No-Rox One- Step reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis.", "Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 .", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region 5 UTR , Open Reading Frames ORFs 1a and 1b, and 3 untranslated region 3 UTR Table 1 . The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps C-C to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay EMSA . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "The binding ability of different TFO RNAs TFO1 to TFO5 against their target regions was determined by EMSA Figure 2 . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "As shown in supplementary Figure 1 Table 3 . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ≤ 0.05 . The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection.", "The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ≥ 0.05 Figure 3 . The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect CPE , the same as virus inoculated cells supplementary Figure 2 . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication.", "Hence, these results illustrated the capacity of circular TFO RNAs except TFO2 to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 .", "The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations Figure 4 . The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ≥ 0.05 supplementary Figure 3 . Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins .", "in the replication process . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA and viral protease inhibitors Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests mean ± SE . * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "In this study, circular Triple Helix Forming Oligonucleotide TFO RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO Figure 1 . The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization Figure 2 . The EMSA result also concurred with the antiviral study, where all circular TFOs except TFO2 were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells Figure 3 . However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value Table 3 , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours .", "However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells data not shown . The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases .", "Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study Figure 4 . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy . Recently, McDonagh et al. developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells .", "The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus and TFO to inhibit the transcription of 1 I collagen in rat fibroblasts , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers Table 1 , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "All designed circular TFOs except TFO2 showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 i.e., 24 mers , which might be insufficient to a triplex formation upon hybridization Figure 2 , be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In vitro antiviral study which showed no antiviral property for unrelated TFO TFO7 and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "BACKGROUND: To date, the use of traditional nucleic acid amplification tests NAAT for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care POC settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification RT-LAMP , exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification NINA heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients.", "The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC. Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required .", "Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus . Rapid tests are currently a key component of HIV screening at the point-of-care POC , significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings. Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks .", "Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated.", "For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay Gen-Probe, Inc., BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466 is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing. To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time ,1 hour is desirable . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity.", "The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity. For increased applicability at the POC, an increasing number of novel isothermal amplification techniques have been developed . Isothermal amplification is an attractive alternative to traditional PCR or RT-PCR since thermalcycling is not required, allowing for greater versatility in terms of heating or amplification devices. One such amplification method, termed Loop-Mediated Isothermal Amplification LAMP , has been optimized for the detection of DNA and/or RNA RT-LAMP from a wide range of bacterial and viral pathogens , including HIV . LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test.", "One such amplification method, termed Loop-Mediated Isothermal Amplification LAMP , has been optimized for the detection of DNA and/or RNA RT-LAMP from a wide range of bacterial and viral pathogens , including HIV . LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test. The amplification reaction requires six primers specific for eight separate regions within the target sequence, contributing to the high specificity of the amplification method. Amplified material can typically be detected within 15-60 minutes when incubated at a constant reaction temperature of 60-65uC . LAMP has also proven to be less sensitive to biological inhibitors than PCR , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 .", "LAMP has also proven to be less sensitive to biological inhibitors than PCR , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction. Several groups have incorporated fluorescent detection methods into the LAMP assay for real-time or immediate naked-eye detection . The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water .", "The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals. Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health PATH, Seattle, WA , as described . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide CaO; Sigma-Aldrich, St. Louis, MO and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids Fig.", "Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide CaO; Sigma-Aldrich, St. Louis, MO and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids Fig. 1 . The lids were modified to contain three sample wells that fit standard 200 ml PCR tubes and were filled with a proprietary phase-change material PCM that was used to buffer the heat derived from the exothermic reaction, thereby providing a constant temperature. Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT .", "Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT . DNA and RNA linearity panels were prepared to determine the sensitivity of the HIV-specific RT-LAMP assay. A DNA panel was generated from DNA extracted from the human monocytic cell line OM-10.1 , using a QIAamp DNA blood mini kit QIAGEN, Valencia, CA . Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml.", "Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially PRD801; SeraCare Life Sciences, Mil- ford, MA and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit QIAGEN . Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD . Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study , or obtained commercially SeraCare Life Sciences .", "Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD . Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study , or obtained commercially SeraCare Life Sciences . All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA Bio-Rad Laboratories, Redmond, WA , GS HIV-1 Western blot Bio-Rad Laboratories , Aptima HIV-1 RNA assay Gen-Probe, Inc., San Diego, CA , and Amplicor HIV-1 DNA assay Roche Diagnostics, Branchburg, NJ . Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood.", "All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL Advanced Biotechnologies Inc. . HIV-1-specific RT-LAMP primers were designed to recognize a conserved sequence within the reverse transcriptase RT gene. The six primers required for the RT-LAMP reaction, forward outer F3 , backward outer B3 , forward inner FIP , backward inner BIP , and the loop primers LoopF and LoopB , were designed using the PrimerExplorer V4 software Eiken Chemical Co. Ltd.; The LAMP primers and amplification cycle have been described in detail by Nagamine et al. .", "The six primers required for the RT-LAMP reaction, forward outer F3 , backward outer B3 , forward inner FIP , backward inner BIP , and the loop primers LoopF and LoopB , were designed using the PrimerExplorer V4 software Eiken Chemical Co. Ltd.; The LAMP primers and amplification cycle have been described in detail by Nagamine et al. . Additional modifications included a linker sequence of four thymidines inserted between the F2 and F1c sequences of the FIP primer, as described , and the addition of the fluorescent molecule HEX to the 59 end of the LoopF primer. The labeled primer, along with a quencher probe, allowed for immediate visual detection of amplified products . The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 BHQ-1 added to the 39 end. The HIV-1 HXB2 sequence GenBank accession number AF033819 was used as the reference for generating the RT-LAMP primers.", "The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 BHQ-1 added to the 39 end. The HIV-1 HXB2 sequence GenBank accession number AF033819 was used as the reference for generating the RT-LAMP primers. The sequences of the HIV-1 RT-specific primers and quencher are listed in Table 1 . The RT-LAMP reaction was performed using the following reaction mix: 0.2 mM final concentration of each F3 and B3 primers, 1.6 mM of each FIP and BIP primers, 0.8 mM of each LoopF and HEX-LoopB primers, 0.8 M betaine Sigma-Aldrich , 10 mM MgSO 4 , 1.4 mM dNTPs, 16 ThermoPol reaction buffer New England Biolabs, Ipswich, MA , 16 U Bst DNA polymerase New England Biolabs and 2 U AMV reverse transcriptase Invitrogen, Carlsbad, CA . The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products.", "The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products. Whole blood was added directly into the reaction at a total volume of 40 ml, following a 1:4 dilution with red blood cell lysis buffer 2.5 mM KHCO 3 , 37.5 mM NH 4 Cl, and 0.025 mM EDTA , as previously described . The reaction mixture was incubated at 60uC for 60 minutes, using a GeneAmpH PCR System Applied Biosystems, Foster City, CA or the NINA heaters. For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction. The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described .", "For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction. The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system Bio-Rad Laboratories, Hercules, CA . Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain Invitrogen , which was subsequently visualized using the ChemiDoc XRS system. To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water.", "To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction. After adding 200 ml of water to each of the sample wells, temperature recording was initiated. Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation.", "Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation. The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300.", "Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300. The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC. To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction.", "To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction. For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates 60% . For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler Table 2 . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters.", "In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler Table 2 . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction. During the 60 minute incubation, the average temperature for each device was 60.2, 59.8, and 59.7 Table 3 . The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree.", "The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree. The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory 28uC; Table 2 . The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC Table 3 .", "The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory 28uC; Table 2 . The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC Table 3 . The average temperature was approximately two degrees lower than the desired reaction temperature of 60uC. Additionally, the temperature of the devices tended to decline from their steady state during the last 20 minutes of the reaction data not shown . The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters.", "The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices Table 4 . Amplification consistency was most evident with two of the patient samples patient #4 and #5 that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes.", "All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes. In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid NINA heaters for amplification of HIV-1 using RT-LAMP. The isothermal amplification reaction coupled with a device that generates heat from an exothermic chemical reaction, as opposed to grid electricity or battery power, comprises a point-of-care NAAT that is practical for use in resource-limited settings. The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler.", "The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler. Since point-of-care testing may refer to an air-conditioned laboratory or a field site with high temperatures and humidity, the stability of the temperature generated by the heating devices must be reliable. Though the temperature profiles at a representative cold temperature of 4uC indicated a loss in reaction temperature towards the end of the 60 minute incubation, the temperature fluctuations were not significant enough to affect the amplification reaction. Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both.", "Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option. We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory. For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume.", "For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction. While the additional heating step is not necessary to observe the amplified products from extracted nucleic acid, the short, high-temperature incubation facilitates the visual observation of the fluorescent label in the whole blood samples. Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the amplification compartment and reinserted into the 80uC compartment.", "Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the amplification compartment and reinserted into the 80uC compartment. Lastly, the DaqPRO data recorder was used in this study for validation purposes only and would not be necessary for the final POC product. The feasibility of using LAMP as a diagnostic method in resource-limited settings has been demonstrated for tuberculosis . To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube.", "To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube. We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification.", "Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification. Future modifications will include optimizing the labeledprimer/quencher sequences so that all components can be added into the reaction mix prior to amplification. Due to availability, the Bio-Rad ChemiDoc system was used as the UV source in this study; however, an inexpensive keychain light would be more suitable for naked-eye detection at the POC. For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes.", "For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device. In summary, the RT-LAMP isothermal amplification method used in conjunction with a simplified, chemical heating device exhibits characteristics that are ideal for a rapid NAAT for POC testing. The simplified, portable assay has the potential to fill an important gap in HIV-1 diagnostics, providing immediate knowledge or confirmation of HIV-1 infection status at the POC." ]

[ "BACKGROUND: To date, the use of traditional nucleic acid amplification tests NAAT for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care POC settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification RT-LAMP , exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification NINA heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients.", "The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC. Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required .", "Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus . Rapid tests are currently a key component of HIV screening at the point-of-care POC , significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings. Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks .", "Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated.", "For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay Gen-Probe, Inc., BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466 is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing. To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time ,1 hour is desirable . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity.", "The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity. For increased applicability at the POC, an increasing number of novel isothermal amplification techniques have been developed . Isothermal amplification is an attractive alternative to traditional PCR or RT-PCR since thermalcycling is not required, allowing for greater versatility in terms of heating or amplification devices. One such amplification method, termed Loop-Mediated Isothermal Amplification LAMP , has been optimized for the detection of DNA and/or RNA RT-LAMP from a wide range of bacterial and viral pathogens , including HIV . LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test.", "One such amplification method, termed Loop-Mediated Isothermal Amplification LAMP , has been optimized for the detection of DNA and/or RNA RT-LAMP from a wide range of bacterial and viral pathogens , including HIV . LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test. The amplification reaction requires six primers specific for eight separate regions within the target sequence, contributing to the high specificity of the amplification method. Amplified material can typically be detected within 15-60 minutes when incubated at a constant reaction temperature of 60-65uC . LAMP has also proven to be less sensitive to biological inhibitors than PCR , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 .", "LAMP has also proven to be less sensitive to biological inhibitors than PCR , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction. Several groups have incorporated fluorescent detection methods into the LAMP assay for real-time or immediate naked-eye detection . The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water .", "The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals. Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health PATH, Seattle, WA , as described . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide CaO; Sigma-Aldrich, St. Louis, MO and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids Fig.", "Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide CaO; Sigma-Aldrich, St. Louis, MO and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids Fig. 1 . The lids were modified to contain three sample wells that fit standard 200 ml PCR tubes and were filled with a proprietary phase-change material PCM that was used to buffer the heat derived from the exothermic reaction, thereby providing a constant temperature. Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT .", "Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT . DNA and RNA linearity panels were prepared to determine the sensitivity of the HIV-specific RT-LAMP assay. A DNA panel was generated from DNA extracted from the human monocytic cell line OM-10.1 , using a QIAamp DNA blood mini kit QIAGEN, Valencia, CA . Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml.", "Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially PRD801; SeraCare Life Sciences, Mil- ford, MA and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit QIAGEN . Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD . Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study , or obtained commercially SeraCare Life Sciences .", "Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD . Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study , or obtained commercially SeraCare Life Sciences . All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA Bio-Rad Laboratories, Redmond, WA , GS HIV-1 Western blot Bio-Rad Laboratories , Aptima HIV-1 RNA assay Gen-Probe, Inc., San Diego, CA , and Amplicor HIV-1 DNA assay Roche Diagnostics, Branchburg, NJ . Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood.", "All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL Advanced Biotechnologies Inc. . HIV-1-specific RT-LAMP primers were designed to recognize a conserved sequence within the reverse transcriptase RT gene. The six primers required for the RT-LAMP reaction, forward outer F3 , backward outer B3 , forward inner FIP , backward inner BIP , and the loop primers LoopF and LoopB , were designed using the PrimerExplorer V4 software Eiken Chemical Co. Ltd.; The LAMP primers and amplification cycle have been described in detail by Nagamine et al. .", "The six primers required for the RT-LAMP reaction, forward outer F3 , backward outer B3 , forward inner FIP , backward inner BIP , and the loop primers LoopF and LoopB , were designed using the PrimerExplorer V4 software Eiken Chemical Co. Ltd.; The LAMP primers and amplification cycle have been described in detail by Nagamine et al. . Additional modifications included a linker sequence of four thymidines inserted between the F2 and F1c sequences of the FIP primer, as described , and the addition of the fluorescent molecule HEX to the 59 end of the LoopF primer. The labeled primer, along with a quencher probe, allowed for immediate visual detection of amplified products . The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 BHQ-1 added to the 39 end. The HIV-1 HXB2 sequence GenBank accession number AF033819 was used as the reference for generating the RT-LAMP primers.", "The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 BHQ-1 added to the 39 end. The HIV-1 HXB2 sequence GenBank accession number AF033819 was used as the reference for generating the RT-LAMP primers. The sequences of the HIV-1 RT-specific primers and quencher are listed in Table 1 . The RT-LAMP reaction was performed using the following reaction mix: 0.2 mM final concentration of each F3 and B3 primers, 1.6 mM of each FIP and BIP primers, 0.8 mM of each LoopF and HEX-LoopB primers, 0.8 M betaine Sigma-Aldrich , 10 mM MgSO 4 , 1.4 mM dNTPs, 16 ThermoPol reaction buffer New England Biolabs, Ipswich, MA , 16 U Bst DNA polymerase New England Biolabs and 2 U AMV reverse transcriptase Invitrogen, Carlsbad, CA . The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products.", "The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products. Whole blood was added directly into the reaction at a total volume of 40 ml, following a 1:4 dilution with red blood cell lysis buffer 2.5 mM KHCO 3 , 37.5 mM NH 4 Cl, and 0.025 mM EDTA , as previously described . The reaction mixture was incubated at 60uC for 60 minutes, using a GeneAmpH PCR System Applied Biosystems, Foster City, CA or the NINA heaters. For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction. The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described .", "For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction. The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system Bio-Rad Laboratories, Hercules, CA . Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain Invitrogen , which was subsequently visualized using the ChemiDoc XRS system. To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water.", "To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction. After adding 200 ml of water to each of the sample wells, temperature recording was initiated. Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation.", "Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation. The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300.", "Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300. The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC. To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction.", "To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction. For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates 60% . For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler Table 2 . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters.", "In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler Table 2 . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction. During the 60 minute incubation, the average temperature for each device was 60.2, 59.8, and 59.7 Table 3 . The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree.", "The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree. The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory 28uC; Table 2 . The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC Table 3 .", "The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory 28uC; Table 2 . The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC Table 3 . The average temperature was approximately two degrees lower than the desired reaction temperature of 60uC. Additionally, the temperature of the devices tended to decline from their steady state during the last 20 minutes of the reaction data not shown . The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters.", "The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices Table 4 . Amplification consistency was most evident with two of the patient samples patient #4 and #5 that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes.", "All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes. In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid NINA heaters for amplification of HIV-1 using RT-LAMP. The isothermal amplification reaction coupled with a device that generates heat from an exothermic chemical reaction, as opposed to grid electricity or battery power, comprises a point-of-care NAAT that is practical for use in resource-limited settings. The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler.", "The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler. Since point-of-care testing may refer to an air-conditioned laboratory or a field site with high temperatures and humidity, the stability of the temperature generated by the heating devices must be reliable. Though the temperature profiles at a representative cold temperature of 4uC indicated a loss in reaction temperature towards the end of the 60 minute incubation, the temperature fluctuations were not significant enough to affect the amplification reaction. Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both.", "Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option. We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory. For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume.", "For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction. While the additional heating step is not necessary to observe the amplified products from extracted nucleic acid, the short, high-temperature incubation facilitates the visual observation of the fluorescent label in the whole blood samples. Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the amplification compartment and reinserted into the 80uC compartment.", "Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the amplification compartment and reinserted into the 80uC compartment. Lastly, the DaqPRO data recorder was used in this study for validation purposes only and would not be necessary for the final POC product. The feasibility of using LAMP as a diagnostic method in resource-limited settings has been demonstrated for tuberculosis . To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube.", "To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube. We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification.", "Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification. Future modifications will include optimizing the labeledprimer/quencher sequences so that all components can be added into the reaction mix prior to amplification. Due to availability, the Bio-Rad ChemiDoc system was used as the UV source in this study; however, an inexpensive keychain light would be more suitable for naked-eye detection at the POC. For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes.", "For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device. In summary, the RT-LAMP isothermal amplification method used in conjunction with a simplified, chemical heating device exhibits characteristics that are ideal for a rapid NAAT for POC testing. The simplified, portable assay has the potential to fill an important gap in HIV-1 diagnostics, providing immediate knowledge or confirmation of HIV-1 infection status at the POC." ]

[ "BACKGROUND: To date, the use of traditional nucleic acid amplification tests NAAT for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care POC settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification RT-LAMP , exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification NINA heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients.", "The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC. Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required .", "Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus . Rapid tests are currently a key component of HIV screening at the point-of-care POC , significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings. Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks .", "Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated.", "For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay Gen-Probe, Inc., BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466 is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing. To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time ,1 hour is desirable . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity.", "The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity. For increased applicability at the POC, an increasing number of novel isothermal amplification techniques have been developed . Isothermal amplification is an attractive alternative to traditional PCR or RT-PCR since thermalcycling is not required, allowing for greater versatility in terms of heating or amplification devices. One such amplification method, termed Loop-Mediated Isothermal Amplification LAMP , has been optimized for the detection of DNA and/or RNA RT-LAMP from a wide range of bacterial and viral pathogens , including HIV . LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test.", "One such amplification method, termed Loop-Mediated Isothermal Amplification LAMP , has been optimized for the detection of DNA and/or RNA RT-LAMP from a wide range of bacterial and viral pathogens , including HIV . LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test. The amplification reaction requires six primers specific for eight separate regions within the target sequence, contributing to the high specificity of the amplification method. Amplified material can typically be detected within 15-60 minutes when incubated at a constant reaction temperature of 60-65uC . LAMP has also proven to be less sensitive to biological inhibitors than PCR , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 .", "LAMP has also proven to be less sensitive to biological inhibitors than PCR , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction. Several groups have incorporated fluorescent detection methods into the LAMP assay for real-time or immediate naked-eye detection . The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water .", "The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals. Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health PATH, Seattle, WA , as described . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide CaO; Sigma-Aldrich, St. Louis, MO and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids Fig.", "Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide CaO; Sigma-Aldrich, St. Louis, MO and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids Fig. 1 . The lids were modified to contain three sample wells that fit standard 200 ml PCR tubes and were filled with a proprietary phase-change material PCM that was used to buffer the heat derived from the exothermic reaction, thereby providing a constant temperature. Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT .", "Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT . DNA and RNA linearity panels were prepared to determine the sensitivity of the HIV-specific RT-LAMP assay. A DNA panel was generated from DNA extracted from the human monocytic cell line OM-10.1 , using a QIAamp DNA blood mini kit QIAGEN, Valencia, CA . Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml.", "Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially PRD801; SeraCare Life Sciences, Mil- ford, MA and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit QIAGEN . Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD . Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study , or obtained commercially SeraCare Life Sciences .", "Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD . Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study , or obtained commercially SeraCare Life Sciences . All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA Bio-Rad Laboratories, Redmond, WA , GS HIV-1 Western blot Bio-Rad Laboratories , Aptima HIV-1 RNA assay Gen-Probe, Inc., San Diego, CA , and Amplicor HIV-1 DNA assay Roche Diagnostics, Branchburg, NJ . Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood.", "All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL Advanced Biotechnologies Inc. . HIV-1-specific RT-LAMP primers were designed to recognize a conserved sequence within the reverse transcriptase RT gene. The six primers required for the RT-LAMP reaction, forward outer F3 , backward outer B3 , forward inner FIP , backward inner BIP , and the loop primers LoopF and LoopB , were designed using the PrimerExplorer V4 software Eiken Chemical Co. Ltd.; The LAMP primers and amplification cycle have been described in detail by Nagamine et al. .", "The six primers required for the RT-LAMP reaction, forward outer F3 , backward outer B3 , forward inner FIP , backward inner BIP , and the loop primers LoopF and LoopB , were designed using the PrimerExplorer V4 software Eiken Chemical Co. Ltd.; The LAMP primers and amplification cycle have been described in detail by Nagamine et al. . Additional modifications included a linker sequence of four thymidines inserted between the F2 and F1c sequences of the FIP primer, as described , and the addition of the fluorescent molecule HEX to the 59 end of the LoopF primer. The labeled primer, along with a quencher probe, allowed for immediate visual detection of amplified products . The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 BHQ-1 added to the 39 end. The HIV-1 HXB2 sequence GenBank accession number AF033819 was used as the reference for generating the RT-LAMP primers.", "The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 BHQ-1 added to the 39 end. The HIV-1 HXB2 sequence GenBank accession number AF033819 was used as the reference for generating the RT-LAMP primers. The sequences of the HIV-1 RT-specific primers and quencher are listed in Table 1 . The RT-LAMP reaction was performed using the following reaction mix: 0.2 mM final concentration of each F3 and B3 primers, 1.6 mM of each FIP and BIP primers, 0.8 mM of each LoopF and HEX-LoopB primers, 0.8 M betaine Sigma-Aldrich , 10 mM MgSO 4 , 1.4 mM dNTPs, 16 ThermoPol reaction buffer New England Biolabs, Ipswich, MA , 16 U Bst DNA polymerase New England Biolabs and 2 U AMV reverse transcriptase Invitrogen, Carlsbad, CA . The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products.", "The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products. Whole blood was added directly into the reaction at a total volume of 40 ml, following a 1:4 dilution with red blood cell lysis buffer 2.5 mM KHCO 3 , 37.5 mM NH 4 Cl, and 0.025 mM EDTA , as previously described . The reaction mixture was incubated at 60uC for 60 minutes, using a GeneAmpH PCR System Applied Biosystems, Foster City, CA or the NINA heaters. For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction. The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described .", "For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction. The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system Bio-Rad Laboratories, Hercules, CA . Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain Invitrogen , which was subsequently visualized using the ChemiDoc XRS system. To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water.", "To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction. After adding 200 ml of water to each of the sample wells, temperature recording was initiated. Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation.", "Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation. The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300.", "Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300. The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC. To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction.", "To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction. For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates 60% . For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler Table 2 . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters.", "In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler Table 2 . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction. During the 60 minute incubation, the average temperature for each device was 60.2, 59.8, and 59.7 Table 3 . The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree.", "The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree. The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory 28uC; Table 2 . The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC Table 3 .", "The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory 28uC; Table 2 . The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC Table 3 . The average temperature was approximately two degrees lower than the desired reaction temperature of 60uC. Additionally, the temperature of the devices tended to decline from their steady state during the last 20 minutes of the reaction data not shown . The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters.", "The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices Table 4 . Amplification consistency was most evident with two of the patient samples patient #4 and #5 that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes.", "All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes. In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid NINA heaters for amplification of HIV-1 using RT-LAMP. The isothermal amplification reaction coupled with a device that generates heat from an exothermic chemical reaction, as opposed to grid electricity or battery power, comprises a point-of-care NAAT that is practical for use in resource-limited settings. The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler.", "The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler. Since point-of-care testing may refer to an air-conditioned laboratory or a field site with high temperatures and humidity, the stability of the temperature generated by the heating devices must be reliable. Though the temperature profiles at a representative cold temperature of 4uC indicated a loss in reaction temperature towards the end of the 60 minute incubation, the temperature fluctuations were not significant enough to affect the amplification reaction. Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both.", "Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option. We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory. For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume.", "For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction. While the additional heating step is not necessary to observe the amplified products from extracted nucleic acid, the short, high-temperature incubation facilitates the visual observation of the fluorescent label in the whole blood samples. Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the amplification compartment and reinserted into the 80uC compartment.", "Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the amplification compartment and reinserted into the 80uC compartment. Lastly, the DaqPRO data recorder was used in this study for validation purposes only and would not be necessary for the final POC product. The feasibility of using LAMP as a diagnostic method in resource-limited settings has been demonstrated for tuberculosis . To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube.", "To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube. We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification.", "Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification. Future modifications will include optimizing the labeledprimer/quencher sequences so that all components can be added into the reaction mix prior to amplification. Due to availability, the Bio-Rad ChemiDoc system was used as the UV source in this study; however, an inexpensive keychain light would be more suitable for naked-eye detection at the POC. For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes.", "For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device. In summary, the RT-LAMP isothermal amplification method used in conjunction with a simplified, chemical heating device exhibits characteristics that are ideal for a rapid NAAT for POC testing. The simplified, portable assay has the potential to fill an important gap in HIV-1 diagnostics, providing immediate knowledge or confirmation of HIV-1 infection status at the POC." ]

[ "BACKGROUND: To date, the use of traditional nucleic acid amplification tests NAAT for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care POC settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification RT-LAMP , exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification NINA heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients.", "The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC. Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required .", "Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus . Rapid tests are currently a key component of HIV screening at the point-of-care POC , significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings. Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks .", "Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated.", "For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay Gen-Probe, Inc., BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466 is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing. To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time ,1 hour is desirable . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity.", "The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity. For increased applicability at the POC, an increasing number of novel isothermal amplification techniques have been developed . Isothermal amplification is an attractive alternative to traditional PCR or RT-PCR since thermalcycling is not required, allowing for greater versatility in terms of heating or amplification devices. One such amplification method, termed Loop-Mediated Isothermal Amplification LAMP , has been optimized for the detection of DNA and/or RNA RT-LAMP from a wide range of bacterial and viral pathogens , including HIV . LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test.", "One such amplification method, termed Loop-Mediated Isothermal Amplification LAMP , has been optimized for the detection of DNA and/or RNA RT-LAMP from a wide range of bacterial and viral pathogens , including HIV . LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test. The amplification reaction requires six primers specific for eight separate regions within the target sequence, contributing to the high specificity of the amplification method. Amplified material can typically be detected within 15-60 minutes when incubated at a constant reaction temperature of 60-65uC . LAMP has also proven to be less sensitive to biological inhibitors than PCR , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 .", "LAMP has also proven to be less sensitive to biological inhibitors than PCR , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction. Several groups have incorporated fluorescent detection methods into the LAMP assay for real-time or immediate naked-eye detection . The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water .", "The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals. Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health PATH, Seattle, WA , as described . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide CaO; Sigma-Aldrich, St. Louis, MO and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids Fig.", "Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide CaO; Sigma-Aldrich, St. Louis, MO and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids Fig. 1 . The lids were modified to contain three sample wells that fit standard 200 ml PCR tubes and were filled with a proprietary phase-change material PCM that was used to buffer the heat derived from the exothermic reaction, thereby providing a constant temperature. Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT .", "Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT . DNA and RNA linearity panels were prepared to determine the sensitivity of the HIV-specific RT-LAMP assay. A DNA panel was generated from DNA extracted from the human monocytic cell line OM-10.1 , using a QIAamp DNA blood mini kit QIAGEN, Valencia, CA . Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml.", "Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially PRD801; SeraCare Life Sciences, Mil- ford, MA and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit QIAGEN . Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD . Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study , or obtained commercially SeraCare Life Sciences .", "Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD . Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study , or obtained commercially SeraCare Life Sciences . All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA Bio-Rad Laboratories, Redmond, WA , GS HIV-1 Western blot Bio-Rad Laboratories , Aptima HIV-1 RNA assay Gen-Probe, Inc., San Diego, CA , and Amplicor HIV-1 DNA assay Roche Diagnostics, Branchburg, NJ . Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood.", "All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL Advanced Biotechnologies Inc. . HIV-1-specific RT-LAMP primers were designed to recognize a conserved sequence within the reverse transcriptase RT gene. The six primers required for the RT-LAMP reaction, forward outer F3 , backward outer B3 , forward inner FIP , backward inner BIP , and the loop primers LoopF and LoopB , were designed using the PrimerExplorer V4 software Eiken Chemical Co. Ltd.; The LAMP primers and amplification cycle have been described in detail by Nagamine et al. .", "The six primers required for the RT-LAMP reaction, forward outer F3 , backward outer B3 , forward inner FIP , backward inner BIP , and the loop primers LoopF and LoopB , were designed using the PrimerExplorer V4 software Eiken Chemical Co. Ltd.; The LAMP primers and amplification cycle have been described in detail by Nagamine et al. . Additional modifications included a linker sequence of four thymidines inserted between the F2 and F1c sequences of the FIP primer, as described , and the addition of the fluorescent molecule HEX to the 59 end of the LoopF primer. The labeled primer, along with a quencher probe, allowed for immediate visual detection of amplified products . The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 BHQ-1 added to the 39 end. The HIV-1 HXB2 sequence GenBank accession number AF033819 was used as the reference for generating the RT-LAMP primers.", "The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 BHQ-1 added to the 39 end. The HIV-1 HXB2 sequence GenBank accession number AF033819 was used as the reference for generating the RT-LAMP primers. The sequences of the HIV-1 RT-specific primers and quencher are listed in Table 1 . The RT-LAMP reaction was performed using the following reaction mix: 0.2 mM final concentration of each F3 and B3 primers, 1.6 mM of each FIP and BIP primers, 0.8 mM of each LoopF and HEX-LoopB primers, 0.8 M betaine Sigma-Aldrich , 10 mM MgSO 4 , 1.4 mM dNTPs, 16 ThermoPol reaction buffer New England Biolabs, Ipswich, MA , 16 U Bst DNA polymerase New England Biolabs and 2 U AMV reverse transcriptase Invitrogen, Carlsbad, CA . The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products.", "The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products. Whole blood was added directly into the reaction at a total volume of 40 ml, following a 1:4 dilution with red blood cell lysis buffer 2.5 mM KHCO 3 , 37.5 mM NH 4 Cl, and 0.025 mM EDTA , as previously described . The reaction mixture was incubated at 60uC for 60 minutes, using a GeneAmpH PCR System Applied Biosystems, Foster City, CA or the NINA heaters. For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction. The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described .", "For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction. The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system Bio-Rad Laboratories, Hercules, CA . Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain Invitrogen , which was subsequently visualized using the ChemiDoc XRS system. To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water.", "To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction. After adding 200 ml of water to each of the sample wells, temperature recording was initiated. Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation.", "Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation. The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300.", "Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300. The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC. To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction.", "To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction. For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates 60% . For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler Table 2 . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters.", "In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler Table 2 . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction. During the 60 minute incubation, the average temperature for each device was 60.2, 59.8, and 59.7 Table 3 . The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree.", "The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree. The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory 28uC; Table 2 . The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC Table 3 .", "The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory 28uC; Table 2 . The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC Table 3 . The average temperature was approximately two degrees lower than the desired reaction temperature of 60uC. Additionally, the temperature of the devices tended to decline from their steady state during the last 20 minutes of the reaction data not shown . The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters.", "The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices Table 4 . Amplification consistency was most evident with two of the patient samples patient #4 and #5 that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes.", "All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes. In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid NINA heaters for amplification of HIV-1 using RT-LAMP. The isothermal amplification reaction coupled with a device that generates heat from an exothermic chemical reaction, as opposed to grid electricity or battery power, comprises a point-of-care NAAT that is practical for use in resource-limited settings. The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler.", "The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler. Since point-of-care testing may refer to an air-conditioned laboratory or a field site with high temperatures and humidity, the stability of the temperature generated by the heating devices must be reliable. Though the temperature profiles at a representative cold temperature of 4uC indicated a loss in reaction temperature towards the end of the 60 minute incubation, the temperature fluctuations were not significant enough to affect the amplification reaction. Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both.", "Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option. We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory. For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume.", "For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction. While the additional heating step is not necessary to observe the amplified products from extracted nucleic acid, the short, high-temperature incubation facilitates the visual observation of the fluorescent label in the whole blood samples. Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the amplification compartment and reinserted into the 80uC compartment.", "Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the amplification compartment and reinserted into the 80uC compartment. Lastly, the DaqPRO data recorder was used in this study for validation purposes only and would not be necessary for the final POC product. The feasibility of using LAMP as a diagnostic method in resource-limited settings has been demonstrated for tuberculosis . To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube.", "To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube. We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification.", "Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification. Future modifications will include optimizing the labeledprimer/quencher sequences so that all components can be added into the reaction mix prior to amplification. Due to availability, the Bio-Rad ChemiDoc system was used as the UV source in this study; however, an inexpensive keychain light would be more suitable for naked-eye detection at the POC. For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes.", "For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device. In summary, the RT-LAMP isothermal amplification method used in conjunction with a simplified, chemical heating device exhibits characteristics that are ideal for a rapid NAAT for POC testing. The simplified, portable assay has the potential to fill an important gap in HIV-1 diagnostics, providing immediate knowledge or confirmation of HIV-1 infection status at the POC." ]

[ "BACKGROUND: To date, the use of traditional nucleic acid amplification tests NAAT for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care POC settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification RT-LAMP , exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification NINA heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients.", "The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC. Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required .", "Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus . Rapid tests are currently a key component of HIV screening at the point-of-care POC , significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings. Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks .", "Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated.", "For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay Gen-Probe, Inc., BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466 is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing. To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time ,1 hour is desirable . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity.", "The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity. For increased applicability at the POC, an increasing number of novel isothermal amplification techniques have been developed . Isothermal amplification is an attractive alternative to traditional PCR or RT-PCR since thermalcycling is not required, allowing for greater versatility in terms of heating or amplification devices. One such amplification method, termed Loop-Mediated Isothermal Amplification LAMP , has been optimized for the detection of DNA and/or RNA RT-LAMP from a wide range of bacterial and viral pathogens , including HIV . LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test.", "One such amplification method, termed Loop-Mediated Isothermal Amplification LAMP , has been optimized for the detection of DNA and/or RNA RT-LAMP from a wide range of bacterial and viral pathogens , including HIV . LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test. The amplification reaction requires six primers specific for eight separate regions within the target sequence, contributing to the high specificity of the amplification method. Amplified material can typically be detected within 15-60 minutes when incubated at a constant reaction temperature of 60-65uC . LAMP has also proven to be less sensitive to biological inhibitors than PCR , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 .", "LAMP has also proven to be less sensitive to biological inhibitors than PCR , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction. Several groups have incorporated fluorescent detection methods into the LAMP assay for real-time or immediate naked-eye detection . The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water .", "The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals. Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health PATH, Seattle, WA , as described . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide CaO; Sigma-Aldrich, St. Louis, MO and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids Fig.", "Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide CaO; Sigma-Aldrich, St. Louis, MO and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids Fig. 1 . The lids were modified to contain three sample wells that fit standard 200 ml PCR tubes and were filled with a proprietary phase-change material PCM that was used to buffer the heat derived from the exothermic reaction, thereby providing a constant temperature. Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT .", "Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT . DNA and RNA linearity panels were prepared to determine the sensitivity of the HIV-specific RT-LAMP assay. A DNA panel was generated from DNA extracted from the human monocytic cell line OM-10.1 , using a QIAamp DNA blood mini kit QIAGEN, Valencia, CA . Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml.", "Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially PRD801; SeraCare Life Sciences, Mil- ford, MA and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit QIAGEN . Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD . Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study , or obtained commercially SeraCare Life Sciences .", "Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD . Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study , or obtained commercially SeraCare Life Sciences . All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA Bio-Rad Laboratories, Redmond, WA , GS HIV-1 Western blot Bio-Rad Laboratories , Aptima HIV-1 RNA assay Gen-Probe, Inc., San Diego, CA , and Amplicor HIV-1 DNA assay Roche Diagnostics, Branchburg, NJ . Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood.", "All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL Advanced Biotechnologies Inc. . HIV-1-specific RT-LAMP primers were designed to recognize a conserved sequence within the reverse transcriptase RT gene. The six primers required for the RT-LAMP reaction, forward outer F3 , backward outer B3 , forward inner FIP , backward inner BIP , and the loop primers LoopF and LoopB , were designed using the PrimerExplorer V4 software Eiken Chemical Co. Ltd.; The LAMP primers and amplification cycle have been described in detail by Nagamine et al. .", "The six primers required for the RT-LAMP reaction, forward outer F3 , backward outer B3 , forward inner FIP , backward inner BIP , and the loop primers LoopF and LoopB , were designed using the PrimerExplorer V4 software Eiken Chemical Co. Ltd.; The LAMP primers and amplification cycle have been described in detail by Nagamine et al. . Additional modifications included a linker sequence of four thymidines inserted between the F2 and F1c sequences of the FIP primer, as described , and the addition of the fluorescent molecule HEX to the 59 end of the LoopF primer. The labeled primer, along with a quencher probe, allowed for immediate visual detection of amplified products . The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 BHQ-1 added to the 39 end. The HIV-1 HXB2 sequence GenBank accession number AF033819 was used as the reference for generating the RT-LAMP primers.", "The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 BHQ-1 added to the 39 end. The HIV-1 HXB2 sequence GenBank accession number AF033819 was used as the reference for generating the RT-LAMP primers. The sequences of the HIV-1 RT-specific primers and quencher are listed in Table 1 . The RT-LAMP reaction was performed using the following reaction mix: 0.2 mM final concentration of each F3 and B3 primers, 1.6 mM of each FIP and BIP primers, 0.8 mM of each LoopF and HEX-LoopB primers, 0.8 M betaine Sigma-Aldrich , 10 mM MgSO 4 , 1.4 mM dNTPs, 16 ThermoPol reaction buffer New England Biolabs, Ipswich, MA , 16 U Bst DNA polymerase New England Biolabs and 2 U AMV reverse transcriptase Invitrogen, Carlsbad, CA . The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products.", "The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products. Whole blood was added directly into the reaction at a total volume of 40 ml, following a 1:4 dilution with red blood cell lysis buffer 2.5 mM KHCO 3 , 37.5 mM NH 4 Cl, and 0.025 mM EDTA , as previously described . The reaction mixture was incubated at 60uC for 60 minutes, using a GeneAmpH PCR System Applied Biosystems, Foster City, CA or the NINA heaters. For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction. The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described .", "For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction. The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system Bio-Rad Laboratories, Hercules, CA . Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain Invitrogen , which was subsequently visualized using the ChemiDoc XRS system. To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water.", "To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction. After adding 200 ml of water to each of the sample wells, temperature recording was initiated. Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation.", "Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation. The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300.", "Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300. The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC. To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction.", "To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction. For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates 60% . For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler Table 2 . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters.", "In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler Table 2 . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction. During the 60 minute incubation, the average temperature for each device was 60.2, 59.8, and 59.7 Table 3 . The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree.", "The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree. The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory 28uC; Table 2 . The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC Table 3 .", "The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory 28uC; Table 2 . The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC Table 3 . The average temperature was approximately two degrees lower than the desired reaction temperature of 60uC. Additionally, the temperature of the devices tended to decline from their steady state during the last 20 minutes of the reaction data not shown . The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters.", "The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices Table 4 . Amplification consistency was most evident with two of the patient samples patient #4 and #5 that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes.", "All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes. In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid NINA heaters for amplification of HIV-1 using RT-LAMP. The isothermal amplification reaction coupled with a device that generates heat from an exothermic chemical reaction, as opposed to grid electricity or battery power, comprises a point-of-care NAAT that is practical for use in resource-limited settings. The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler.", "The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler. Since point-of-care testing may refer to an air-conditioned laboratory or a field site with high temperatures and humidity, the stability of the temperature generated by the heating devices must be reliable. Though the temperature profiles at a representative cold temperature of 4uC indicated a loss in reaction temperature towards the end of the 60 minute incubation, the temperature fluctuations were not significant enough to affect the amplification reaction. Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both.", "Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option. We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory. For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume.", "For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction. While the additional heating step is not necessary to observe the amplified products from extracted nucleic acid, the short, high-temperature incubation facilitates the visual observation of the fluorescent label in the whole blood samples. Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the amplification compartment and reinserted into the 80uC compartment.", "Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the amplification compartment and reinserted into the 80uC compartment. Lastly, the DaqPRO data recorder was used in this study for validation purposes only and would not be necessary for the final POC product. The feasibility of using LAMP as a diagnostic method in resource-limited settings has been demonstrated for tuberculosis . To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube.", "To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube. We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification.", "Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification. Future modifications will include optimizing the labeledprimer/quencher sequences so that all components can be added into the reaction mix prior to amplification. Due to availability, the Bio-Rad ChemiDoc system was used as the UV source in this study; however, an inexpensive keychain light would be more suitable for naked-eye detection at the POC. For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes.", "For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device. In summary, the RT-LAMP isothermal amplification method used in conjunction with a simplified, chemical heating device exhibits characteristics that are ideal for a rapid NAAT for POC testing. The simplified, portable assay has the potential to fill an important gap in HIV-1 diagnostics, providing immediate knowledge or confirmation of HIV-1 infection status at the POC." ]

[ "A series of nine substituted 2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides was assessed as prospective bactericidal agents against three clinical isolates of methicillin-resistant Staphylococcus aureus MRSA and S. aureus ATCC 29213 as the reference and quality control strain. The minimum bactericidal concentration was determined by subculturing aliquots from MIC determination onto substance-free agar plates. The bactericidal kinetics of compounds 5-chloro-2-hydroxy-N- 2S -3-methyl-1-oxo-1-{ 4- trifluoromethyl phenyl amino}butan-2-yl benzamide 1f , N-{ 2S -1- 4-bromophenyl amino -3-methyl-1-oxobutan-2-yl}-4-chloro-2-hydroxybenzamide 1g , and 4-chloro-N-{ 2S -1- 3,4-dichlorophenyl amino -3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide 1h was established by time-kill assay with a final concentration of the compound equal to 1x, 2x, and 4x MIC; aliquots were removed at 0, 4, 6, 8, and 24 h time points. The most potent bactericidal agent was compound 1f exhibiting remarkable rapid concentration-dependent bactericidal effect even at 2x MIC at 4, 6, and 8 h with a reduction in bacterial count ranging from 3.08 to 3.75 log. CFU/mL and at 4x MIC at 4, 6, 8, and 24 h 5.30 log. CFU/mL reduction in bacterial count after incubation against MRSA 63718.", "CFU/mL and at 4x MIC at 4, 6, 8, and 24 h 5.30 log. CFU/mL reduction in bacterial count after incubation against MRSA 63718. Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 . The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration MIC values i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism within the susceptible range was described previously .", "The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 . The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration MIC values i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism within the susceptible range was described previously . Thus, the emergence of MRSA and vancomycin-resistant S. aureus in the recent years as well makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria . However, for the treatment of S. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs . This fact should be considered during the development of effective and safe treatment options for MRSA infections. The history of clinical usage of salicylanilides 2-hydroxy-N-phenylbenzamides dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s .", "This fact should be considered during the development of effective and safe treatment options for MRSA infections. The history of clinical usage of salicylanilides 2-hydroxy-N-phenylbenzamides dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s . Nowadays, salicylanilides SALs are a class of aromatic compounds possessing a wide range of interesting pharmacological activities, such as anthelmintic , antibacterial , antimycobacterial , antifungal , and antiviral , among others. Despite being studied since the 1960s, the mechanism of action responsible for biological activities of these compounds has not been explained so far. SALs have been found to inhibit the two-component regulatory systems TCS of bacteria . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis .", "SALs have been found to inhibit the two-component regulatory systems TCS of bacteria . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis . Furthermore, salicylanilides have been recognised as inhibitors of some bacterial enzymes, such as sortase A from S. aureus , d-alanine-d-alanine ligase , or transglycosylases from S. aureus but not from M. tuberculosis . These enzymes participate in secretion of various proteins or in biosynthesis of bacterial cell wall. Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action.", "Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges. This study is a follow-up paper to a recently published article . The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides, called diamides due to their skeleton for general structure see Table 1 , was described previously , and their antimycobacterial and antibacterial activities against various bacterial species were reported . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA.", "The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides, called diamides due to their skeleton for general structure see Table 1 , was described previously , and their antimycobacterial and antibacterial activities against various bacterial species were reported . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA. The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis . ; and MRSA SA 3202 National Institute of Public Health, Prague, Czech Republic both of human origin.", "The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis . ; and MRSA SA 3202 National Institute of Public Health, Prague, Czech Republic both of human origin. Suspected colonies were confirmed by PCR; a 108 bp fragment specific for S. aureus was detected . All isolates were tested for the presence of the mecA gene encoding methicillin resistance . These three clinical isolates were classified as vancomycin-susceptible but with higher MIC of vancomycin equal to 2 g/mL VA2-MRSA within the susceptible range for MRSA 63718 methicillinresistant S. aureus VS-MRSA . For the MICs of vancomycin, see Table 1 .", "These three clinical isolates were classified as vancomycin-susceptible but with higher MIC of vancomycin equal to 2 g/mL VA2-MRSA within the susceptible range for MRSA 63718 methicillinresistant S. aureus VS-MRSA . For the MICs of vancomycin, see Table 1 . Vancomycin-susceptible methicillin-susceptible Staphylococcus aureus VS-MSSA ATCC 29213, obtained from the American Type Culture Collection, was used as the reference and quality control strain. The bacteria were stored at −80 ∘ C and were kept on blood agar plates Columbia agar base with 5% ovine blood between experiments. MBCs . The MBCs i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium were determined by subculturing aliquots 20 L from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar MHA plates.", "MBCs . The MBCs i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium were determined by subculturing aliquots 20 L from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar MHA plates. The plates were incubated aerobically at 37 ∘ C for 24 h for colony count. The MBC was defined as the lowest concentration of substance, which produced ≥99.9% killing Table 1 : Chemical structures and in vitro MIC and MBC g/mL values of tested 5-and 4-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides bactericidal effect of individual compounds against particular strains marked in bold . after 24 h of incubation as compared to the colony count of the starting inoculum . To ensure reproducibility, each MBC assay was performed in at least triplicate on separate occasions.", "after 24 h of incubation as compared to the colony count of the starting inoculum . To ensure reproducibility, each MBC assay was performed in at least triplicate on separate occasions. N H O H N O OH 1 2 R 1 R 3 R 2 Comp. R 1 R 2 R 3 MIC g/mL MBC g/mL 1 2 3 4 1 2 3 4 1a 5-Cl 4-CH 3 S -CH 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-Cl 4-CH 3 S -CH CH 3 2 >256 >256 32 32 >256 >256 128 >256 1c 5-Cl 4-CH 3 S -benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-Cl 4-CH 3 R -CH 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-Cl 4-OCH 3 S -CH CH 3 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-Cl 4-CF 3 S -CH CH 3 2 4 2 2 2 4 4 8 4 1g 4-Cl 4-Br S -CH CH 3 2 8 4 4 4 1 6 8 8 8 1h 4-Cl 3,4-Cl S -CH CH 3 2 2 1 1 1 4 1 4 2 1i 4-Cl 3,4-Cl S -benzyl 1 1 0.5 0.5 8 1 8 1 AMP - - - >16 >16 >16 0.25 >16 >16 >16 0.25 CPX - - - >16 >16 >16 0.5 >16 >16 >16 0.5 VAN - - - 2 1 1 1 2 1 1 1 Time-kill assays were performed by the broth macrodilution method according to previously described methodology with some modifications. Briefly, flasks containing sterile fresh Mueller-Hinton broth MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots 20 L were plated on MHA plates in duplicate.", "Briefly, flasks containing sterile fresh Mueller-Hinton broth MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots 20 L were plated on MHA plates in duplicate. Colony counts were performed on plates yielding 6 to 60 colonies, and the mean was calculated. Antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments.", "The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments. The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included. Time-kill curves were constructed by plotting the log 10 CFU per millilitre versus time over 24 h , and the change in bacterial concentration was determined. The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum. Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently .", "The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum. Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently . In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 methicillin-susceptible as the reference and quality control strain. Since SALs and their analogues are known as compounds with bacteriostatic effect , this is the first study where SAL-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. Recently MIC values of diamides expressed as molar concentrations in mol/L were published .", "Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. Recently MIC values of diamides expressed as molar concentrations in mol/L were published . To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. Potential bactericidal activity of diamides was assessed using MBC assay . MBC values of all tested compounds are recorded in Table 1 as well. Based on the obtained results, all compounds assessed as active according to MIC values in our previous study 1f-i showed low or moderate MBC values against all four strains.", "MBC values of all tested compounds are recorded in Table 1 as well. Based on the obtained results, all compounds assessed as active according to MIC values in our previous study 1f-i showed low or moderate MBC values against all four strains. The MBC values of these compounds did not exceed the highest tested drug concentration and ranged from 1 to 16 g/mL. In all cases, there were comparable MBC values for the clinical isolates of MRSA and the S. aureus reference strain. Bactericidal activity is defined as a ratio of MBC to MIC of ≤4 . Table 1 bactericidal activity is expressed in bold.", "Bactericidal activity is defined as a ratio of MBC to MIC of ≤4 . Table 1 bactericidal activity is expressed in bold. As mentioned above, SALs are known to exhibit a bacteriostatic effect , so it was very interesting to discover that diamides possess bactericidal activity. The amide bond -CONH- can cause interactions with a variety of enzymes ; therefore the presence of two amide bonds could be responsible for the bactericidal effect of diamides against MRSA. The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus including MRSA cell wall biosynthesis . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane .", "The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus including MRSA cell wall biosynthesis . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane . Since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents , the failure in the cell wall biosynthesis due to the inhibition of transglycosylases could be responsible for bactericidal activity of diamides against MRSA. Based on these findings, antibacterial active diamides with bactericidal effect against all four tested strains as prospective bactericidal agents were chosen for subsequent timekill curve studies to determine the real dependence of bactericidal effect on concentration over time. 1-oxobutan-2-yl}-2-hydroxybenzamide 1h were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain. The antibacterial effect of DMSO used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control.", "1-oxobutan-2-yl}-2-hydroxybenzamide 1h were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain. The antibacterial effect of DMSO used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control. The extent of bacterial killing was estimated by the number of these strains showing a decrease ranging from 1 to 3 log 10 CFU/mL in viable cell count at different times after incubation. A summary of these data is presented in Table 2 . Based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. No bactericidal activity i.e., ≥3 log 10 CFU/mL decrease was observed at 1x MIC for any strain and time after incubation tested.", "Based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. No bactericidal activity i.e., ≥3 log 10 CFU/mL decrease was observed at 1x MIC for any strain and time after incubation tested. At 4x MIC from the four strains, compounds 1f, 1 g, and 1h killed 2, 1, and 2 strains, respectively, at 8 h after incubation and 4, 2, and 2 strains, respectively, at 24 h after incubation. The findings of time-kill studies for each of the four staphylococci strains at exposure to compounds 1f, 1g, and 1h are summarized in Table 3 . Bactericidal activity i.e., ≥3 log 10 CFU/mL decrease is expressed in bold. For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718.", "Bactericidal activity i.e., ≥3 log 10 CFU/mL decrease is expressed in bold. For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718. Time was not the predictive factor influencing the antibacterial activity because log 10 differences in CFU/mL from the starting inoculum were the same for 4x MIC with the highest efficiency with a reduction in bacterial count of 5.30 log 10 CFU/mL or very similar for 2x MIC with a moderate regrowth after 24 h causing a loss of bactericidal activity over 24 h. The bactericidal effect was maintained even at 2x MIC at 4 h after incubation for this strain reduction of 3.08 log 10 CFU/mL . For the remaining strains, clinical isolates of MRSA SA 630, MRSA SA 3202, and S. aureus ATCC 29213, reliable bactericidal effect was recorded at 4x MIC at 24 h after incubation for all these strains with a reduction in bacterial count of 3.22, 3.30, and 3.65 log 10 CFU/mL, respectively. For compound 1g bactericidal effect against MRSA 63718 was noticed at 2x MIC at 6 and 8 h after incubation and at 4x MIC at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 CFU/mL. The most effective killing was achieved at 6 h for both concentrations.", "For compound 1g bactericidal effect against MRSA 63718 was noticed at 2x MIC at 6 and 8 h after incubation and at 4x MIC at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 CFU/mL. The most effective killing was achieved at 6 h for both concentrations. As in the case of compound 1f, a regrowth was observed after 24 h after incubation. For the remaining isolates of MRSA, SA 630 and SA 3202, bactericidal effect occurred only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.38 and 4.01 log 10 CFU/mL, respectively. The highest bactericidal effect was recorded for MRSA SA 3202 at 4x MIC at 24 h after incubation. A reduction consistent with bacteriostatic effect 0.03 to 2.37 log 10 CFU/mL was observed at other concentrations over time for both isolates.", "The highest bactericidal effect was recorded for MRSA SA 3202 at 4x MIC at 24 h after incubation. A reduction consistent with bacteriostatic effect 0.03 to 2.37 log 10 CFU/mL was observed at other concentrations over time for both isolates. No bactericidal effect was observed for the S. aureus reference strain; compound 1g demonstrated a pattern of bacteriostatic activity against this strain with a reduction in bacterial count ranging from 0.07 to 2.33 log 10 CFU/mL at 4x MIC over time. In other cases, a slight increase in bacterial counts i.e., overgrowth compared with the starting inoculum was observed with values ranging from 0.10 to 1.57 log 10 CFU/mL for this reference strain. For compound 1h bactericidal effect against MRSA 63718 was maintained at 4x MIC at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 CFU/mL, respectively. The same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation.", "For compound 1h bactericidal effect against MRSA 63718 was maintained at 4x MIC at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 CFU/mL, respectively. The same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation. Regrowth at 24 h after incubation causing a loss of bactericidal activity was recorded similarly as with previous compounds. The reason for regrowth of the test organism at 24 h in the experiment is unknown. Most probably, selection of resistant mutants is responsible for this phenomenon ; degradation of the drug in the growth medium is not assumed, as regrowth was Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain. For MRSA SA 630 concentration-dependent killing was recorded at 4x MIC at 6, 8, and 24 h after incubation with log 10 differences in CFU/mL from the starting inoculum being very similar over time ranging from 3.18 to 3.39 log 10 CFU/mL .", "Most probably, selection of resistant mutants is responsible for this phenomenon ; degradation of the drug in the growth medium is not assumed, as regrowth was Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain. For MRSA SA 630 concentration-dependent killing was recorded at 4x MIC at 6, 8, and 24 h after incubation with log 10 differences in CFU/mL from the starting inoculum being very similar over time ranging from 3.18 to 3.39 log 10 CFU/mL . For MRSA SA 3202 reliable bactericidal effect was maintained only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.02 log 10 CFU/mL. As for compound 1g, bacteriostatic activity against S. aureus reference strain was observed with a reduction in bacterial count ranging from 0.34 to 2.62 log 10 CFU/mL at 2x and 4x MIC. Overgrowth values ranging from 0.04 to 1.43 log 10 CFU/mL was recorded at 1x MIC for this strain. It is of note that in all staphylococci strains with similar MICs and MBCs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied with clinical strains of MRSA being effectively killed and the reference strain remaining unaffected at 4x MIC.", "Overgrowth values ranging from 0.04 to 1.43 log 10 CFU/mL was recorded at 1x MIC for this strain. It is of note that in all staphylococci strains with similar MICs and MBCs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied with clinical strains of MRSA being effectively killed and the reference strain remaining unaffected at 4x MIC. There is a discrepancy between bactericidal results of MBC assay compared with time-kill kinetics. This difference could be caused by comparing microtiter MBC assay to macrobroth time-kill assay dilutions . Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents . Concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then .", "Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents . Concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then . However, bactericidal therapy could produce a better treatment result by rapid reduction of the bacterial load . Moreover, in the case of an immune system disorder e.g., immunosuppressive therapy, AIDS patients, etc. bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds .", "bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds . The clinical outcome of MRSA bacteraemia is significantly influenced by vancomycin MIC. Treatment failure exceeding 60% for S. aureus with vancomycin MIC of 4 g/mL resulted in the change of susceptibility breakpoint from 4 g/mL to 2 g/mL by the Clinical and Laboratory Standards Institute CLSI in 2006 as well as by the US Food and Drug Administration FDA in 2008 . It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs 2 g/mL , alternative therapy should be considered . It is of note that based on time-kill assays in the present study, all tested diamides particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL.", "It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs 2 g/mL , alternative therapy should be considered . It is of note that based on time-kill assays in the present study, all tested diamides particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL. The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. The present study is the first evidence of bactericidal effect of SAL analogues.", "Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. The present study is the first evidence of bactericidal effect of SAL analogues. Against other strains, reliable bactericidal effect was maintained at 4x MIC at 24 h after incubation. Considering the necessity to broaden the spectrum of bactericidal agents, diamides from the current study with a novel mechanism of action could present a very promising and interesting solution to this challenge for the future." ]

[ "A series of nine substituted 2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides was assessed as prospective bactericidal agents against three clinical isolates of methicillin-resistant Staphylococcus aureus MRSA and S. aureus ATCC 29213 as the reference and quality control strain. The minimum bactericidal concentration was determined by subculturing aliquots from MIC determination onto substance-free agar plates. The bactericidal kinetics of compounds 5-chloro-2-hydroxy-N- 2S -3-methyl-1-oxo-1-{ 4- trifluoromethyl phenyl amino}butan-2-yl benzamide 1f , N-{ 2S -1- 4-bromophenyl amino -3-methyl-1-oxobutan-2-yl}-4-chloro-2-hydroxybenzamide 1g , and 4-chloro-N-{ 2S -1- 3,4-dichlorophenyl amino -3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide 1h was established by time-kill assay with a final concentration of the compound equal to 1x, 2x, and 4x MIC; aliquots were removed at 0, 4, 6, 8, and 24 h time points. The most potent bactericidal agent was compound 1f exhibiting remarkable rapid concentration-dependent bactericidal effect even at 2x MIC at 4, 6, and 8 h with a reduction in bacterial count ranging from 3.08 to 3.75 log. CFU/mL and at 4x MIC at 4, 6, 8, and 24 h 5.30 log. CFU/mL reduction in bacterial count after incubation against MRSA 63718.", "CFU/mL and at 4x MIC at 4, 6, 8, and 24 h 5.30 log. CFU/mL reduction in bacterial count after incubation against MRSA 63718. Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 . The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration MIC values i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism within the susceptible range was described previously .", "The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 . The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration MIC values i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism within the susceptible range was described previously . Thus, the emergence of MRSA and vancomycin-resistant S. aureus in the recent years as well makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria . However, for the treatment of S. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs . This fact should be considered during the development of effective and safe treatment options for MRSA infections. The history of clinical usage of salicylanilides 2-hydroxy-N-phenylbenzamides dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s .", "This fact should be considered during the development of effective and safe treatment options for MRSA infections. The history of clinical usage of salicylanilides 2-hydroxy-N-phenylbenzamides dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s . Nowadays, salicylanilides SALs are a class of aromatic compounds possessing a wide range of interesting pharmacological activities, such as anthelmintic , antibacterial , antimycobacterial , antifungal , and antiviral , among others. Despite being studied since the 1960s, the mechanism of action responsible for biological activities of these compounds has not been explained so far. SALs have been found to inhibit the two-component regulatory systems TCS of bacteria . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis .", "SALs have been found to inhibit the two-component regulatory systems TCS of bacteria . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis . Furthermore, salicylanilides have been recognised as inhibitors of some bacterial enzymes, such as sortase A from S. aureus , d-alanine-d-alanine ligase , or transglycosylases from S. aureus but not from M. tuberculosis . These enzymes participate in secretion of various proteins or in biosynthesis of bacterial cell wall. Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action.", "Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges. This study is a follow-up paper to a recently published article . The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides, called diamides due to their skeleton for general structure see Table 1 , was described previously , and their antimycobacterial and antibacterial activities against various bacterial species were reported . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA.", "The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides, called diamides due to their skeleton for general structure see Table 1 , was described previously , and their antimycobacterial and antibacterial activities against various bacterial species were reported . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA. The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis . ; and MRSA SA 3202 National Institute of Public Health, Prague, Czech Republic both of human origin.", "The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis . ; and MRSA SA 3202 National Institute of Public Health, Prague, Czech Republic both of human origin. Suspected colonies were confirmed by PCR; a 108 bp fragment specific for S. aureus was detected . All isolates were tested for the presence of the mecA gene encoding methicillin resistance . These three clinical isolates were classified as vancomycin-susceptible but with higher MIC of vancomycin equal to 2 g/mL VA2-MRSA within the susceptible range for MRSA 63718 methicillinresistant S. aureus VS-MRSA . For the MICs of vancomycin, see Table 1 .", "These three clinical isolates were classified as vancomycin-susceptible but with higher MIC of vancomycin equal to 2 g/mL VA2-MRSA within the susceptible range for MRSA 63718 methicillinresistant S. aureus VS-MRSA . For the MICs of vancomycin, see Table 1 . Vancomycin-susceptible methicillin-susceptible Staphylococcus aureus VS-MSSA ATCC 29213, obtained from the American Type Culture Collection, was used as the reference and quality control strain. The bacteria were stored at −80 ∘ C and were kept on blood agar plates Columbia agar base with 5% ovine blood between experiments. MBCs . The MBCs i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium were determined by subculturing aliquots 20 L from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar MHA plates.", "MBCs . The MBCs i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium were determined by subculturing aliquots 20 L from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar MHA plates. The plates were incubated aerobically at 37 ∘ C for 24 h for colony count. The MBC was defined as the lowest concentration of substance, which produced ≥99.9% killing Table 1 : Chemical structures and in vitro MIC and MBC g/mL values of tested 5-and 4-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides bactericidal effect of individual compounds against particular strains marked in bold . after 24 h of incubation as compared to the colony count of the starting inoculum . To ensure reproducibility, each MBC assay was performed in at least triplicate on separate occasions.", "after 24 h of incubation as compared to the colony count of the starting inoculum . To ensure reproducibility, each MBC assay was performed in at least triplicate on separate occasions. N H O H N O OH 1 2 R 1 R 3 R 2 Comp. R 1 R 2 R 3 MIC g/mL MBC g/mL 1 2 3 4 1 2 3 4 1a 5-Cl 4-CH 3 S -CH 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-Cl 4-CH 3 S -CH CH 3 2 >256 >256 32 32 >256 >256 128 >256 1c 5-Cl 4-CH 3 S -benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-Cl 4-CH 3 R -CH 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-Cl 4-OCH 3 S -CH CH 3 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-Cl 4-CF 3 S -CH CH 3 2 4 2 2 2 4 4 8 4 1g 4-Cl 4-Br S -CH CH 3 2 8 4 4 4 1 6 8 8 8 1h 4-Cl 3,4-Cl S -CH CH 3 2 2 1 1 1 4 1 4 2 1i 4-Cl 3,4-Cl S -benzyl 1 1 0.5 0.5 8 1 8 1 AMP - - - >16 >16 >16 0.25 >16 >16 >16 0.25 CPX - - - >16 >16 >16 0.5 >16 >16 >16 0.5 VAN - - - 2 1 1 1 2 1 1 1 Time-kill assays were performed by the broth macrodilution method according to previously described methodology with some modifications. Briefly, flasks containing sterile fresh Mueller-Hinton broth MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots 20 L were plated on MHA plates in duplicate.", "Briefly, flasks containing sterile fresh Mueller-Hinton broth MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots 20 L were plated on MHA plates in duplicate. Colony counts were performed on plates yielding 6 to 60 colonies, and the mean was calculated. Antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments.", "The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments. The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included. Time-kill curves were constructed by plotting the log 10 CFU per millilitre versus time over 24 h , and the change in bacterial concentration was determined. The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum. Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently .", "The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum. Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently . In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 methicillin-susceptible as the reference and quality control strain. Since SALs and their analogues are known as compounds with bacteriostatic effect , this is the first study where SAL-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. Recently MIC values of diamides expressed as molar concentrations in mol/L were published .", "Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. Recently MIC values of diamides expressed as molar concentrations in mol/L were published . To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. Potential bactericidal activity of diamides was assessed using MBC assay . MBC values of all tested compounds are recorded in Table 1 as well. Based on the obtained results, all compounds assessed as active according to MIC values in our previous study 1f-i showed low or moderate MBC values against all four strains.", "MBC values of all tested compounds are recorded in Table 1 as well. Based on the obtained results, all compounds assessed as active according to MIC values in our previous study 1f-i showed low or moderate MBC values against all four strains. The MBC values of these compounds did not exceed the highest tested drug concentration and ranged from 1 to 16 g/mL. In all cases, there were comparable MBC values for the clinical isolates of MRSA and the S. aureus reference strain. Bactericidal activity is defined as a ratio of MBC to MIC of ≤4 . Table 1 bactericidal activity is expressed in bold.", "Bactericidal activity is defined as a ratio of MBC to MIC of ≤4 . Table 1 bactericidal activity is expressed in bold. As mentioned above, SALs are known to exhibit a bacteriostatic effect , so it was very interesting to discover that diamides possess bactericidal activity. The amide bond -CONH- can cause interactions with a variety of enzymes ; therefore the presence of two amide bonds could be responsible for the bactericidal effect of diamides against MRSA. The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus including MRSA cell wall biosynthesis . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane .", "The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus including MRSA cell wall biosynthesis . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane . Since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents , the failure in the cell wall biosynthesis due to the inhibition of transglycosylases could be responsible for bactericidal activity of diamides against MRSA. Based on these findings, antibacterial active diamides with bactericidal effect against all four tested strains as prospective bactericidal agents were chosen for subsequent timekill curve studies to determine the real dependence of bactericidal effect on concentration over time. 1-oxobutan-2-yl}-2-hydroxybenzamide 1h were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain. The antibacterial effect of DMSO used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control.", "1-oxobutan-2-yl}-2-hydroxybenzamide 1h were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain. The antibacterial effect of DMSO used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control. The extent of bacterial killing was estimated by the number of these strains showing a decrease ranging from 1 to 3 log 10 CFU/mL in viable cell count at different times after incubation. A summary of these data is presented in Table 2 . Based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. No bactericidal activity i.e., ≥3 log 10 CFU/mL decrease was observed at 1x MIC for any strain and time after incubation tested.", "Based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. No bactericidal activity i.e., ≥3 log 10 CFU/mL decrease was observed at 1x MIC for any strain and time after incubation tested. At 4x MIC from the four strains, compounds 1f, 1 g, and 1h killed 2, 1, and 2 strains, respectively, at 8 h after incubation and 4, 2, and 2 strains, respectively, at 24 h after incubation. The findings of time-kill studies for each of the four staphylococci strains at exposure to compounds 1f, 1g, and 1h are summarized in Table 3 . Bactericidal activity i.e., ≥3 log 10 CFU/mL decrease is expressed in bold. For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718.", "Bactericidal activity i.e., ≥3 log 10 CFU/mL decrease is expressed in bold. For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718. Time was not the predictive factor influencing the antibacterial activity because log 10 differences in CFU/mL from the starting inoculum were the same for 4x MIC with the highest efficiency with a reduction in bacterial count of 5.30 log 10 CFU/mL or very similar for 2x MIC with a moderate regrowth after 24 h causing a loss of bactericidal activity over 24 h. The bactericidal effect was maintained even at 2x MIC at 4 h after incubation for this strain reduction of 3.08 log 10 CFU/mL . For the remaining strains, clinical isolates of MRSA SA 630, MRSA SA 3202, and S. aureus ATCC 29213, reliable bactericidal effect was recorded at 4x MIC at 24 h after incubation for all these strains with a reduction in bacterial count of 3.22, 3.30, and 3.65 log 10 CFU/mL, respectively. For compound 1g bactericidal effect against MRSA 63718 was noticed at 2x MIC at 6 and 8 h after incubation and at 4x MIC at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 CFU/mL. The most effective killing was achieved at 6 h for both concentrations.", "For compound 1g bactericidal effect against MRSA 63718 was noticed at 2x MIC at 6 and 8 h after incubation and at 4x MIC at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 CFU/mL. The most effective killing was achieved at 6 h for both concentrations. As in the case of compound 1f, a regrowth was observed after 24 h after incubation. For the remaining isolates of MRSA, SA 630 and SA 3202, bactericidal effect occurred only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.38 and 4.01 log 10 CFU/mL, respectively. The highest bactericidal effect was recorded for MRSA SA 3202 at 4x MIC at 24 h after incubation. A reduction consistent with bacteriostatic effect 0.03 to 2.37 log 10 CFU/mL was observed at other concentrations over time for both isolates.", "The highest bactericidal effect was recorded for MRSA SA 3202 at 4x MIC at 24 h after incubation. A reduction consistent with bacteriostatic effect 0.03 to 2.37 log 10 CFU/mL was observed at other concentrations over time for both isolates. No bactericidal effect was observed for the S. aureus reference strain; compound 1g demonstrated a pattern of bacteriostatic activity against this strain with a reduction in bacterial count ranging from 0.07 to 2.33 log 10 CFU/mL at 4x MIC over time. In other cases, a slight increase in bacterial counts i.e., overgrowth compared with the starting inoculum was observed with values ranging from 0.10 to 1.57 log 10 CFU/mL for this reference strain. For compound 1h bactericidal effect against MRSA 63718 was maintained at 4x MIC at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 CFU/mL, respectively. The same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation.", "For compound 1h bactericidal effect against MRSA 63718 was maintained at 4x MIC at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 CFU/mL, respectively. The same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation. Regrowth at 24 h after incubation causing a loss of bactericidal activity was recorded similarly as with previous compounds. The reason for regrowth of the test organism at 24 h in the experiment is unknown. Most probably, selection of resistant mutants is responsible for this phenomenon ; degradation of the drug in the growth medium is not assumed, as regrowth was Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain. For MRSA SA 630 concentration-dependent killing was recorded at 4x MIC at 6, 8, and 24 h after incubation with log 10 differences in CFU/mL from the starting inoculum being very similar over time ranging from 3.18 to 3.39 log 10 CFU/mL .", "Most probably, selection of resistant mutants is responsible for this phenomenon ; degradation of the drug in the growth medium is not assumed, as regrowth was Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain. For MRSA SA 630 concentration-dependent killing was recorded at 4x MIC at 6, 8, and 24 h after incubation with log 10 differences in CFU/mL from the starting inoculum being very similar over time ranging from 3.18 to 3.39 log 10 CFU/mL . For MRSA SA 3202 reliable bactericidal effect was maintained only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.02 log 10 CFU/mL. As for compound 1g, bacteriostatic activity against S. aureus reference strain was observed with a reduction in bacterial count ranging from 0.34 to 2.62 log 10 CFU/mL at 2x and 4x MIC. Overgrowth values ranging from 0.04 to 1.43 log 10 CFU/mL was recorded at 1x MIC for this strain. It is of note that in all staphylococci strains with similar MICs and MBCs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied with clinical strains of MRSA being effectively killed and the reference strain remaining unaffected at 4x MIC.", "Overgrowth values ranging from 0.04 to 1.43 log 10 CFU/mL was recorded at 1x MIC for this strain. It is of note that in all staphylococci strains with similar MICs and MBCs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied with clinical strains of MRSA being effectively killed and the reference strain remaining unaffected at 4x MIC. There is a discrepancy between bactericidal results of MBC assay compared with time-kill kinetics. This difference could be caused by comparing microtiter MBC assay to macrobroth time-kill assay dilutions . Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents . Concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then .", "Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents . Concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then . However, bactericidal therapy could produce a better treatment result by rapid reduction of the bacterial load . Moreover, in the case of an immune system disorder e.g., immunosuppressive therapy, AIDS patients, etc. bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds .", "bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds . The clinical outcome of MRSA bacteraemia is significantly influenced by vancomycin MIC. Treatment failure exceeding 60% for S. aureus with vancomycin MIC of 4 g/mL resulted in the change of susceptibility breakpoint from 4 g/mL to 2 g/mL by the Clinical and Laboratory Standards Institute CLSI in 2006 as well as by the US Food and Drug Administration FDA in 2008 . It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs 2 g/mL , alternative therapy should be considered . It is of note that based on time-kill assays in the present study, all tested diamides particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL.", "It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs 2 g/mL , alternative therapy should be considered . It is of note that based on time-kill assays in the present study, all tested diamides particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL. The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. The present study is the first evidence of bactericidal effect of SAL analogues.", "Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. The present study is the first evidence of bactericidal effect of SAL analogues. Against other strains, reliable bactericidal effect was maintained at 4x MIC at 24 h after incubation. Considering the necessity to broaden the spectrum of bactericidal agents, diamides from the current study with a novel mechanism of action could present a very promising and interesting solution to this challenge for the future." ]

[ "A series of nine substituted 2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides was assessed as prospective bactericidal agents against three clinical isolates of methicillin-resistant Staphylococcus aureus MRSA and S. aureus ATCC 29213 as the reference and quality control strain. The minimum bactericidal concentration was determined by subculturing aliquots from MIC determination onto substance-free agar plates. The bactericidal kinetics of compounds 5-chloro-2-hydroxy-N- 2S -3-methyl-1-oxo-1-{ 4- trifluoromethyl phenyl amino}butan-2-yl benzamide 1f , N-{ 2S -1- 4-bromophenyl amino -3-methyl-1-oxobutan-2-yl}-4-chloro-2-hydroxybenzamide 1g , and 4-chloro-N-{ 2S -1- 3,4-dichlorophenyl amino -3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide 1h was established by time-kill assay with a final concentration of the compound equal to 1x, 2x, and 4x MIC; aliquots were removed at 0, 4, 6, 8, and 24 h time points. The most potent bactericidal agent was compound 1f exhibiting remarkable rapid concentration-dependent bactericidal effect even at 2x MIC at 4, 6, and 8 h with a reduction in bacterial count ranging from 3.08 to 3.75 log. CFU/mL and at 4x MIC at 4, 6, 8, and 24 h 5.30 log. CFU/mL reduction in bacterial count after incubation against MRSA 63718.", "CFU/mL and at 4x MIC at 4, 6, 8, and 24 h 5.30 log. CFU/mL reduction in bacterial count after incubation against MRSA 63718. Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 . The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration MIC values i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism within the susceptible range was described previously .", "The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 . The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration MIC values i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism within the susceptible range was described previously . Thus, the emergence of MRSA and vancomycin-resistant S. aureus in the recent years as well makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria . However, for the treatment of S. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs . This fact should be considered during the development of effective and safe treatment options for MRSA infections. The history of clinical usage of salicylanilides 2-hydroxy-N-phenylbenzamides dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s .", "This fact should be considered during the development of effective and safe treatment options for MRSA infections. The history of clinical usage of salicylanilides 2-hydroxy-N-phenylbenzamides dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s . Nowadays, salicylanilides SALs are a class of aromatic compounds possessing a wide range of interesting pharmacological activities, such as anthelmintic , antibacterial , antimycobacterial , antifungal , and antiviral , among others. Despite being studied since the 1960s, the mechanism of action responsible for biological activities of these compounds has not been explained so far. SALs have been found to inhibit the two-component regulatory systems TCS of bacteria . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis .", "SALs have been found to inhibit the two-component regulatory systems TCS of bacteria . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis . Furthermore, salicylanilides have been recognised as inhibitors of some bacterial enzymes, such as sortase A from S. aureus , d-alanine-d-alanine ligase , or transglycosylases from S. aureus but not from M. tuberculosis . These enzymes participate in secretion of various proteins or in biosynthesis of bacterial cell wall. Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action.", "Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges. This study is a follow-up paper to a recently published article . The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides, called diamides due to their skeleton for general structure see Table 1 , was described previously , and their antimycobacterial and antibacterial activities against various bacterial species were reported . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA.", "The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides, called diamides due to their skeleton for general structure see Table 1 , was described previously , and their antimycobacterial and antibacterial activities against various bacterial species were reported . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA. The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis . ; and MRSA SA 3202 National Institute of Public Health, Prague, Czech Republic both of human origin.", "The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis . ; and MRSA SA 3202 National Institute of Public Health, Prague, Czech Republic both of human origin. Suspected colonies were confirmed by PCR; a 108 bp fragment specific for S. aureus was detected . All isolates were tested for the presence of the mecA gene encoding methicillin resistance . These three clinical isolates were classified as vancomycin-susceptible but with higher MIC of vancomycin equal to 2 g/mL VA2-MRSA within the susceptible range for MRSA 63718 methicillinresistant S. aureus VS-MRSA . For the MICs of vancomycin, see Table 1 .", "These three clinical isolates were classified as vancomycin-susceptible but with higher MIC of vancomycin equal to 2 g/mL VA2-MRSA within the susceptible range for MRSA 63718 methicillinresistant S. aureus VS-MRSA . For the MICs of vancomycin, see Table 1 . Vancomycin-susceptible methicillin-susceptible Staphylococcus aureus VS-MSSA ATCC 29213, obtained from the American Type Culture Collection, was used as the reference and quality control strain. The bacteria were stored at −80 ∘ C and were kept on blood agar plates Columbia agar base with 5% ovine blood between experiments. MBCs . The MBCs i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium were determined by subculturing aliquots 20 L from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar MHA plates.", "MBCs . The MBCs i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium were determined by subculturing aliquots 20 L from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar MHA plates. The plates were incubated aerobically at 37 ∘ C for 24 h for colony count. The MBC was defined as the lowest concentration of substance, which produced ≥99.9% killing Table 1 : Chemical structures and in vitro MIC and MBC g/mL values of tested 5-and 4-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides bactericidal effect of individual compounds against particular strains marked in bold . after 24 h of incubation as compared to the colony count of the starting inoculum . To ensure reproducibility, each MBC assay was performed in at least triplicate on separate occasions.", "after 24 h of incubation as compared to the colony count of the starting inoculum . To ensure reproducibility, each MBC assay was performed in at least triplicate on separate occasions. N H O H N O OH 1 2 R 1 R 3 R 2 Comp. R 1 R 2 R 3 MIC g/mL MBC g/mL 1 2 3 4 1 2 3 4 1a 5-Cl 4-CH 3 S -CH 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-Cl 4-CH 3 S -CH CH 3 2 >256 >256 32 32 >256 >256 128 >256 1c 5-Cl 4-CH 3 S -benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-Cl 4-CH 3 R -CH 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-Cl 4-OCH 3 S -CH CH 3 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-Cl 4-CF 3 S -CH CH 3 2 4 2 2 2 4 4 8 4 1g 4-Cl 4-Br S -CH CH 3 2 8 4 4 4 1 6 8 8 8 1h 4-Cl 3,4-Cl S -CH CH 3 2 2 1 1 1 4 1 4 2 1i 4-Cl 3,4-Cl S -benzyl 1 1 0.5 0.5 8 1 8 1 AMP - - - >16 >16 >16 0.25 >16 >16 >16 0.25 CPX - - - >16 >16 >16 0.5 >16 >16 >16 0.5 VAN - - - 2 1 1 1 2 1 1 1 Time-kill assays were performed by the broth macrodilution method according to previously described methodology with some modifications. Briefly, flasks containing sterile fresh Mueller-Hinton broth MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots 20 L were plated on MHA plates in duplicate.", "Briefly, flasks containing sterile fresh Mueller-Hinton broth MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots 20 L were plated on MHA plates in duplicate. Colony counts were performed on plates yielding 6 to 60 colonies, and the mean was calculated. Antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments.", "The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments. The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included. Time-kill curves were constructed by plotting the log 10 CFU per millilitre versus time over 24 h , and the change in bacterial concentration was determined. The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum. Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently .", "The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum. Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently . In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 methicillin-susceptible as the reference and quality control strain. Since SALs and their analogues are known as compounds with bacteriostatic effect , this is the first study where SAL-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. Recently MIC values of diamides expressed as molar concentrations in mol/L were published .", "Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. Recently MIC values of diamides expressed as molar concentrations in mol/L were published . To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. Potential bactericidal activity of diamides was assessed using MBC assay . MBC values of all tested compounds are recorded in Table 1 as well. Based on the obtained results, all compounds assessed as active according to MIC values in our previous study 1f-i showed low or moderate MBC values against all four strains.", "MBC values of all tested compounds are recorded in Table 1 as well. Based on the obtained results, all compounds assessed as active according to MIC values in our previous study 1f-i showed low or moderate MBC values against all four strains. The MBC values of these compounds did not exceed the highest tested drug concentration and ranged from 1 to 16 g/mL. In all cases, there were comparable MBC values for the clinical isolates of MRSA and the S. aureus reference strain. Bactericidal activity is defined as a ratio of MBC to MIC of ≤4 . Table 1 bactericidal activity is expressed in bold.", "Bactericidal activity is defined as a ratio of MBC to MIC of ≤4 . Table 1 bactericidal activity is expressed in bold. As mentioned above, SALs are known to exhibit a bacteriostatic effect , so it was very interesting to discover that diamides possess bactericidal activity. The amide bond -CONH- can cause interactions with a variety of enzymes ; therefore the presence of two amide bonds could be responsible for the bactericidal effect of diamides against MRSA. The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus including MRSA cell wall biosynthesis . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane .", "The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus including MRSA cell wall biosynthesis . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane . Since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents , the failure in the cell wall biosynthesis due to the inhibition of transglycosylases could be responsible for bactericidal activity of diamides against MRSA. Based on these findings, antibacterial active diamides with bactericidal effect against all four tested strains as prospective bactericidal agents were chosen for subsequent timekill curve studies to determine the real dependence of bactericidal effect on concentration over time. 1-oxobutan-2-yl}-2-hydroxybenzamide 1h were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain. The antibacterial effect of DMSO used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control.", "1-oxobutan-2-yl}-2-hydroxybenzamide 1h were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain. The antibacterial effect of DMSO used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control. The extent of bacterial killing was estimated by the number of these strains showing a decrease ranging from 1 to 3 log 10 CFU/mL in viable cell count at different times after incubation. A summary of these data is presented in Table 2 . Based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. No bactericidal activity i.e., ≥3 log 10 CFU/mL decrease was observed at 1x MIC for any strain and time after incubation tested.", "Based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. No bactericidal activity i.e., ≥3 log 10 CFU/mL decrease was observed at 1x MIC for any strain and time after incubation tested. At 4x MIC from the four strains, compounds 1f, 1 g, and 1h killed 2, 1, and 2 strains, respectively, at 8 h after incubation and 4, 2, and 2 strains, respectively, at 24 h after incubation. The findings of time-kill studies for each of the four staphylococci strains at exposure to compounds 1f, 1g, and 1h are summarized in Table 3 . Bactericidal activity i.e., ≥3 log 10 CFU/mL decrease is expressed in bold. For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718.", "Bactericidal activity i.e., ≥3 log 10 CFU/mL decrease is expressed in bold. For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718. Time was not the predictive factor influencing the antibacterial activity because log 10 differences in CFU/mL from the starting inoculum were the same for 4x MIC with the highest efficiency with a reduction in bacterial count of 5.30 log 10 CFU/mL or very similar for 2x MIC with a moderate regrowth after 24 h causing a loss of bactericidal activity over 24 h. The bactericidal effect was maintained even at 2x MIC at 4 h after incubation for this strain reduction of 3.08 log 10 CFU/mL . For the remaining strains, clinical isolates of MRSA SA 630, MRSA SA 3202, and S. aureus ATCC 29213, reliable bactericidal effect was recorded at 4x MIC at 24 h after incubation for all these strains with a reduction in bacterial count of 3.22, 3.30, and 3.65 log 10 CFU/mL, respectively. For compound 1g bactericidal effect against MRSA 63718 was noticed at 2x MIC at 6 and 8 h after incubation and at 4x MIC at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 CFU/mL. The most effective killing was achieved at 6 h for both concentrations.", "For compound 1g bactericidal effect against MRSA 63718 was noticed at 2x MIC at 6 and 8 h after incubation and at 4x MIC at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 CFU/mL. The most effective killing was achieved at 6 h for both concentrations. As in the case of compound 1f, a regrowth was observed after 24 h after incubation. For the remaining isolates of MRSA, SA 630 and SA 3202, bactericidal effect occurred only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.38 and 4.01 log 10 CFU/mL, respectively. The highest bactericidal effect was recorded for MRSA SA 3202 at 4x MIC at 24 h after incubation. A reduction consistent with bacteriostatic effect 0.03 to 2.37 log 10 CFU/mL was observed at other concentrations over time for both isolates.", "The highest bactericidal effect was recorded for MRSA SA 3202 at 4x MIC at 24 h after incubation. A reduction consistent with bacteriostatic effect 0.03 to 2.37 log 10 CFU/mL was observed at other concentrations over time for both isolates. No bactericidal effect was observed for the S. aureus reference strain; compound 1g demonstrated a pattern of bacteriostatic activity against this strain with a reduction in bacterial count ranging from 0.07 to 2.33 log 10 CFU/mL at 4x MIC over time. In other cases, a slight increase in bacterial counts i.e., overgrowth compared with the starting inoculum was observed with values ranging from 0.10 to 1.57 log 10 CFU/mL for this reference strain. For compound 1h bactericidal effect against MRSA 63718 was maintained at 4x MIC at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 CFU/mL, respectively. The same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation.", "For compound 1h bactericidal effect against MRSA 63718 was maintained at 4x MIC at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 CFU/mL, respectively. The same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation. Regrowth at 24 h after incubation causing a loss of bactericidal activity was recorded similarly as with previous compounds. The reason for regrowth of the test organism at 24 h in the experiment is unknown. Most probably, selection of resistant mutants is responsible for this phenomenon ; degradation of the drug in the growth medium is not assumed, as regrowth was Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain. For MRSA SA 630 concentration-dependent killing was recorded at 4x MIC at 6, 8, and 24 h after incubation with log 10 differences in CFU/mL from the starting inoculum being very similar over time ranging from 3.18 to 3.39 log 10 CFU/mL .", "Most probably, selection of resistant mutants is responsible for this phenomenon ; degradation of the drug in the growth medium is not assumed, as regrowth was Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain. For MRSA SA 630 concentration-dependent killing was recorded at 4x MIC at 6, 8, and 24 h after incubation with log 10 differences in CFU/mL from the starting inoculum being very similar over time ranging from 3.18 to 3.39 log 10 CFU/mL . For MRSA SA 3202 reliable bactericidal effect was maintained only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.02 log 10 CFU/mL. As for compound 1g, bacteriostatic activity against S. aureus reference strain was observed with a reduction in bacterial count ranging from 0.34 to 2.62 log 10 CFU/mL at 2x and 4x MIC. Overgrowth values ranging from 0.04 to 1.43 log 10 CFU/mL was recorded at 1x MIC for this strain. It is of note that in all staphylococci strains with similar MICs and MBCs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied with clinical strains of MRSA being effectively killed and the reference strain remaining unaffected at 4x MIC.", "Overgrowth values ranging from 0.04 to 1.43 log 10 CFU/mL was recorded at 1x MIC for this strain. It is of note that in all staphylococci strains with similar MICs and MBCs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied with clinical strains of MRSA being effectively killed and the reference strain remaining unaffected at 4x MIC. There is a discrepancy between bactericidal results of MBC assay compared with time-kill kinetics. This difference could be caused by comparing microtiter MBC assay to macrobroth time-kill assay dilutions . Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents . Concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then .", "Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents . Concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then . However, bactericidal therapy could produce a better treatment result by rapid reduction of the bacterial load . Moreover, in the case of an immune system disorder e.g., immunosuppressive therapy, AIDS patients, etc. bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds .", "bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds . The clinical outcome of MRSA bacteraemia is significantly influenced by vancomycin MIC. Treatment failure exceeding 60% for S. aureus with vancomycin MIC of 4 g/mL resulted in the change of susceptibility breakpoint from 4 g/mL to 2 g/mL by the Clinical and Laboratory Standards Institute CLSI in 2006 as well as by the US Food and Drug Administration FDA in 2008 . It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs 2 g/mL , alternative therapy should be considered . It is of note that based on time-kill assays in the present study, all tested diamides particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL.", "It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs 2 g/mL , alternative therapy should be considered . It is of note that based on time-kill assays in the present study, all tested diamides particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL. The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. The present study is the first evidence of bactericidal effect of SAL analogues.", "Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. The present study is the first evidence of bactericidal effect of SAL analogues. Against other strains, reliable bactericidal effect was maintained at 4x MIC at 24 h after incubation. Considering the necessity to broaden the spectrum of bactericidal agents, diamides from the current study with a novel mechanism of action could present a very promising and interesting solution to this challenge for the future." ]

[ "A series of nine substituted 2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides was assessed as prospective bactericidal agents against three clinical isolates of methicillin-resistant Staphylococcus aureus MRSA and S. aureus ATCC 29213 as the reference and quality control strain. The minimum bactericidal concentration was determined by subculturing aliquots from MIC determination onto substance-free agar plates. The bactericidal kinetics of compounds 5-chloro-2-hydroxy-N- 2S -3-methyl-1-oxo-1-{ 4- trifluoromethyl phenyl amino}butan-2-yl benzamide 1f , N-{ 2S -1- 4-bromophenyl amino -3-methyl-1-oxobutan-2-yl}-4-chloro-2-hydroxybenzamide 1g , and 4-chloro-N-{ 2S -1- 3,4-dichlorophenyl amino -3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide 1h was established by time-kill assay with a final concentration of the compound equal to 1x, 2x, and 4x MIC; aliquots were removed at 0, 4, 6, 8, and 24 h time points. The most potent bactericidal agent was compound 1f exhibiting remarkable rapid concentration-dependent bactericidal effect even at 2x MIC at 4, 6, and 8 h with a reduction in bacterial count ranging from 3.08 to 3.75 log. CFU/mL and at 4x MIC at 4, 6, 8, and 24 h 5.30 log. CFU/mL reduction in bacterial count after incubation against MRSA 63718.", "CFU/mL and at 4x MIC at 4, 6, 8, and 24 h 5.30 log. CFU/mL reduction in bacterial count after incubation against MRSA 63718. Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 . The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration MIC values i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism within the susceptible range was described previously .", "The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 . The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration MIC values i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism within the susceptible range was described previously . Thus, the emergence of MRSA and vancomycin-resistant S. aureus in the recent years as well makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria . However, for the treatment of S. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs . This fact should be considered during the development of effective and safe treatment options for MRSA infections. The history of clinical usage of salicylanilides 2-hydroxy-N-phenylbenzamides dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s .", "This fact should be considered during the development of effective and safe treatment options for MRSA infections. The history of clinical usage of salicylanilides 2-hydroxy-N-phenylbenzamides dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s . Nowadays, salicylanilides SALs are a class of aromatic compounds possessing a wide range of interesting pharmacological activities, such as anthelmintic , antibacterial , antimycobacterial , antifungal , and antiviral , among others. Despite being studied since the 1960s, the mechanism of action responsible for biological activities of these compounds has not been explained so far. SALs have been found to inhibit the two-component regulatory systems TCS of bacteria . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis .", "SALs have been found to inhibit the two-component regulatory systems TCS of bacteria . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis . Furthermore, salicylanilides have been recognised as inhibitors of some bacterial enzymes, such as sortase A from S. aureus , d-alanine-d-alanine ligase , or transglycosylases from S. aureus but not from M. tuberculosis . These enzymes participate in secretion of various proteins or in biosynthesis of bacterial cell wall. Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action.", "Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges. This study is a follow-up paper to a recently published article . The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides, called diamides due to their skeleton for general structure see Table 1 , was described previously , and their antimycobacterial and antibacterial activities against various bacterial species were reported . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA.", "The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides, called diamides due to their skeleton for general structure see Table 1 , was described previously , and their antimycobacterial and antibacterial activities against various bacterial species were reported . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA. The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis . ; and MRSA SA 3202 National Institute of Public Health, Prague, Czech Republic both of human origin.", "The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis . ; and MRSA SA 3202 National Institute of Public Health, Prague, Czech Republic both of human origin. Suspected colonies were confirmed by PCR; a 108 bp fragment specific for S. aureus was detected . All isolates were tested for the presence of the mecA gene encoding methicillin resistance . These three clinical isolates were classified as vancomycin-susceptible but with higher MIC of vancomycin equal to 2 g/mL VA2-MRSA within the susceptible range for MRSA 63718 methicillinresistant S. aureus VS-MRSA . For the MICs of vancomycin, see Table 1 .", "These three clinical isolates were classified as vancomycin-susceptible but with higher MIC of vancomycin equal to 2 g/mL VA2-MRSA within the susceptible range for MRSA 63718 methicillinresistant S. aureus VS-MRSA . For the MICs of vancomycin, see Table 1 . Vancomycin-susceptible methicillin-susceptible Staphylococcus aureus VS-MSSA ATCC 29213, obtained from the American Type Culture Collection, was used as the reference and quality control strain. The bacteria were stored at −80 ∘ C and were kept on blood agar plates Columbia agar base with 5% ovine blood between experiments. MBCs . The MBCs i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium were determined by subculturing aliquots 20 L from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar MHA plates.", "MBCs . The MBCs i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium were determined by subculturing aliquots 20 L from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar MHA plates. The plates were incubated aerobically at 37 ∘ C for 24 h for colony count. The MBC was defined as the lowest concentration of substance, which produced ≥99.9% killing Table 1 : Chemical structures and in vitro MIC and MBC g/mL values of tested 5-and 4-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides bactericidal effect of individual compounds against particular strains marked in bold . after 24 h of incubation as compared to the colony count of the starting inoculum . To ensure reproducibility, each MBC assay was performed in at least triplicate on separate occasions.", "after 24 h of incubation as compared to the colony count of the starting inoculum . To ensure reproducibility, each MBC assay was performed in at least triplicate on separate occasions. N H O H N O OH 1 2 R 1 R 3 R 2 Comp. R 1 R 2 R 3 MIC g/mL MBC g/mL 1 2 3 4 1 2 3 4 1a 5-Cl 4-CH 3 S -CH 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-Cl 4-CH 3 S -CH CH 3 2 >256 >256 32 32 >256 >256 128 >256 1c 5-Cl 4-CH 3 S -benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-Cl 4-CH 3 R -CH 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-Cl 4-OCH 3 S -CH CH 3 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-Cl 4-CF 3 S -CH CH 3 2 4 2 2 2 4 4 8 4 1g 4-Cl 4-Br S -CH CH 3 2 8 4 4 4 1 6 8 8 8 1h 4-Cl 3,4-Cl S -CH CH 3 2 2 1 1 1 4 1 4 2 1i 4-Cl 3,4-Cl S -benzyl 1 1 0.5 0.5 8 1 8 1 AMP - - - >16 >16 >16 0.25 >16 >16 >16 0.25 CPX - - - >16 >16 >16 0.5 >16 >16 >16 0.5 VAN - - - 2 1 1 1 2 1 1 1 Time-kill assays were performed by the broth macrodilution method according to previously described methodology with some modifications. Briefly, flasks containing sterile fresh Mueller-Hinton broth MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots 20 L were plated on MHA plates in duplicate.", "Briefly, flasks containing sterile fresh Mueller-Hinton broth MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots 20 L were plated on MHA plates in duplicate. Colony counts were performed on plates yielding 6 to 60 colonies, and the mean was calculated. Antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments.", "The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments. The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included. Time-kill curves were constructed by plotting the log 10 CFU per millilitre versus time over 24 h , and the change in bacterial concentration was determined. The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum. Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently .", "The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum. Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently . In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 methicillin-susceptible as the reference and quality control strain. Since SALs and their analogues are known as compounds with bacteriostatic effect , this is the first study where SAL-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. Recently MIC values of diamides expressed as molar concentrations in mol/L were published .", "Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. Recently MIC values of diamides expressed as molar concentrations in mol/L were published . To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. Potential bactericidal activity of diamides was assessed using MBC assay . MBC values of all tested compounds are recorded in Table 1 as well. Based on the obtained results, all compounds assessed as active according to MIC values in our previous study 1f-i showed low or moderate MBC values against all four strains.", "MBC values of all tested compounds are recorded in Table 1 as well. Based on the obtained results, all compounds assessed as active according to MIC values in our previous study 1f-i showed low or moderate MBC values against all four strains. The MBC values of these compounds did not exceed the highest tested drug concentration and ranged from 1 to 16 g/mL. In all cases, there were comparable MBC values for the clinical isolates of MRSA and the S. aureus reference strain. Bactericidal activity is defined as a ratio of MBC to MIC of ≤4 . Table 1 bactericidal activity is expressed in bold.", "Bactericidal activity is defined as a ratio of MBC to MIC of ≤4 . Table 1 bactericidal activity is expressed in bold. As mentioned above, SALs are known to exhibit a bacteriostatic effect , so it was very interesting to discover that diamides possess bactericidal activity. The amide bond -CONH- can cause interactions with a variety of enzymes ; therefore the presence of two amide bonds could be responsible for the bactericidal effect of diamides against MRSA. The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus including MRSA cell wall biosynthesis . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane .", "The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus including MRSA cell wall biosynthesis . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane . Since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents , the failure in the cell wall biosynthesis due to the inhibition of transglycosylases could be responsible for bactericidal activity of diamides against MRSA. Based on these findings, antibacterial active diamides with bactericidal effect against all four tested strains as prospective bactericidal agents were chosen for subsequent timekill curve studies to determine the real dependence of bactericidal effect on concentration over time. 1-oxobutan-2-yl}-2-hydroxybenzamide 1h were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain. The antibacterial effect of DMSO used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control.", "1-oxobutan-2-yl}-2-hydroxybenzamide 1h were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain. The antibacterial effect of DMSO used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control. The extent of bacterial killing was estimated by the number of these strains showing a decrease ranging from 1 to 3 log 10 CFU/mL in viable cell count at different times after incubation. A summary of these data is presented in Table 2 . Based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. No bactericidal activity i.e., ≥3 log 10 CFU/mL decrease was observed at 1x MIC for any strain and time after incubation tested.", "Based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. No bactericidal activity i.e., ≥3 log 10 CFU/mL decrease was observed at 1x MIC for any strain and time after incubation tested. At 4x MIC from the four strains, compounds 1f, 1 g, and 1h killed 2, 1, and 2 strains, respectively, at 8 h after incubation and 4, 2, and 2 strains, respectively, at 24 h after incubation. The findings of time-kill studies for each of the four staphylococci strains at exposure to compounds 1f, 1g, and 1h are summarized in Table 3 . Bactericidal activity i.e., ≥3 log 10 CFU/mL decrease is expressed in bold. For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718.", "Bactericidal activity i.e., ≥3 log 10 CFU/mL decrease is expressed in bold. For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718. Time was not the predictive factor influencing the antibacterial activity because log 10 differences in CFU/mL from the starting inoculum were the same for 4x MIC with the highest efficiency with a reduction in bacterial count of 5.30 log 10 CFU/mL or very similar for 2x MIC with a moderate regrowth after 24 h causing a loss of bactericidal activity over 24 h. The bactericidal effect was maintained even at 2x MIC at 4 h after incubation for this strain reduction of 3.08 log 10 CFU/mL . For the remaining strains, clinical isolates of MRSA SA 630, MRSA SA 3202, and S. aureus ATCC 29213, reliable bactericidal effect was recorded at 4x MIC at 24 h after incubation for all these strains with a reduction in bacterial count of 3.22, 3.30, and 3.65 log 10 CFU/mL, respectively. For compound 1g bactericidal effect against MRSA 63718 was noticed at 2x MIC at 6 and 8 h after incubation and at 4x MIC at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 CFU/mL. The most effective killing was achieved at 6 h for both concentrations.", "For compound 1g bactericidal effect against MRSA 63718 was noticed at 2x MIC at 6 and 8 h after incubation and at 4x MIC at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 CFU/mL. The most effective killing was achieved at 6 h for both concentrations. As in the case of compound 1f, a regrowth was observed after 24 h after incubation. For the remaining isolates of MRSA, SA 630 and SA 3202, bactericidal effect occurred only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.38 and 4.01 log 10 CFU/mL, respectively. The highest bactericidal effect was recorded for MRSA SA 3202 at 4x MIC at 24 h after incubation. A reduction consistent with bacteriostatic effect 0.03 to 2.37 log 10 CFU/mL was observed at other concentrations over time for both isolates.", "The highest bactericidal effect was recorded for MRSA SA 3202 at 4x MIC at 24 h after incubation. A reduction consistent with bacteriostatic effect 0.03 to 2.37 log 10 CFU/mL was observed at other concentrations over time for both isolates. No bactericidal effect was observed for the S. aureus reference strain; compound 1g demonstrated a pattern of bacteriostatic activity against this strain with a reduction in bacterial count ranging from 0.07 to 2.33 log 10 CFU/mL at 4x MIC over time. In other cases, a slight increase in bacterial counts i.e., overgrowth compared with the starting inoculum was observed with values ranging from 0.10 to 1.57 log 10 CFU/mL for this reference strain. For compound 1h bactericidal effect against MRSA 63718 was maintained at 4x MIC at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 CFU/mL, respectively. The same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation.", "For compound 1h bactericidal effect against MRSA 63718 was maintained at 4x MIC at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 CFU/mL, respectively. The same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation. Regrowth at 24 h after incubation causing a loss of bactericidal activity was recorded similarly as with previous compounds. The reason for regrowth of the test organism at 24 h in the experiment is unknown. Most probably, selection of resistant mutants is responsible for this phenomenon ; degradation of the drug in the growth medium is not assumed, as regrowth was Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain. For MRSA SA 630 concentration-dependent killing was recorded at 4x MIC at 6, 8, and 24 h after incubation with log 10 differences in CFU/mL from the starting inoculum being very similar over time ranging from 3.18 to 3.39 log 10 CFU/mL .", "Most probably, selection of resistant mutants is responsible for this phenomenon ; degradation of the drug in the growth medium is not assumed, as regrowth was Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain. For MRSA SA 630 concentration-dependent killing was recorded at 4x MIC at 6, 8, and 24 h after incubation with log 10 differences in CFU/mL from the starting inoculum being very similar over time ranging from 3.18 to 3.39 log 10 CFU/mL . For MRSA SA 3202 reliable bactericidal effect was maintained only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.02 log 10 CFU/mL. As for compound 1g, bacteriostatic activity against S. aureus reference strain was observed with a reduction in bacterial count ranging from 0.34 to 2.62 log 10 CFU/mL at 2x and 4x MIC. Overgrowth values ranging from 0.04 to 1.43 log 10 CFU/mL was recorded at 1x MIC for this strain. It is of note that in all staphylococci strains with similar MICs and MBCs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied with clinical strains of MRSA being effectively killed and the reference strain remaining unaffected at 4x MIC.", "Overgrowth values ranging from 0.04 to 1.43 log 10 CFU/mL was recorded at 1x MIC for this strain. It is of note that in all staphylococci strains with similar MICs and MBCs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied with clinical strains of MRSA being effectively killed and the reference strain remaining unaffected at 4x MIC. There is a discrepancy between bactericidal results of MBC assay compared with time-kill kinetics. This difference could be caused by comparing microtiter MBC assay to macrobroth time-kill assay dilutions . Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents . Concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then .", "Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents . Concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then . However, bactericidal therapy could produce a better treatment result by rapid reduction of the bacterial load . Moreover, in the case of an immune system disorder e.g., immunosuppressive therapy, AIDS patients, etc. bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds .", "bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds . The clinical outcome of MRSA bacteraemia is significantly influenced by vancomycin MIC. Treatment failure exceeding 60% for S. aureus with vancomycin MIC of 4 g/mL resulted in the change of susceptibility breakpoint from 4 g/mL to 2 g/mL by the Clinical and Laboratory Standards Institute CLSI in 2006 as well as by the US Food and Drug Administration FDA in 2008 . It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs 2 g/mL , alternative therapy should be considered . It is of note that based on time-kill assays in the present study, all tested diamides particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL.", "It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs 2 g/mL , alternative therapy should be considered . It is of note that based on time-kill assays in the present study, all tested diamides particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL. The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. The present study is the first evidence of bactericidal effect of SAL analogues.", "Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. The present study is the first evidence of bactericidal effect of SAL analogues. Against other strains, reliable bactericidal effect was maintained at 4x MIC at 24 h after incubation. Considering the necessity to broaden the spectrum of bactericidal agents, diamides from the current study with a novel mechanism of action could present a very promising and interesting solution to this challenge for the future." ]

[ "The common marmoset Callithrix jacchus is increasingly being utilised as a nonhuman primate model for human disease, ranging from autoimmune to infectious disease. In order to fully exploit these models, meaningful comparison to the human host response is necessary. Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals. Single cell suspensions of blood, spleen, and lung were examined. Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values.", "Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values. Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most obvious in cell types active in the innate immune response. This work significantly enhances the ability to understand the immune response in these animals and fortifies their use as models of infectious disease. Text: The common marmoset Callithrix jacchus , a New World monkey NWM species is a small, arboreal nonhuman primate NHP , native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans .", "Text: The common marmoset Callithrix jacchus , a New World monkey NWM species is a small, arboreal nonhuman primate NHP , native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans . In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys OWM e.g., rhesus and cynomolgus macaques . Evolutionarily, both NWM and OWM sit within the simiiformes infraorder of the suborder Haplorhini of primates . Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate.", "Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate. Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus , Hepatitis C virus , Dengue virus , Herpesvirus , Junin virus Rift Valley Fever , and SARS . Marmosets have also been used to model a number of biodefense pathogens including Eastern Equine Encephalitis virus , Bacillus anthracis , Francisella tularensis , Burkholderia pseudomallei , Marburg haemorrhagic fever virus , Ebola haemorrhagic fever virus , and Variola virus . The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood.", "The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood. Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases. There is a high level of gene homology between humans and NHPs which underlies physiological and biochemical similarities. Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man . Immunologically, there is a high degree of homology between humans and marmosets .", "Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man . Immunologically, there is a high degree of homology between humans and marmosets . The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels. There is at least 95% homology between human costimulatory molecules e.g., CD80, CD86 etc. and those of marmosets . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology .", "and those of marmosets . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology . Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood . However, these reagents have not been exploited to investigate the immune response to infectious disease. To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model ; neutrophils and macrophages in the Herpes model ; or CD3+ and CD20+ cells in the Lassa model . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease.", "To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model ; neutrophils and macrophages in the Herpes model ; or CD3+ and CD20+ cells in the Lassa model . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease. The aim of this work is to determine key changes and identify correlates of infection or protection. Healthy sexually mature common marmosets C. jacchus were obtained from the Dstl Porton Down breeding colony and housed in vasectomized male and female pairs. The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment.", "The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment. All animal studies were carried out in accordance with the UK Animals Scientific Procedures Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989. Animals were challenged with an intracellular pathogen by either the subcutaneous or inhalational route and were humanely killed at various time points after challenge. Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic.", "Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic. Populations. Blood and tissue samples were homogenised to provide single cell suspensions . Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 SP34-2 , CD8 LT8 , CD11c SHCL3 , CD14 M5E2 , CD16 3G8 , CD20 Bly1 , CD45RA 5H9 , CD54 HCD54 , CD56 B159 , CD69 FN50 , CD163 GHI/61 , and MCHII L243 BD Bioscience, Insight Bioscience, AbD serotec . Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry FACScanto II BD within 72 hours of staining.", "Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 SP34-2 , CD8 LT8 , CD11c SHCL3 , CD14 M5E2 , CD16 3G8 , CD20 Bly1 , CD45RA 5H9 , CD54 HCD54 , CD56 B159 , CD69 FN50 , CD163 GHI/61 , and MCHII L243 BD Bioscience, Insight Bioscience, AbD serotec . Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry FACScanto II BD within 72 hours of staining. Levels of circulating cytokines and chemokines were also quantified in the blood of marmosets from the Dstl colony using human multiplex kits available commercially BD cytokine flex beads and the Luminex system . These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 . However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells.", "However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells. These tissue homogenates are of particular interest in relation to target sites of infection: the lung as the site of initial infection following an inhalational challenge and the spleen as a representative organ following a parental challenge. Cell types targeted during this analysis include cells important in the innate response e.g., neutrophils, macrophages, and NK cells and the adaptive response T and B cells with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection. Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size forward and granularity side scatter Figure 1 a .", "Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size forward and granularity side scatter Figure 1 a . Identification of cell types in tissue samples was more difficult as the scatter profiles are less clearly compartmentalized. The common leukocyte antigen CD45 normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples. Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments data not shown . Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin Figure 1 b .", "Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments data not shown . Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin Figure 1 b . Figure 1 shows the basic division of lymphocytes between T, B, and NK cells from a healthy blood sample. Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets Figure 2 a , Table 1 ; approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells. The variability of the data is depicted in Figure 2 a with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals.", "The variability of the data is depicted in Figure 2 a with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals. This analysis was also applied to lung and spleen homogenates from naive marmosets Figures 2 b and 2 c . Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals. As expected, low numbers of neutrophils are found in naive spleen or lung tissue 8% both . Healthy mouse spleens typically have approximately 1-2% granulocytes .", "As expected, low numbers of neutrophils are found in naive spleen or lung tissue 8% both . Healthy mouse spleens typically have approximately 1-2% granulocytes . Understandably, there are few reports on the typical cell percentages expected in healthy human individuals for these tissues. However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells . In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples and information available for human blood Table 1 .", "In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples and information available for human blood Table 1 . However, one report found the proportion of CD8+ T-cells was almost three times greater in marmosets than humans, 61% to 21% respectively compared to the 30% observed in this study and the work previously reported by Brok et al. . Brok's study involved a small number of animals eight and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils , although these differences are highly strain specific.", "Brok's study involved a small number of animals eight and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils , although these differences are highly strain specific. C57BL/6J mice are reported to have 67% B cells and BALB/C mice 46%; both of which are consistently higher than the percentage found in marmosets and humans of approximately 25% Table 1 . The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is lower than the 35% found in marmosets and the 40-75% expected for healthy human blood. This is encouraging as neutrophils play a pivotal role in the innate response to infection . A cross-species comparison suggests that monocytes comprise 3% of leukocytes Table 1 .", "This is encouraging as neutrophils play a pivotal role in the innate response to infection . A cross-species comparison suggests that monocytes comprise 3% of leukocytes Table 1 . Levels of circulating cytokines and chemokines IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN were also quantified in the blood, lung, and spleen of naïve marmosets from the Dstl colony. None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue. Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus .", "Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus . Following challenge with F. tularensis, increasing levels of NK cells, neutrophils, T cells, and macrophages were observed, peaking at 48 hours after challenge before rapidly declining. This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood. Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset present data Marmoset Mouse 4 Human Asian Human Caucasian Number the lungs, spleen, and blood as the disease progressed TNF and IFN were not measured in this study .", "Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset present data Marmoset Mouse 4 Human Asian Human Caucasian Number the lungs, spleen, and blood as the disease progressed TNF and IFN were not measured in this study . Following inhalational challenge of marmosets with B. pseudomallei, an increase in the number of neutrophils was observed in the blood at 36 hours after challenge, followed by a rapid decline that was associated with an influx of neutrophils into the lung at 46 hours after challenge. A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed. There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge.", "There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge. The levels of various cell types and cytokines were also measured in the blood of animals following inhalational challenge with Marburg virus . In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time TNF was not measured . In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells .", "In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells . More recent work at Dstl has reported further cross-reactivity between marmoset cells and human cytokines to induce activity in marmoset T cells . These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs e.g., NIH NHP reagent resource, has expanded the ability to assess activation markers for disease. Detection of the following cell surface markers with human antibodies was trialed: CD54 ICAM-1 associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II HLA-DR . CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets .", "Detection of the following cell surface markers with human antibodies was trialed: CD54 ICAM-1 associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II HLA-DR . CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets . These markers have been used to expand on our previously published work to determine changes in the activation status of basic cell types in response to an acute bacterial infection. Animals were challenged with bacteria at a comparable dose either by inhalation = 22 or by a systemic route = 12 and humanely killed once they had reached a humane endpoint between day 4 and day 5 after challenge . Figure 3 illustrates the cellular activity in representative tissues following inhalational Figures 3 b and 3 e or systemic challenge Figures 3 c and 3 f and in naïve samples Figures 3 a and 3 d . Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16.", "Figure 3 illustrates the cellular activity in representative tissues following inhalational Figures 3 b and 3 e or systemic challenge Figures 3 c and 3 f and in naïve samples Figures 3 a and 3 d . Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16. In response to disease, the proportions of the cell types appear to remain relativity constant; however, the activation markers provide more detailed information and show involvement of all the cell types explored. Extensive activation was to be expected considering that the samples were taken at the humane endpoint. There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection.", "There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection. Levels of circulating cytokines and chemokines IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN were also quantified in the lung and spleen samples. All of the cytokines with the exception of Rantes were expressed at high levels ng/mg in all samples, which was expected as the animals had succumbed to terminal disease. The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease.", "The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease. In conclusion, the immune response in marmosets to infectious disease can be characterised in terms of the phenotype and activation status of all the major immune cells and key cytokine and chemokine expression. This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies. This information can also potentially be used for pivotal studies to support licensure of products under the FDA Animal Rule. This, in conjunction with the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment, makes the marmoset an appropriate animal model for biodefense-related pathogens." ]

[ "The common marmoset Callithrix jacchus is increasingly being utilised as a nonhuman primate model for human disease, ranging from autoimmune to infectious disease. In order to fully exploit these models, meaningful comparison to the human host response is necessary. Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals. Single cell suspensions of blood, spleen, and lung were examined. Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values.", "Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values. Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most obvious in cell types active in the innate immune response. This work significantly enhances the ability to understand the immune response in these animals and fortifies their use as models of infectious disease. Text: The common marmoset Callithrix jacchus , a New World monkey NWM species is a small, arboreal nonhuman primate NHP , native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans .", "Text: The common marmoset Callithrix jacchus , a New World monkey NWM species is a small, arboreal nonhuman primate NHP , native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans . In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys OWM e.g., rhesus and cynomolgus macaques . Evolutionarily, both NWM and OWM sit within the simiiformes infraorder of the suborder Haplorhini of primates . Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate.", "Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate. Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus , Hepatitis C virus , Dengue virus , Herpesvirus , Junin virus Rift Valley Fever , and SARS . Marmosets have also been used to model a number of biodefense pathogens including Eastern Equine Encephalitis virus , Bacillus anthracis , Francisella tularensis , Burkholderia pseudomallei , Marburg haemorrhagic fever virus , Ebola haemorrhagic fever virus , and Variola virus . The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood.", "The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood. Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases. There is a high level of gene homology between humans and NHPs which underlies physiological and biochemical similarities. Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man . Immunologically, there is a high degree of homology between humans and marmosets .", "Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man . Immunologically, there is a high degree of homology between humans and marmosets . The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels. There is at least 95% homology between human costimulatory molecules e.g., CD80, CD86 etc. and those of marmosets . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology .", "and those of marmosets . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology . Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood . However, these reagents have not been exploited to investigate the immune response to infectious disease. To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model ; neutrophils and macrophages in the Herpes model ; or CD3+ and CD20+ cells in the Lassa model . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease.", "To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model ; neutrophils and macrophages in the Herpes model ; or CD3+ and CD20+ cells in the Lassa model . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease. The aim of this work is to determine key changes and identify correlates of infection or protection. Healthy sexually mature common marmosets C. jacchus were obtained from the Dstl Porton Down breeding colony and housed in vasectomized male and female pairs. The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment.", "The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment. All animal studies were carried out in accordance with the UK Animals Scientific Procedures Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989. Animals were challenged with an intracellular pathogen by either the subcutaneous or inhalational route and were humanely killed at various time points after challenge. Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic.", "Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic. Populations. Blood and tissue samples were homogenised to provide single cell suspensions . Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 SP34-2 , CD8 LT8 , CD11c SHCL3 , CD14 M5E2 , CD16 3G8 , CD20 Bly1 , CD45RA 5H9 , CD54 HCD54 , CD56 B159 , CD69 FN50 , CD163 GHI/61 , and MCHII L243 BD Bioscience, Insight Bioscience, AbD serotec . Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry FACScanto II BD within 72 hours of staining.", "Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 SP34-2 , CD8 LT8 , CD11c SHCL3 , CD14 M5E2 , CD16 3G8 , CD20 Bly1 , CD45RA 5H9 , CD54 HCD54 , CD56 B159 , CD69 FN50 , CD163 GHI/61 , and MCHII L243 BD Bioscience, Insight Bioscience, AbD serotec . Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry FACScanto II BD within 72 hours of staining. Levels of circulating cytokines and chemokines were also quantified in the blood of marmosets from the Dstl colony using human multiplex kits available commercially BD cytokine flex beads and the Luminex system . These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 . However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells.", "However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells. These tissue homogenates are of particular interest in relation to target sites of infection: the lung as the site of initial infection following an inhalational challenge and the spleen as a representative organ following a parental challenge. Cell types targeted during this analysis include cells important in the innate response e.g., neutrophils, macrophages, and NK cells and the adaptive response T and B cells with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection. Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size forward and granularity side scatter Figure 1 a .", "Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size forward and granularity side scatter Figure 1 a . Identification of cell types in tissue samples was more difficult as the scatter profiles are less clearly compartmentalized. The common leukocyte antigen CD45 normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples. Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments data not shown . Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin Figure 1 b .", "Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments data not shown . Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin Figure 1 b . Figure 1 shows the basic division of lymphocytes between T, B, and NK cells from a healthy blood sample. Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets Figure 2 a , Table 1 ; approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells. The variability of the data is depicted in Figure 2 a with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals.", "The variability of the data is depicted in Figure 2 a with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals. This analysis was also applied to lung and spleen homogenates from naive marmosets Figures 2 b and 2 c . Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals. As expected, low numbers of neutrophils are found in naive spleen or lung tissue 8% both . Healthy mouse spleens typically have approximately 1-2% granulocytes .", "As expected, low numbers of neutrophils are found in naive spleen or lung tissue 8% both . Healthy mouse spleens typically have approximately 1-2% granulocytes . Understandably, there are few reports on the typical cell percentages expected in healthy human individuals for these tissues. However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells . In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples and information available for human blood Table 1 .", "In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples and information available for human blood Table 1 . However, one report found the proportion of CD8+ T-cells was almost three times greater in marmosets than humans, 61% to 21% respectively compared to the 30% observed in this study and the work previously reported by Brok et al. . Brok's study involved a small number of animals eight and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils , although these differences are highly strain specific.", "Brok's study involved a small number of animals eight and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils , although these differences are highly strain specific. C57BL/6J mice are reported to have 67% B cells and BALB/C mice 46%; both of which are consistently higher than the percentage found in marmosets and humans of approximately 25% Table 1 . The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is lower than the 35% found in marmosets and the 40-75% expected for healthy human blood. This is encouraging as neutrophils play a pivotal role in the innate response to infection . A cross-species comparison suggests that monocytes comprise 3% of leukocytes Table 1 .", "This is encouraging as neutrophils play a pivotal role in the innate response to infection . A cross-species comparison suggests that monocytes comprise 3% of leukocytes Table 1 . Levels of circulating cytokines and chemokines IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN were also quantified in the blood, lung, and spleen of naïve marmosets from the Dstl colony. None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue. Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus .", "Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus . Following challenge with F. tularensis, increasing levels of NK cells, neutrophils, T cells, and macrophages were observed, peaking at 48 hours after challenge before rapidly declining. This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood. Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset present data Marmoset Mouse 4 Human Asian Human Caucasian Number the lungs, spleen, and blood as the disease progressed TNF and IFN were not measured in this study .", "Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset present data Marmoset Mouse 4 Human Asian Human Caucasian Number the lungs, spleen, and blood as the disease progressed TNF and IFN were not measured in this study . Following inhalational challenge of marmosets with B. pseudomallei, an increase in the number of neutrophils was observed in the blood at 36 hours after challenge, followed by a rapid decline that was associated with an influx of neutrophils into the lung at 46 hours after challenge. A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed. There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge.", "There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge. The levels of various cell types and cytokines were also measured in the blood of animals following inhalational challenge with Marburg virus . In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time TNF was not measured . In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells .", "In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells . More recent work at Dstl has reported further cross-reactivity between marmoset cells and human cytokines to induce activity in marmoset T cells . These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs e.g., NIH NHP reagent resource, has expanded the ability to assess activation markers for disease. Detection of the following cell surface markers with human antibodies was trialed: CD54 ICAM-1 associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II HLA-DR . CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets .", "Detection of the following cell surface markers with human antibodies was trialed: CD54 ICAM-1 associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II HLA-DR . CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets . These markers have been used to expand on our previously published work to determine changes in the activation status of basic cell types in response to an acute bacterial infection. Animals were challenged with bacteria at a comparable dose either by inhalation = 22 or by a systemic route = 12 and humanely killed once they had reached a humane endpoint between day 4 and day 5 after challenge . Figure 3 illustrates the cellular activity in representative tissues following inhalational Figures 3 b and 3 e or systemic challenge Figures 3 c and 3 f and in naïve samples Figures 3 a and 3 d . Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16.", "Figure 3 illustrates the cellular activity in representative tissues following inhalational Figures 3 b and 3 e or systemic challenge Figures 3 c and 3 f and in naïve samples Figures 3 a and 3 d . Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16. In response to disease, the proportions of the cell types appear to remain relativity constant; however, the activation markers provide more detailed information and show involvement of all the cell types explored. Extensive activation was to be expected considering that the samples were taken at the humane endpoint. There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection.", "There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection. Levels of circulating cytokines and chemokines IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN were also quantified in the lung and spleen samples. All of the cytokines with the exception of Rantes were expressed at high levels ng/mg in all samples, which was expected as the animals had succumbed to terminal disease. The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease.", "The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease. In conclusion, the immune response in marmosets to infectious disease can be characterised in terms of the phenotype and activation status of all the major immune cells and key cytokine and chemokine expression. This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies. This information can also potentially be used for pivotal studies to support licensure of products under the FDA Animal Rule. This, in conjunction with the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment, makes the marmoset an appropriate animal model for biodefense-related pathogens." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 HAdV-55 in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome ARDS caused by HAdV-55 requiring invasive mechanical ventilation IMV and/or extracorporeal membrane oxygenation ECMO are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays CXRs and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10. copies in three patients and was 1 × 10. in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation NPPV failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men.", "Four patients received venovenous ECMO. Four 80% of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012 Text: Human adenoviruses HAdVs are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients . Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 .", "Human adenovirus type 55 HAdV-55 , which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 and in Singapore in 2005 . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia . This pathogen was fully characterized by whole-genome sequencing . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 .", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes HAdV-3, HAdV-7 and HAdV-14 . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome ARDS requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation ECMO support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 .", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation NPPV in ARDS patients was carried out in our center ClinicalTrials.gov ID: NCT01585922 . From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: . developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; .", "developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; . bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; . respiratory failure not fully explained by cardiac failure or fluid overload; . partial oxygen pressure/ fraction of inspired oxygen PaO 2 /FiO 2 ≤100 mmHg with positive end-expiratory pressure PEEP ≥5 cmH 2 O; and . a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation IMV were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously.", "This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital LLKYPJ2012031 . Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics age and sex , comorbidities, clinical symptoms fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache , signs body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs , laboratory tests whole-blood cell count and blood chemistry and microbiological findings and images of the lung chest X-ray CXR and computed tomography . Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains.", "All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS.", "Continuous variables were summarized as mean ± standard deviation SD or median interquartile range . During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years .", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years range, 28 to 40 years . All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay Oxford Immunotec, Marlborough, MA, USA and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C range, 39.0°C to 40.0°C , which persisted for 8 days range, 6 to 11 days . Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days range, 1 to 10 days . After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 .", "The mean time from onset to ICU admission was 9.6 days range, 8 to 11 days Table 1 . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute range = 38 to 52 . Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 range = 49 to 62.5 . White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range.", "All patients had elevated serum aspartate aminotransferase AST , lactate dehydrogenase LDH and hydroxybutyrate dehydrogenase HBDH Table 1 . At admission, all patients' levels of immunoglobulin serum immunoglobulins G and M and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts Table 2 . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission Figure 1 . Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Three patients were examined by highresolution computed tomography HRCT . Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT Figure 2 . The mean duration from onset to a single-lobe consolidation on CXRs was 2 days range = 1 to 5 days . The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia.", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days range = 4 to 7 days . All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate ETA samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days Table 3 . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy Figure 3 . Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "The mean duration until NPPV failure was 30.8 hours range = 22 to 48 hours , and the mean time until IMV failure was 6.2 days range 2 = to 13 days Table 1 . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir 10 mg/kg, every 8 hours, intravenous drip , ganciclovir 5 mg/kg, every 12 hours, intravenous drip and ribavirin 250 mg, twice daily, intravenous drip . Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 .", "Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient Table 3 . Four 80% of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. . HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. .", ". HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. . Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. . Viral load monitoring may help predict disease severity and patient outcome. . The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. . HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support.", ". HAdV-55-induced severe ARDS has a very high mortality rate 80% despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 , serotype 7 and, more recently, serotype 14 in the general population and in military trainees . HAdV-55 was first completely characterized in Shaanxi, China and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al.", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Community-acquired pneumonia CAP requires prompt treatment, but its diagnosis is complex. Improvement of bacterial CAP diagnosis by biomarkers has been evaluated using chest X-ray infiltrate as the CAP gold standard, producing conflicting results. We analyzed the diagnostic accuracy of biomarkers in suspected CAP adults visiting emergency departments for whom CAP diagnosis was established by an adjudication committee which founded its judgment on a systematic multidetector thoracic CT scan. METHODS: In an ancillary study of a multi-center prospective study evaluating the impact of systematic thoracic CT scan on CAP diagnosis, sensitivity and specificity of C-reactive protein CRP and procalcitonin PCT were evaluated. Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion. An adjudication committee classified CAP diagnostic probability on a 4-level Likert scale, based on all available data.", "Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion. An adjudication committee classified CAP diagnostic probability on a 4-level Likert scale, based on all available data. RESULTS: Two hundred patients with suspected CAP were analyzed. The adjudication committee classified 98 patients 49.0 % as definite CAP, 8 4.0 % as probable, 23 11.5 % as possible and excluded in 71 35.5 %, including 29 patients with pulmonary infiltrates on chest X-ray . Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded. Viruses were identified by PCR in 29 % of patients classified as definite.", "Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded. Viruses were identified by PCR in 29 % of patients classified as definite. Area under the curve was 0.787 95 % confidence interval 95 % CI , 0.717 to 0.857 for CRP and 0.655 95 % CI, 0.570 to 0.739 for PCT to detect definite CAP. CRP threshold at 50 mg/L resulted in a positive predictive value of 0.76 and a negative predictive value of 0.75. No PCT cut-off resulted in satisfactory positive or negative predictive values. CRP and PCT accuracy was not improved by exclusion of the 25 25.5 % definite viral CAP cases.", "No PCT cut-off resulted in satisfactory positive or negative predictive values. CRP and PCT accuracy was not improved by exclusion of the 25 25.5 % definite viral CAP cases. CONCLUSIONS: For patients with suspected CAP visiting emergency departments, diagnostic accuracy of CRP and PCT are insufficient to confirm the CAP diagnosis established using a gold standard that includes thoracic CT scan. Diagnostic accuracy of these biomarkers is also insufficient to distinguish bacterial CAP from viral CAP. TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 February 7, 2012 ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s13054-015-1083-6 contains supplementary material, which is available to authorized users. Text: Community-acquired pneumonia CAP is a frequently seen disease, with high morbidity and mortality, accounting for 600,000 hospitalizations each year.", "TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 February 7, 2012 ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s13054-015-1083-6 contains supplementary material, which is available to authorized users. Text: Community-acquired pneumonia CAP is a frequently seen disease, with high morbidity and mortality, accounting for 600,000 hospitalizations each year. It represents the seventh leading cause of death in the USA . CAP prognosis depends on the rapidity of specific treatment, which should ideally be initiated within four hours and no later than eight hours after diagnosis . CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms , an increase in biomarkers reflecting systemic inflammatory response syndrome SIRS , and the presence of new parenchymal infiltrates on chest X-ray. However, CAP diagnosis remains uncertain in many cases with alternative diagnoses, such as cardiac failure, acute bronchitis, chronic obstructive pulmonary disease COPD exacerbations, pulmonary embolism, neoplasia, and sepsis .", "CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms , an increase in biomarkers reflecting systemic inflammatory response syndrome SIRS , and the presence of new parenchymal infiltrates on chest X-ray. However, CAP diagnosis remains uncertain in many cases with alternative diagnoses, such as cardiac failure, acute bronchitis, chronic obstructive pulmonary disease COPD exacerbations, pulmonary embolism, neoplasia, and sepsis . Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis ; over diagnosis of CAP is frequent when infiltrates of noninfectious origin coexist with pulmonary or general symptoms, and the diagnosis of CAP is often ignored when the lung infiltrates are at the limit of visibility or are hidden due to superposition . We recently published a study in which thoracic CT scan was systematically performed in a population of clinically suspected CAP patients visiting the emergency department for CAP the ESCAPED study . We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan. We also reported the isolation of viruses in one-third of patients .", "We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan. We also reported the isolation of viruses in one-third of patients . Several attempts have been made to improve CAP diagnosis based on biomarkers, such as C-reactive protein CRP and procalcitonin PCT ; however, there are conflicting data on their reliability . This could be due to the consideration of CAP diagnosis based on chest X-ray as establishing pulmonary infection. In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up. We also analyzed whether the viral etiology of definite CAP based on polymerase chain reaction PCR multiplex naso-pharyngeal swab interfered with the accuracy of the biomarkers.", "In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up. We also analyzed whether the viral etiology of definite CAP based on polymerase chain reaction PCR multiplex naso-pharyngeal swab interfered with the accuracy of the biomarkers. Setting ESCAPED was a multicenter, prospective, interventional study, entitled \"Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department ESCAPED \" , conducted from November 2011 to January 2013, in four emergency departments EDs of four tertiary teaching hospitals in Paris, France, designed to measure the impact of thoracic CT scan on clinical decision. The study was sponsored and monitored by the Paris public health hospitals, and funded by the French Ministry of Health. The French health authorities Agence nationale de sécurité des medicaments et produits de santé, ANSM and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures. All enrolled patients provided written informed consent for inclusion.", "The French health authorities Agence nationale de sécurité des medicaments et produits de santé, ANSM and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures. All enrolled patients provided written informed consent for inclusion. The protocol was registered in the clinicaltrial.gov website under the PACSCAN acronym, the French translation of the English ESCAPED acronym NCT01574066 . The Ethics Committee of Ile de France Comité de Protection des Personnes. Paris N°2 011-oct-12749 approved the study protocol. The primary objective was to compare CRP and PCT values in the four different categories of CAP level of certainty using the day-28 adjudication committee classification.", "Paris N°2 011-oct-12749 approved the study protocol. The primary objective was to compare CRP and PCT values in the four different categories of CAP level of certainty using the day-28 adjudication committee classification. The four categories were: 1 absence of CAP hereafter referred to as excluded CAP diagnosis; 2 possible CAP; 3 probable CAP; and 4 definite CAP. The secondary objectives were to assess whether CRP and PCT were associated with CAP diagnosis using sensitivity analyses in three successive subgroups chosen a priori; 1 when specifically considering patients classified as having excluded CAP diagnosis and definite CAP i.e., the patients for whom the level of certainty was the highest ; 2 when patients with excluded CAP diagnosis and diagnosed extra-pulmonary infectious disease which may increase biomarker values were not taken into account, in the excluded CAP group; and 3 when patients classified as viral CAP were not taken into account in the definite CAP group, as PCT has been reported to be lower in viral infections as compared to bacterial infections . Consecutive adults . Multidetector thoracic CT-scan was performed after chest X-ray, ideally within the four hours following inclusion.", "Consecutive adults . Multidetector thoracic CT-scan was performed after chest X-ray, ideally within the four hours following inclusion. Chest X-ray and thoracic CT-scan were performed using a standardized protocol. The four levels of CAP probability according to CT scan were defined as definite systematic alveolar condensation, alveolar condensation with peripheral and localized ground glass opacities, bronchiolar focal or multifocal micronodules , probable peripheral alveolar condensation, retractile systematic alveolar condensation, or diffuse ground glass opacities , possible pulmonary infarct , or excluded pulmonary mass, other abnormalities, or normal images . Scan views were recorded on a DVD. Based on data collected from baseline standardized case report forms, DVD recorded pictures of X-ray and CTscan, and blinded to local interpretations, an adjudication committee consisting of three independent senior experts in infectious diseases, pneumology and radiology retrospectively assigned the probability of CAP diagnosis using the same 4-level Likert scale, with all available data including patients' discharge summary, and follow-up data obtained by assistant investigators who contacted by phone either the patient, relatives or general practitioners at day 28.", "Scan views were recorded on a DVD. Based on data collected from baseline standardized case report forms, DVD recorded pictures of X-ray and CTscan, and blinded to local interpretations, an adjudication committee consisting of three independent senior experts in infectious diseases, pneumology and radiology retrospectively assigned the probability of CAP diagnosis using the same 4-level Likert scale, with all available data including patients' discharge summary, and follow-up data obtained by assistant investigators who contacted by phone either the patient, relatives or general practitioners at day 28. For this study, the gold standard of CAP was the diagnosis assessed by this adjudication committee. Alternative diagnoses were established for excluded CAP and classified as non-CAP pulmonary diseases and extra-pulmonary infectious diseases and others. Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study. CRP and PCT concentrations were measured a posteriori on plasma collection see Additional file 1 for methodology , except for patients in whom marker dosage was performed by the emergency practitioner on his own initiative.", "Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study. CRP and PCT concentrations were measured a posteriori on plasma collection see Additional file 1 for methodology , except for patients in whom marker dosage was performed by the emergency practitioner on his own initiative. Naso-pharyngeal swabs were collected at enrollment and placed in a Middle Virocult MWE Sigma® transport medium. Samples were kept at room temperature and sent to the virology laboratory of Bichat -Claude Bernard Hospital Paris as soon as possible after collection. The samples were not frozen and thawed. Multiplex PCR RespiFinder-19 assay Pathofinder®, Maastricht, Netherlands was performed on naso-pharyngeal swabs to detect 15 respiratory viruses -coronavirus 229E, NL63, OC43, human metapneumovirus hMPV , influenza A, A H1N1 pdm2009 and B viruses, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus RSV A and B, rhinovirus, adenovirus, and 4 intracellular bacteria -Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, in one reaction.", "The samples were not frozen and thawed. Multiplex PCR RespiFinder-19 assay Pathofinder®, Maastricht, Netherlands was performed on naso-pharyngeal swabs to detect 15 respiratory viruses -coronavirus 229E, NL63, OC43, human metapneumovirus hMPV , influenza A, A H1N1 pdm2009 and B viruses, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus RSV A and B, rhinovirus, adenovirus, and 4 intracellular bacteria -Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, in one reaction. The multiplex PCR results were not available to the adjudication committee. Routine microbiological examinations were also performed at the discretion of the emergency physicians and included blood culture, sputum culture, and antigenuria see Additional file 1 for methodology . CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples sputum, blood culture, antigenuria when performed. Baseline and follow-up characteristics were described by means and standard deviations SD or by median and interquartile range IQR for continuous variables normally distributed or with skewed distribution, respectively, and by percentages for categorical variables, for the total study population and for the study groups.", "CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples sputum, blood culture, antigenuria when performed. Baseline and follow-up characteristics were described by means and standard deviations SD or by median and interquartile range IQR for continuous variables normally distributed or with skewed distribution, respectively, and by percentages for categorical variables, for the total study population and for the study groups. We performed chi-square or Fisher exact tests when appropriate for qualitative variables, and the Student or Mann-Whitney tests for continuous variables with skewed distributions to compare baseline patient characteristics and study outcomes between study groups. The distribution values of the biomarkers were determined in the different populations of patients using boxplots. The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis definite CAP vs excluded CAP . CRP was evaluated at several cut-off points of 20 mg/L, 30 mg/L, 50 mg/L, 70 mg/L, and 100 mg/L, values used in previous studies .", "The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis definite CAP vs excluded CAP . CRP was evaluated at several cut-off points of 20 mg/L, 30 mg/L, 50 mg/L, 70 mg/L, and 100 mg/L, values used in previous studies . Several cut-off points for PCT were chosen at the level of 0.10 μg/L , and at the two levels for suspected bacterial infection as stated by the manufacturer, i.e., 0.25 μg/L and 0.50 μg/L. Sensitivities, specificities, positive predictive values PPVs , negative predictive values NPVs , and likelihood ratio were calculated. Receiver operating characteristic ROC curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP. From these optimal cut-offs for CRP and PCT, sensitivity analyses were performed combining the CRP and PCT cut-offs.", "Receiver operating characteristic ROC curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP. From these optimal cut-offs for CRP and PCT, sensitivity analyses were performed combining the CRP and PCT cut-offs. A multivariate logistic regression model was built to identify factors associated with having definite CAP as compared to having an excluded CAP diagnosis. We excluded from the excluded CAP diagnosis group, patients with an extra-pulmonary infectious disease. All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval 95 % CI and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test. All tests were two-sided, and p-values below 0.05 were considered to denote statistical significance.", "All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval 95 % CI and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test. All tests were two-sided, and p-values below 0.05 were considered to denote statistical significance. All statistical analyses were performed using SPSS statistical software version 21.0 SPSS Inc., Chicago, IL, USA . Two hundred patients with suspected CAP out of the 319 in the ESCAPED study were included in the present study, for which CRP and PCT assays and nasopharyngeal swab for multiplex PCR were available Fig. 1 . Characteristics of the 200 patients age, age more than 65, gender, probability of CAP diagnosis by adjudication committee were not significantly different from those of the 119 other patients of the ESCAPED study and are summarized in Table 1 .", "1 . Characteristics of the 200 patients age, age more than 65, gender, probability of CAP diagnosis by adjudication committee were not significantly different from those of the 119 other patients of the ESCAPED study and are summarized in Table 1 . CRP and PCT assays were performed based on the emergency practitioner's own initiative in 70 patients for CRP and 131 for PCT, or performed a posteriori on plasma samples of the remaining patients. Sex ratio was approximately 1. More than half of the patients 54 % were 65 years of age or older. The Pulmonary infiltrates were seen on chest X-ray in 127 63.5 % patients.", "More than half of the patients 54 % were 65 years of age or older. The Pulmonary infiltrates were seen on chest X-ray in 127 63.5 % patients. Thoracic CT-scan excluded a CAP diagnosis in 16.5 % of these 127 patients; on the contrary, thoracic CT-scan revealed a parenchymal infiltrate in 27 % of the 73 patients without infiltrate on chest X-ray. Based on all available data including multidetector CT scan results but excluding PCR results , the adjudication The CRP and PCT distributions in the 200 patients are presented in Fig. 2 A statistically significant difference between the two groups excluded CAP vs definite CAP was demonstrated for several cut-off points for CRP and PCT Table 2 . For CRP, the value of 50 mg/L resulted in a PPV of 0.76 and a NPV of 0.75.", "2 A statistically significant difference between the two groups excluded CAP vs definite CAP was demonstrated for several cut-off points for CRP and PCT Table 2 . For CRP, the value of 50 mg/L resulted in a PPV of 0.76 and a NPV of 0.75. For PCT, no value resulted in a satisfactory PPV or NPV. For these two biochemical markers, the ability to predict CAP was evaluated by a ROC curve. The AUC was 0.787 95 % CI 0.717-0.857 , optimal cut-off = 45.9 mg/L for CRP Fig. 3 and 0.655 95 % CI 0.570-0.739 , optimal cut-off = 0.13 μg/ L for PCT Fig. 4 .", "3 and 0.655 95 % CI 0.570-0.739 , optimal cut-off = 0.13 μg/ L for PCT Fig. 4 . Sensitivity analyses for the combination of CRP and PCT, using these optimal cut-offs, resulted in a PPV of 0.74 and a NPV of 0.58. Use of the other PCT cut-offs did not result in better PPV or NPV Table 2 . The present study is novel as patients prospectively benefited from extensive investigation to determine the diagnosis of CAP in the ED, including both early multidetector thoracic CT-scan and day-28 adjudication committee. This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients.", "The present study is novel as patients prospectively benefited from extensive investigation to determine the diagnosis of CAP in the ED, including both early multidetector thoracic CT-scan and day-28 adjudication committee. This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients. In these extensively characterized patients, both CRP and PCT lacked operational precision to allow the decisionmaking process to rule out or confirm diagnosis of CAP even in selected subgroups. The clinical characteristics of the patients included in this sub-study are consistent with those in the current literature. As previously reported, patients frequently had a history of respiratory disorders, cancer and congestive heart failure . The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65.", "As previously reported, patients frequently had a history of respiratory disorders, cancer and congestive heart failure . The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65. This may explain why the mean age of our patients 64 years falls within the lower values of those reported elsewhere . Data to identify the microbial agent responsible for the disease were collected by the usual techniques and multiplex PCR. Viral identification using naso-pharyngeal PCR that revealed viral respiratory infection in approximately one-third of cases was concordant with values reported in the literature . Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP.", "Viral identification using naso-pharyngeal PCR that revealed viral respiratory infection in approximately one-third of cases was concordant with values reported in the literature . Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP. In the present study, patients were recruited on the basis of initial clinical assessment for the diagnosis of CAP. Therefore, we believe that the characteristics of the patients closely correspond to those that lead practitioners to consider a possible diagnosis of CAP. In these patients, the design of our study allowed us to confirm or refute CAP diagnosis with a high level of certainty. Results confirmed the poor predictive value of clinical symptoms new onset of systemic features and symptoms of an acute lower respiratory tract illness in identifying CAP patients .", "In these patients, the design of our study allowed us to confirm or refute CAP diagnosis with a high level of certainty. Results confirmed the poor predictive value of clinical symptoms new onset of systemic features and symptoms of an acute lower respiratory tract illness in identifying CAP patients . Indeed, clinical presentation of excluded CAP patients was similar to that of definite CAP patients except for fever and cough that were more frequent in definite CAP patients. Furthermore, the design also revealed that the combination of clinical symptoms and chest X-ray results led to CAP misdiagnosis in a high number of patients, including the 98 whose CAP diagnosis was excluded by the adjudication committee and who would have been considered as possible, probable or definite CAP without the use of the CT scan. This low specificity of clinical-standard radiological evaluation led to the consideration of either non-infectious pulmonary diseases such as, cardiac failure, pulmonary embolism, pulmonary neoplasia or bronchitis or extra-pulmonary infectious diseases as CAP. Of note, some of these diseases are also associated with increased biomarker values.", "This low specificity of clinical-standard radiological evaluation led to the consideration of either non-infectious pulmonary diseases such as, cardiac failure, pulmonary embolism, pulmonary neoplasia or bronchitis or extra-pulmonary infectious diseases as CAP. Of note, some of these diseases are also associated with increased biomarker values. This raises concerns about previous evaluations of biomarkers in CAP-suspected patients, which used clinical and standard radiological chest X-ray evaluations as the gold standard for CAP diagnosis . The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections . However, this issue is still unresolved , with conflicting positions . In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis, we were unable to establish discriminating values for PCT.", "However, this issue is still unresolved , with conflicting positions . In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis, we were unable to establish discriminating values for PCT. Recent data suggested that CRP could be of more help in assisting in the diagnosis of lower respiratory tract infections LRTI . In our study, although CRP seems more discriminating than PCT, neither the experimental exclusion of extra-pulmonary bacterial infections from the excluded CAP group, nor the exclusion of viral CAP from the definite CAP patients group, made possible the determination of a discriminant cutoff. The combination of CRP and PCT was not more discriminating than each biomarker separately. An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy .", "The combination of CRP and PCT was not more discriminating than each biomarker separately. An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy . According to this strategy, a PCT concentration higher than 0.25 μg/L should prompt administration of antibiotics to patients with suspected LRTI. In our study, this value was associated with poor performance. Additionally, mean PCT levels remained above this threshold both in excluded CAP patients without infectious disorders and in definite CAP presumably related to virus. Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting. This study has some limitations.", "Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting. This study has some limitations. First, the adjudication committee was not blinded to the value of biomarkers measured at bedside in some patients 70 for CRP and 131 for PCT and its CAP classification could thus have been influenced by these results. However, the lack of statistically significant differences in the mean CRP and PCT values in the definite CAP cases, whether or not these biomarkers were available for the adjudication committee, argues against a major impact of these results on adjudication committee classification. Second, another critical point is the prescription of antibiotic therapy 34 % previous to inclusion. We cannot exclude that these previously-treated CAP patients may have altered biomarker performance and reduced the yield of bacterial cultures, although such a population reflects the usual emergency department practice.", "Second, another critical point is the prescription of antibiotic therapy 34 % previous to inclusion. We cannot exclude that these previously-treated CAP patients may have altered biomarker performance and reduced the yield of bacterial cultures, although such a population reflects the usual emergency department practice. Third, multiplex PCR was performed on naso-pharyngeal sampling and not on lower respiratory tract samples, which does not allow definite confirmation of the viral origin of CAP. However, a recent large study on CAP patients which reported a viral etiology of CAP at a comparable rate, did not find upper respiratory tract shedding in a control population without CAP explored during the same year and season . Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty. Given the diversity of the clinical and radiological CAP presentations, CAP diagnosis is often uncertain.", "Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty. Given the diversity of the clinical and radiological CAP presentations, CAP diagnosis is often uncertain. In our population of patients treated in the emergency room with clinical symptoms evoking CAP, neither CRP nor PCT cut-off values carried sufficient weight to confirm or refute CAP diagnosis at bedside; this underlines that these biomarkers are telltales of the host inflammatory response to the intrusion of microorganisms independent of the site of infection. These results, based on a systematic thoracic CT scan evaluation of CAP-suspected patients, do not argue for the use of CRP and PCT in routine care to diagnose CAP with certainty in patients visiting the ED for suspected CAP." ]

[ "INTRODUCTION: Community-acquired pneumonia CAP requires prompt treatment, but its diagnosis is complex. Improvement of bacterial CAP diagnosis by biomarkers has been evaluated using chest X-ray infiltrate as the CAP gold standard, producing conflicting results. We analyzed the diagnostic accuracy of biomarkers in suspected CAP adults visiting emergency departments for whom CAP diagnosis was established by an adjudication committee which founded its judgment on a systematic multidetector thoracic CT scan. METHODS: In an ancillary study of a multi-center prospective study evaluating the impact of systematic thoracic CT scan on CAP diagnosis, sensitivity and specificity of C-reactive protein CRP and procalcitonin PCT were evaluated. Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion. An adjudication committee classified CAP diagnostic probability on a 4-level Likert scale, based on all available data.", "Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion. An adjudication committee classified CAP diagnostic probability on a 4-level Likert scale, based on all available data. RESULTS: Two hundred patients with suspected CAP were analyzed. The adjudication committee classified 98 patients 49.0 % as definite CAP, 8 4.0 % as probable, 23 11.5 % as possible and excluded in 71 35.5 %, including 29 patients with pulmonary infiltrates on chest X-ray . Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded. Viruses were identified by PCR in 29 % of patients classified as definite.", "Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded. Viruses were identified by PCR in 29 % of patients classified as definite. Area under the curve was 0.787 95 % confidence interval 95 % CI , 0.717 to 0.857 for CRP and 0.655 95 % CI, 0.570 to 0.739 for PCT to detect definite CAP. CRP threshold at 50 mg/L resulted in a positive predictive value of 0.76 and a negative predictive value of 0.75. No PCT cut-off resulted in satisfactory positive or negative predictive values. CRP and PCT accuracy was not improved by exclusion of the 25 25.5 % definite viral CAP cases.", "No PCT cut-off resulted in satisfactory positive or negative predictive values. CRP and PCT accuracy was not improved by exclusion of the 25 25.5 % definite viral CAP cases. CONCLUSIONS: For patients with suspected CAP visiting emergency departments, diagnostic accuracy of CRP and PCT are insufficient to confirm the CAP diagnosis established using a gold standard that includes thoracic CT scan. Diagnostic accuracy of these biomarkers is also insufficient to distinguish bacterial CAP from viral CAP. TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 February 7, 2012 ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s13054-015-1083-6 contains supplementary material, which is available to authorized users. Text: Community-acquired pneumonia CAP is a frequently seen disease, with high morbidity and mortality, accounting for 600,000 hospitalizations each year.", "TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 February 7, 2012 ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s13054-015-1083-6 contains supplementary material, which is available to authorized users. Text: Community-acquired pneumonia CAP is a frequently seen disease, with high morbidity and mortality, accounting for 600,000 hospitalizations each year. It represents the seventh leading cause of death in the USA . CAP prognosis depends on the rapidity of specific treatment, which should ideally be initiated within four hours and no later than eight hours after diagnosis . CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms , an increase in biomarkers reflecting systemic inflammatory response syndrome SIRS , and the presence of new parenchymal infiltrates on chest X-ray. However, CAP diagnosis remains uncertain in many cases with alternative diagnoses, such as cardiac failure, acute bronchitis, chronic obstructive pulmonary disease COPD exacerbations, pulmonary embolism, neoplasia, and sepsis .", "CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms , an increase in biomarkers reflecting systemic inflammatory response syndrome SIRS , and the presence of new parenchymal infiltrates on chest X-ray. However, CAP diagnosis remains uncertain in many cases with alternative diagnoses, such as cardiac failure, acute bronchitis, chronic obstructive pulmonary disease COPD exacerbations, pulmonary embolism, neoplasia, and sepsis . Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis ; over diagnosis of CAP is frequent when infiltrates of noninfectious origin coexist with pulmonary or general symptoms, and the diagnosis of CAP is often ignored when the lung infiltrates are at the limit of visibility or are hidden due to superposition . We recently published a study in which thoracic CT scan was systematically performed in a population of clinically suspected CAP patients visiting the emergency department for CAP the ESCAPED study . We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan. We also reported the isolation of viruses in one-third of patients .", "We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan. We also reported the isolation of viruses in one-third of patients . Several attempts have been made to improve CAP diagnosis based on biomarkers, such as C-reactive protein CRP and procalcitonin PCT ; however, there are conflicting data on their reliability . This could be due to the consideration of CAP diagnosis based on chest X-ray as establishing pulmonary infection. In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up. We also analyzed whether the viral etiology of definite CAP based on polymerase chain reaction PCR multiplex naso-pharyngeal swab interfered with the accuracy of the biomarkers.", "In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up. We also analyzed whether the viral etiology of definite CAP based on polymerase chain reaction PCR multiplex naso-pharyngeal swab interfered with the accuracy of the biomarkers. Setting ESCAPED was a multicenter, prospective, interventional study, entitled \"Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department ESCAPED \" , conducted from November 2011 to January 2013, in four emergency departments EDs of four tertiary teaching hospitals in Paris, France, designed to measure the impact of thoracic CT scan on clinical decision. The study was sponsored and monitored by the Paris public health hospitals, and funded by the French Ministry of Health. The French health authorities Agence nationale de sécurité des medicaments et produits de santé, ANSM and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures. All enrolled patients provided written informed consent for inclusion.", "The French health authorities Agence nationale de sécurité des medicaments et produits de santé, ANSM and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures. All enrolled patients provided written informed consent for inclusion. The protocol was registered in the clinicaltrial.gov website under the PACSCAN acronym, the French translation of the English ESCAPED acronym NCT01574066 . The Ethics Committee of Ile de France Comité de Protection des Personnes. Paris N°2 011-oct-12749 approved the study protocol. The primary objective was to compare CRP and PCT values in the four different categories of CAP level of certainty using the day-28 adjudication committee classification.", "Paris N°2 011-oct-12749 approved the study protocol. The primary objective was to compare CRP and PCT values in the four different categories of CAP level of certainty using the day-28 adjudication committee classification. The four categories were: 1 absence of CAP hereafter referred to as excluded CAP diagnosis; 2 possible CAP; 3 probable CAP; and 4 definite CAP. The secondary objectives were to assess whether CRP and PCT were associated with CAP diagnosis using sensitivity analyses in three successive subgroups chosen a priori; 1 when specifically considering patients classified as having excluded CAP diagnosis and definite CAP i.e., the patients for whom the level of certainty was the highest ; 2 when patients with excluded CAP diagnosis and diagnosed extra-pulmonary infectious disease which may increase biomarker values were not taken into account, in the excluded CAP group; and 3 when patients classified as viral CAP were not taken into account in the definite CAP group, as PCT has been reported to be lower in viral infections as compared to bacterial infections . Consecutive adults . Multidetector thoracic CT-scan was performed after chest X-ray, ideally within the four hours following inclusion.", "Consecutive adults . Multidetector thoracic CT-scan was performed after chest X-ray, ideally within the four hours following inclusion. Chest X-ray and thoracic CT-scan were performed using a standardized protocol. The four levels of CAP probability according to CT scan were defined as definite systematic alveolar condensation, alveolar condensation with peripheral and localized ground glass opacities, bronchiolar focal or multifocal micronodules , probable peripheral alveolar condensation, retractile systematic alveolar condensation, or diffuse ground glass opacities , possible pulmonary infarct , or excluded pulmonary mass, other abnormalities, or normal images . Scan views were recorded on a DVD. Based on data collected from baseline standardized case report forms, DVD recorded pictures of X-ray and CTscan, and blinded to local interpretations, an adjudication committee consisting of three independent senior experts in infectious diseases, pneumology and radiology retrospectively assigned the probability of CAP diagnosis using the same 4-level Likert scale, with all available data including patients' discharge summary, and follow-up data obtained by assistant investigators who contacted by phone either the patient, relatives or general practitioners at day 28.", "Scan views were recorded on a DVD. Based on data collected from baseline standardized case report forms, DVD recorded pictures of X-ray and CTscan, and blinded to local interpretations, an adjudication committee consisting of three independent senior experts in infectious diseases, pneumology and radiology retrospectively assigned the probability of CAP diagnosis using the same 4-level Likert scale, with all available data including patients' discharge summary, and follow-up data obtained by assistant investigators who contacted by phone either the patient, relatives or general practitioners at day 28. For this study, the gold standard of CAP was the diagnosis assessed by this adjudication committee. Alternative diagnoses were established for excluded CAP and classified as non-CAP pulmonary diseases and extra-pulmonary infectious diseases and others. Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study. CRP and PCT concentrations were measured a posteriori on plasma collection see Additional file 1 for methodology , except for patients in whom marker dosage was performed by the emergency practitioner on his own initiative.", "Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study. CRP and PCT concentrations were measured a posteriori on plasma collection see Additional file 1 for methodology , except for patients in whom marker dosage was performed by the emergency practitioner on his own initiative. Naso-pharyngeal swabs were collected at enrollment and placed in a Middle Virocult MWE Sigma® transport medium. Samples were kept at room temperature and sent to the virology laboratory of Bichat -Claude Bernard Hospital Paris as soon as possible after collection. The samples were not frozen and thawed. Multiplex PCR RespiFinder-19 assay Pathofinder®, Maastricht, Netherlands was performed on naso-pharyngeal swabs to detect 15 respiratory viruses -coronavirus 229E, NL63, OC43, human metapneumovirus hMPV , influenza A, A H1N1 pdm2009 and B viruses, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus RSV A and B, rhinovirus, adenovirus, and 4 intracellular bacteria -Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, in one reaction.", "The samples were not frozen and thawed. Multiplex PCR RespiFinder-19 assay Pathofinder®, Maastricht, Netherlands was performed on naso-pharyngeal swabs to detect 15 respiratory viruses -coronavirus 229E, NL63, OC43, human metapneumovirus hMPV , influenza A, A H1N1 pdm2009 and B viruses, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus RSV A and B, rhinovirus, adenovirus, and 4 intracellular bacteria -Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, in one reaction. The multiplex PCR results were not available to the adjudication committee. Routine microbiological examinations were also performed at the discretion of the emergency physicians and included blood culture, sputum culture, and antigenuria see Additional file 1 for methodology . CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples sputum, blood culture, antigenuria when performed. Baseline and follow-up characteristics were described by means and standard deviations SD or by median and interquartile range IQR for continuous variables normally distributed or with skewed distribution, respectively, and by percentages for categorical variables, for the total study population and for the study groups.", "CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples sputum, blood culture, antigenuria when performed. Baseline and follow-up characteristics were described by means and standard deviations SD or by median and interquartile range IQR for continuous variables normally distributed or with skewed distribution, respectively, and by percentages for categorical variables, for the total study population and for the study groups. We performed chi-square or Fisher exact tests when appropriate for qualitative variables, and the Student or Mann-Whitney tests for continuous variables with skewed distributions to compare baseline patient characteristics and study outcomes between study groups. The distribution values of the biomarkers were determined in the different populations of patients using boxplots. The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis definite CAP vs excluded CAP . CRP was evaluated at several cut-off points of 20 mg/L, 30 mg/L, 50 mg/L, 70 mg/L, and 100 mg/L, values used in previous studies .", "The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis definite CAP vs excluded CAP . CRP was evaluated at several cut-off points of 20 mg/L, 30 mg/L, 50 mg/L, 70 mg/L, and 100 mg/L, values used in previous studies . Several cut-off points for PCT were chosen at the level of 0.10 μg/L , and at the two levels for suspected bacterial infection as stated by the manufacturer, i.e., 0.25 μg/L and 0.50 μg/L. Sensitivities, specificities, positive predictive values PPVs , negative predictive values NPVs , and likelihood ratio were calculated. Receiver operating characteristic ROC curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP. From these optimal cut-offs for CRP and PCT, sensitivity analyses were performed combining the CRP and PCT cut-offs.", "Receiver operating characteristic ROC curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP. From these optimal cut-offs for CRP and PCT, sensitivity analyses were performed combining the CRP and PCT cut-offs. A multivariate logistic regression model was built to identify factors associated with having definite CAP as compared to having an excluded CAP diagnosis. We excluded from the excluded CAP diagnosis group, patients with an extra-pulmonary infectious disease. All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval 95 % CI and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test. All tests were two-sided, and p-values below 0.05 were considered to denote statistical significance.", "All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval 95 % CI and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test. All tests were two-sided, and p-values below 0.05 were considered to denote statistical significance. All statistical analyses were performed using SPSS statistical software version 21.0 SPSS Inc., Chicago, IL, USA . Two hundred patients with suspected CAP out of the 319 in the ESCAPED study were included in the present study, for which CRP and PCT assays and nasopharyngeal swab for multiplex PCR were available Fig. 1 . Characteristics of the 200 patients age, age more than 65, gender, probability of CAP diagnosis by adjudication committee were not significantly different from those of the 119 other patients of the ESCAPED study and are summarized in Table 1 .", "1 . Characteristics of the 200 patients age, age more than 65, gender, probability of CAP diagnosis by adjudication committee were not significantly different from those of the 119 other patients of the ESCAPED study and are summarized in Table 1 . CRP and PCT assays were performed based on the emergency practitioner's own initiative in 70 patients for CRP and 131 for PCT, or performed a posteriori on plasma samples of the remaining patients. Sex ratio was approximately 1. More than half of the patients 54 % were 65 years of age or older. The Pulmonary infiltrates were seen on chest X-ray in 127 63.5 % patients.", "More than half of the patients 54 % were 65 years of age or older. The Pulmonary infiltrates were seen on chest X-ray in 127 63.5 % patients. Thoracic CT-scan excluded a CAP diagnosis in 16.5 % of these 127 patients; on the contrary, thoracic CT-scan revealed a parenchymal infiltrate in 27 % of the 73 patients without infiltrate on chest X-ray. Based on all available data including multidetector CT scan results but excluding PCR results , the adjudication The CRP and PCT distributions in the 200 patients are presented in Fig. 2 A statistically significant difference between the two groups excluded CAP vs definite CAP was demonstrated for several cut-off points for CRP and PCT Table 2 . For CRP, the value of 50 mg/L resulted in a PPV of 0.76 and a NPV of 0.75.", "2 A statistically significant difference between the two groups excluded CAP vs definite CAP was demonstrated for several cut-off points for CRP and PCT Table 2 . For CRP, the value of 50 mg/L resulted in a PPV of 0.76 and a NPV of 0.75. For PCT, no value resulted in a satisfactory PPV or NPV. For these two biochemical markers, the ability to predict CAP was evaluated by a ROC curve. The AUC was 0.787 95 % CI 0.717-0.857 , optimal cut-off = 45.9 mg/L for CRP Fig. 3 and 0.655 95 % CI 0.570-0.739 , optimal cut-off = 0.13 μg/ L for PCT Fig. 4 .", "3 and 0.655 95 % CI 0.570-0.739 , optimal cut-off = 0.13 μg/ L for PCT Fig. 4 . Sensitivity analyses for the combination of CRP and PCT, using these optimal cut-offs, resulted in a PPV of 0.74 and a NPV of 0.58. Use of the other PCT cut-offs did not result in better PPV or NPV Table 2 . The present study is novel as patients prospectively benefited from extensive investigation to determine the diagnosis of CAP in the ED, including both early multidetector thoracic CT-scan and day-28 adjudication committee. This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients.", "The present study is novel as patients prospectively benefited from extensive investigation to determine the diagnosis of CAP in the ED, including both early multidetector thoracic CT-scan and day-28 adjudication committee. This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients. In these extensively characterized patients, both CRP and PCT lacked operational precision to allow the decisionmaking process to rule out or confirm diagnosis of CAP even in selected subgroups. The clinical characteristics of the patients included in this sub-study are consistent with those in the current literature. As previously reported, patients frequently had a history of respiratory disorders, cancer and congestive heart failure . The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65.", "As previously reported, patients frequently had a history of respiratory disorders, cancer and congestive heart failure . The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65. This may explain why the mean age of our patients 64 years falls within the lower values of those reported elsewhere . Data to identify the microbial agent responsible for the disease were collected by the usual techniques and multiplex PCR. Viral identification using naso-pharyngeal PCR that revealed viral respiratory infection in approximately one-third of cases was concordant with values reported in the literature . Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP.", "Viral identification using naso-pharyngeal PCR that revealed viral respiratory infection in approximately one-third of cases was concordant with values reported in the literature . Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP. In the present study, patients were recruited on the basis of initial clinical assessment for the diagnosis of CAP. Therefore, we believe that the characteristics of the patients closely correspond to those that lead practitioners to consider a possible diagnosis of CAP. In these patients, the design of our study allowed us to confirm or refute CAP diagnosis with a high level of certainty. Results confirmed the poor predictive value of clinical symptoms new onset of systemic features and symptoms of an acute lower respiratory tract illness in identifying CAP patients .", "In these patients, the design of our study allowed us to confirm or refute CAP diagnosis with a high level of certainty. Results confirmed the poor predictive value of clinical symptoms new onset of systemic features and symptoms of an acute lower respiratory tract illness in identifying CAP patients . Indeed, clinical presentation of excluded CAP patients was similar to that of definite CAP patients except for fever and cough that were more frequent in definite CAP patients. Furthermore, the design also revealed that the combination of clinical symptoms and chest X-ray results led to CAP misdiagnosis in a high number of patients, including the 98 whose CAP diagnosis was excluded by the adjudication committee and who would have been considered as possible, probable or definite CAP without the use of the CT scan. This low specificity of clinical-standard radiological evaluation led to the consideration of either non-infectious pulmonary diseases such as, cardiac failure, pulmonary embolism, pulmonary neoplasia or bronchitis or extra-pulmonary infectious diseases as CAP. Of note, some of these diseases are also associated with increased biomarker values.", "This low specificity of clinical-standard radiological evaluation led to the consideration of either non-infectious pulmonary diseases such as, cardiac failure, pulmonary embolism, pulmonary neoplasia or bronchitis or extra-pulmonary infectious diseases as CAP. Of note, some of these diseases are also associated with increased biomarker values. This raises concerns about previous evaluations of biomarkers in CAP-suspected patients, which used clinical and standard radiological chest X-ray evaluations as the gold standard for CAP diagnosis . The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections . However, this issue is still unresolved , with conflicting positions . In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis, we were unable to establish discriminating values for PCT.", "However, this issue is still unresolved , with conflicting positions . In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis, we were unable to establish discriminating values for PCT. Recent data suggested that CRP could be of more help in assisting in the diagnosis of lower respiratory tract infections LRTI . In our study, although CRP seems more discriminating than PCT, neither the experimental exclusion of extra-pulmonary bacterial infections from the excluded CAP group, nor the exclusion of viral CAP from the definite CAP patients group, made possible the determination of a discriminant cutoff. The combination of CRP and PCT was not more discriminating than each biomarker separately. An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy .", "The combination of CRP and PCT was not more discriminating than each biomarker separately. An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy . According to this strategy, a PCT concentration higher than 0.25 μg/L should prompt administration of antibiotics to patients with suspected LRTI. In our study, this value was associated with poor performance. Additionally, mean PCT levels remained above this threshold both in excluded CAP patients without infectious disorders and in definite CAP presumably related to virus. Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting. This study has some limitations.", "Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting. This study has some limitations. First, the adjudication committee was not blinded to the value of biomarkers measured at bedside in some patients 70 for CRP and 131 for PCT and its CAP classification could thus have been influenced by these results. However, the lack of statistically significant differences in the mean CRP and PCT values in the definite CAP cases, whether or not these biomarkers were available for the adjudication committee, argues against a major impact of these results on adjudication committee classification. Second, another critical point is the prescription of antibiotic therapy 34 % previous to inclusion. We cannot exclude that these previously-treated CAP patients may have altered biomarker performance and reduced the yield of bacterial cultures, although such a population reflects the usual emergency department practice.", "Second, another critical point is the prescription of antibiotic therapy 34 % previous to inclusion. We cannot exclude that these previously-treated CAP patients may have altered biomarker performance and reduced the yield of bacterial cultures, although such a population reflects the usual emergency department practice. Third, multiplex PCR was performed on naso-pharyngeal sampling and not on lower respiratory tract samples, which does not allow definite confirmation of the viral origin of CAP. However, a recent large study on CAP patients which reported a viral etiology of CAP at a comparable rate, did not find upper respiratory tract shedding in a control population without CAP explored during the same year and season . Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty. Given the diversity of the clinical and radiological CAP presentations, CAP diagnosis is often uncertain.", "Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty. Given the diversity of the clinical and radiological CAP presentations, CAP diagnosis is often uncertain. In our population of patients treated in the emergency room with clinical symptoms evoking CAP, neither CRP nor PCT cut-off values carried sufficient weight to confirm or refute CAP diagnosis at bedside; this underlines that these biomarkers are telltales of the host inflammatory response to the intrusion of microorganisms independent of the site of infection. These results, based on a systematic thoracic CT scan evaluation of CAP-suspected patients, do not argue for the use of CRP and PCT in routine care to diagnose CAP with certainty in patients visiting the ED for suspected CAP." ]

[ "INTRODUCTION: Community-acquired pneumonia CAP requires prompt treatment, but its diagnosis is complex. Improvement of bacterial CAP diagnosis by biomarkers has been evaluated using chest X-ray infiltrate as the CAP gold standard, producing conflicting results. We analyzed the diagnostic accuracy of biomarkers in suspected CAP adults visiting emergency departments for whom CAP diagnosis was established by an adjudication committee which founded its judgment on a systematic multidetector thoracic CT scan. METHODS: In an ancillary study of a multi-center prospective study evaluating the impact of systematic thoracic CT scan on CAP diagnosis, sensitivity and specificity of C-reactive protein CRP and procalcitonin PCT were evaluated. Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion. An adjudication committee classified CAP diagnostic probability on a 4-level Likert scale, based on all available data.", "Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion. An adjudication committee classified CAP diagnostic probability on a 4-level Likert scale, based on all available data. RESULTS: Two hundred patients with suspected CAP were analyzed. The adjudication committee classified 98 patients 49.0 % as definite CAP, 8 4.0 % as probable, 23 11.5 % as possible and excluded in 71 35.5 %, including 29 patients with pulmonary infiltrates on chest X-ray . Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded. Viruses were identified by PCR in 29 % of patients classified as definite.", "Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded. Viruses were identified by PCR in 29 % of patients classified as definite. Area under the curve was 0.787 95 % confidence interval 95 % CI , 0.717 to 0.857 for CRP and 0.655 95 % CI, 0.570 to 0.739 for PCT to detect definite CAP. CRP threshold at 50 mg/L resulted in a positive predictive value of 0.76 and a negative predictive value of 0.75. No PCT cut-off resulted in satisfactory positive or negative predictive values. CRP and PCT accuracy was not improved by exclusion of the 25 25.5 % definite viral CAP cases.", "No PCT cut-off resulted in satisfactory positive or negative predictive values. CRP and PCT accuracy was not improved by exclusion of the 25 25.5 % definite viral CAP cases. CONCLUSIONS: For patients with suspected CAP visiting emergency departments, diagnostic accuracy of CRP and PCT are insufficient to confirm the CAP diagnosis established using a gold standard that includes thoracic CT scan. Diagnostic accuracy of these biomarkers is also insufficient to distinguish bacterial CAP from viral CAP. TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 February 7, 2012 ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s13054-015-1083-6 contains supplementary material, which is available to authorized users. Text: Community-acquired pneumonia CAP is a frequently seen disease, with high morbidity and mortality, accounting for 600,000 hospitalizations each year.", "TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 February 7, 2012 ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s13054-015-1083-6 contains supplementary material, which is available to authorized users. Text: Community-acquired pneumonia CAP is a frequently seen disease, with high morbidity and mortality, accounting for 600,000 hospitalizations each year. It represents the seventh leading cause of death in the USA . CAP prognosis depends on the rapidity of specific treatment, which should ideally be initiated within four hours and no later than eight hours after diagnosis . CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms , an increase in biomarkers reflecting systemic inflammatory response syndrome SIRS , and the presence of new parenchymal infiltrates on chest X-ray. However, CAP diagnosis remains uncertain in many cases with alternative diagnoses, such as cardiac failure, acute bronchitis, chronic obstructive pulmonary disease COPD exacerbations, pulmonary embolism, neoplasia, and sepsis .", "CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms , an increase in biomarkers reflecting systemic inflammatory response syndrome SIRS , and the presence of new parenchymal infiltrates on chest X-ray. However, CAP diagnosis remains uncertain in many cases with alternative diagnoses, such as cardiac failure, acute bronchitis, chronic obstructive pulmonary disease COPD exacerbations, pulmonary embolism, neoplasia, and sepsis . Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis ; over diagnosis of CAP is frequent when infiltrates of noninfectious origin coexist with pulmonary or general symptoms, and the diagnosis of CAP is often ignored when the lung infiltrates are at the limit of visibility or are hidden due to superposition . We recently published a study in which thoracic CT scan was systematically performed in a population of clinically suspected CAP patients visiting the emergency department for CAP the ESCAPED study . We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan. We also reported the isolation of viruses in one-third of patients .", "We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan. We also reported the isolation of viruses in one-third of patients . Several attempts have been made to improve CAP diagnosis based on biomarkers, such as C-reactive protein CRP and procalcitonin PCT ; however, there are conflicting data on their reliability . This could be due to the consideration of CAP diagnosis based on chest X-ray as establishing pulmonary infection. In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up. We also analyzed whether the viral etiology of definite CAP based on polymerase chain reaction PCR multiplex naso-pharyngeal swab interfered with the accuracy of the biomarkers.", "In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up. We also analyzed whether the viral etiology of definite CAP based on polymerase chain reaction PCR multiplex naso-pharyngeal swab interfered with the accuracy of the biomarkers. Setting ESCAPED was a multicenter, prospective, interventional study, entitled \"Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department ESCAPED \" , conducted from November 2011 to January 2013, in four emergency departments EDs of four tertiary teaching hospitals in Paris, France, designed to measure the impact of thoracic CT scan on clinical decision. The study was sponsored and monitored by the Paris public health hospitals, and funded by the French Ministry of Health. The French health authorities Agence nationale de sécurité des medicaments et produits de santé, ANSM and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures. All enrolled patients provided written informed consent for inclusion.", "The French health authorities Agence nationale de sécurité des medicaments et produits de santé, ANSM and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures. All enrolled patients provided written informed consent for inclusion. The protocol was registered in the clinicaltrial.gov website under the PACSCAN acronym, the French translation of the English ESCAPED acronym NCT01574066 . The Ethics Committee of Ile de France Comité de Protection des Personnes. Paris N°2 011-oct-12749 approved the study protocol. The primary objective was to compare CRP and PCT values in the four different categories of CAP level of certainty using the day-28 adjudication committee classification.", "Paris N°2 011-oct-12749 approved the study protocol. The primary objective was to compare CRP and PCT values in the four different categories of CAP level of certainty using the day-28 adjudication committee classification. The four categories were: 1 absence of CAP hereafter referred to as excluded CAP diagnosis; 2 possible CAP; 3 probable CAP; and 4 definite CAP. The secondary objectives were to assess whether CRP and PCT were associated with CAP diagnosis using sensitivity analyses in three successive subgroups chosen a priori; 1 when specifically considering patients classified as having excluded CAP diagnosis and definite CAP i.e., the patients for whom the level of certainty was the highest ; 2 when patients with excluded CAP diagnosis and diagnosed extra-pulmonary infectious disease which may increase biomarker values were not taken into account, in the excluded CAP group; and 3 when patients classified as viral CAP were not taken into account in the definite CAP group, as PCT has been reported to be lower in viral infections as compared to bacterial infections . Consecutive adults . Multidetector thoracic CT-scan was performed after chest X-ray, ideally within the four hours following inclusion.", "Consecutive adults . Multidetector thoracic CT-scan was performed after chest X-ray, ideally within the four hours following inclusion. Chest X-ray and thoracic CT-scan were performed using a standardized protocol. The four levels of CAP probability according to CT scan were defined as definite systematic alveolar condensation, alveolar condensation with peripheral and localized ground glass opacities, bronchiolar focal or multifocal micronodules , probable peripheral alveolar condensation, retractile systematic alveolar condensation, or diffuse ground glass opacities , possible pulmonary infarct , or excluded pulmonary mass, other abnormalities, or normal images . Scan views were recorded on a DVD. Based on data collected from baseline standardized case report forms, DVD recorded pictures of X-ray and CTscan, and blinded to local interpretations, an adjudication committee consisting of three independent senior experts in infectious diseases, pneumology and radiology retrospectively assigned the probability of CAP diagnosis using the same 4-level Likert scale, with all available data including patients' discharge summary, and follow-up data obtained by assistant investigators who contacted by phone either the patient, relatives or general practitioners at day 28.", "Scan views were recorded on a DVD. Based on data collected from baseline standardized case report forms, DVD recorded pictures of X-ray and CTscan, and blinded to local interpretations, an adjudication committee consisting of three independent senior experts in infectious diseases, pneumology and radiology retrospectively assigned the probability of CAP diagnosis using the same 4-level Likert scale, with all available data including patients' discharge summary, and follow-up data obtained by assistant investigators who contacted by phone either the patient, relatives or general practitioners at day 28. For this study, the gold standard of CAP was the diagnosis assessed by this adjudication committee. Alternative diagnoses were established for excluded CAP and classified as non-CAP pulmonary diseases and extra-pulmonary infectious diseases and others. Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study. CRP and PCT concentrations were measured a posteriori on plasma collection see Additional file 1 for methodology , except for patients in whom marker dosage was performed by the emergency practitioner on his own initiative.", "Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study. CRP and PCT concentrations were measured a posteriori on plasma collection see Additional file 1 for methodology , except for patients in whom marker dosage was performed by the emergency practitioner on his own initiative. Naso-pharyngeal swabs were collected at enrollment and placed in a Middle Virocult MWE Sigma® transport medium. Samples were kept at room temperature and sent to the virology laboratory of Bichat -Claude Bernard Hospital Paris as soon as possible after collection. The samples were not frozen and thawed. Multiplex PCR RespiFinder-19 assay Pathofinder®, Maastricht, Netherlands was performed on naso-pharyngeal swabs to detect 15 respiratory viruses -coronavirus 229E, NL63, OC43, human metapneumovirus hMPV , influenza A, A H1N1 pdm2009 and B viruses, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus RSV A and B, rhinovirus, adenovirus, and 4 intracellular bacteria -Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, in one reaction.", "The samples were not frozen and thawed. Multiplex PCR RespiFinder-19 assay Pathofinder®, Maastricht, Netherlands was performed on naso-pharyngeal swabs to detect 15 respiratory viruses -coronavirus 229E, NL63, OC43, human metapneumovirus hMPV , influenza A, A H1N1 pdm2009 and B viruses, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus RSV A and B, rhinovirus, adenovirus, and 4 intracellular bacteria -Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, in one reaction. The multiplex PCR results were not available to the adjudication committee. Routine microbiological examinations were also performed at the discretion of the emergency physicians and included blood culture, sputum culture, and antigenuria see Additional file 1 for methodology . CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples sputum, blood culture, antigenuria when performed. Baseline and follow-up characteristics were described by means and standard deviations SD or by median and interquartile range IQR for continuous variables normally distributed or with skewed distribution, respectively, and by percentages for categorical variables, for the total study population and for the study groups.", "CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples sputum, blood culture, antigenuria when performed. Baseline and follow-up characteristics were described by means and standard deviations SD or by median and interquartile range IQR for continuous variables normally distributed or with skewed distribution, respectively, and by percentages for categorical variables, for the total study population and for the study groups. We performed chi-square or Fisher exact tests when appropriate for qualitative variables, and the Student or Mann-Whitney tests for continuous variables with skewed distributions to compare baseline patient characteristics and study outcomes between study groups. The distribution values of the biomarkers were determined in the different populations of patients using boxplots. The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis definite CAP vs excluded CAP . CRP was evaluated at several cut-off points of 20 mg/L, 30 mg/L, 50 mg/L, 70 mg/L, and 100 mg/L, values used in previous studies .", "The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis definite CAP vs excluded CAP . CRP was evaluated at several cut-off points of 20 mg/L, 30 mg/L, 50 mg/L, 70 mg/L, and 100 mg/L, values used in previous studies . Several cut-off points for PCT were chosen at the level of 0.10 μg/L , and at the two levels for suspected bacterial infection as stated by the manufacturer, i.e., 0.25 μg/L and 0.50 μg/L. Sensitivities, specificities, positive predictive values PPVs , negative predictive values NPVs , and likelihood ratio were calculated. Receiver operating characteristic ROC curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP. From these optimal cut-offs for CRP and PCT, sensitivity analyses were performed combining the CRP and PCT cut-offs.", "Receiver operating characteristic ROC curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP. From these optimal cut-offs for CRP and PCT, sensitivity analyses were performed combining the CRP and PCT cut-offs. A multivariate logistic regression model was built to identify factors associated with having definite CAP as compared to having an excluded CAP diagnosis. We excluded from the excluded CAP diagnosis group, patients with an extra-pulmonary infectious disease. All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval 95 % CI and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test. All tests were two-sided, and p-values below 0.05 were considered to denote statistical significance.", "All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval 95 % CI and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test. All tests were two-sided, and p-values below 0.05 were considered to denote statistical significance. All statistical analyses were performed using SPSS statistical software version 21.0 SPSS Inc., Chicago, IL, USA . Two hundred patients with suspected CAP out of the 319 in the ESCAPED study were included in the present study, for which CRP and PCT assays and nasopharyngeal swab for multiplex PCR were available Fig. 1 . Characteristics of the 200 patients age, age more than 65, gender, probability of CAP diagnosis by adjudication committee were not significantly different from those of the 119 other patients of the ESCAPED study and are summarized in Table 1 .", "1 . Characteristics of the 200 patients age, age more than 65, gender, probability of CAP diagnosis by adjudication committee were not significantly different from those of the 119 other patients of the ESCAPED study and are summarized in Table 1 . CRP and PCT assays were performed based on the emergency practitioner's own initiative in 70 patients for CRP and 131 for PCT, or performed a posteriori on plasma samples of the remaining patients. Sex ratio was approximately 1. More than half of the patients 54 % were 65 years of age or older. The Pulmonary infiltrates were seen on chest X-ray in 127 63.5 % patients.", "More than half of the patients 54 % were 65 years of age or older. The Pulmonary infiltrates were seen on chest X-ray in 127 63.5 % patients. Thoracic CT-scan excluded a CAP diagnosis in 16.5 % of these 127 patients; on the contrary, thoracic CT-scan revealed a parenchymal infiltrate in 27 % of the 73 patients without infiltrate on chest X-ray. Based on all available data including multidetector CT scan results but excluding PCR results , the adjudication The CRP and PCT distributions in the 200 patients are presented in Fig. 2 A statistically significant difference between the two groups excluded CAP vs definite CAP was demonstrated for several cut-off points for CRP and PCT Table 2 . For CRP, the value of 50 mg/L resulted in a PPV of 0.76 and a NPV of 0.75.", "2 A statistically significant difference between the two groups excluded CAP vs definite CAP was demonstrated for several cut-off points for CRP and PCT Table 2 . For CRP, the value of 50 mg/L resulted in a PPV of 0.76 and a NPV of 0.75. For PCT, no value resulted in a satisfactory PPV or NPV. For these two biochemical markers, the ability to predict CAP was evaluated by a ROC curve. The AUC was 0.787 95 % CI 0.717-0.857 , optimal cut-off = 45.9 mg/L for CRP Fig. 3 and 0.655 95 % CI 0.570-0.739 , optimal cut-off = 0.13 μg/ L for PCT Fig. 4 .", "3 and 0.655 95 % CI 0.570-0.739 , optimal cut-off = 0.13 μg/ L for PCT Fig. 4 . Sensitivity analyses for the combination of CRP and PCT, using these optimal cut-offs, resulted in a PPV of 0.74 and a NPV of 0.58. Use of the other PCT cut-offs did not result in better PPV or NPV Table 2 . The present study is novel as patients prospectively benefited from extensive investigation to determine the diagnosis of CAP in the ED, including both early multidetector thoracic CT-scan and day-28 adjudication committee. This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients.", "The present study is novel as patients prospectively benefited from extensive investigation to determine the diagnosis of CAP in the ED, including both early multidetector thoracic CT-scan and day-28 adjudication committee. This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients. In these extensively characterized patients, both CRP and PCT lacked operational precision to allow the decisionmaking process to rule out or confirm diagnosis of CAP even in selected subgroups. The clinical characteristics of the patients included in this sub-study are consistent with those in the current literature. As previously reported, patients frequently had a history of respiratory disorders, cancer and congestive heart failure . The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65.", "As previously reported, patients frequently had a history of respiratory disorders, cancer and congestive heart failure . The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65. This may explain why the mean age of our patients 64 years falls within the lower values of those reported elsewhere . Data to identify the microbial agent responsible for the disease were collected by the usual techniques and multiplex PCR. Viral identification using naso-pharyngeal PCR that revealed viral respiratory infection in approximately one-third of cases was concordant with values reported in the literature . Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP.", "Viral identification using naso-pharyngeal PCR that revealed viral respiratory infection in approximately one-third of cases was concordant with values reported in the literature . Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP. In the present study, patients were recruited on the basis of initial clinical assessment for the diagnosis of CAP. Therefore, we believe that the characteristics of the patients closely correspond to those that lead practitioners to consider a possible diagnosis of CAP. In these patients, the design of our study allowed us to confirm or refute CAP diagnosis with a high level of certainty. Results confirmed the poor predictive value of clinical symptoms new onset of systemic features and symptoms of an acute lower respiratory tract illness in identifying CAP patients .", "In these patients, the design of our study allowed us to confirm or refute CAP diagnosis with a high level of certainty. Results confirmed the poor predictive value of clinical symptoms new onset of systemic features and symptoms of an acute lower respiratory tract illness in identifying CAP patients . Indeed, clinical presentation of excluded CAP patients was similar to that of definite CAP patients except for fever and cough that were more frequent in definite CAP patients. Furthermore, the design also revealed that the combination of clinical symptoms and chest X-ray results led to CAP misdiagnosis in a high number of patients, including the 98 whose CAP diagnosis was excluded by the adjudication committee and who would have been considered as possible, probable or definite CAP without the use of the CT scan. This low specificity of clinical-standard radiological evaluation led to the consideration of either non-infectious pulmonary diseases such as, cardiac failure, pulmonary embolism, pulmonary neoplasia or bronchitis or extra-pulmonary infectious diseases as CAP. Of note, some of these diseases are also associated with increased biomarker values.", "This low specificity of clinical-standard radiological evaluation led to the consideration of either non-infectious pulmonary diseases such as, cardiac failure, pulmonary embolism, pulmonary neoplasia or bronchitis or extra-pulmonary infectious diseases as CAP. Of note, some of these diseases are also associated with increased biomarker values. This raises concerns about previous evaluations of biomarkers in CAP-suspected patients, which used clinical and standard radiological chest X-ray evaluations as the gold standard for CAP diagnosis . The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections . However, this issue is still unresolved , with conflicting positions . In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis, we were unable to establish discriminating values for PCT.", "However, this issue is still unresolved , with conflicting positions . In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis, we were unable to establish discriminating values for PCT. Recent data suggested that CRP could be of more help in assisting in the diagnosis of lower respiratory tract infections LRTI . In our study, although CRP seems more discriminating than PCT, neither the experimental exclusion of extra-pulmonary bacterial infections from the excluded CAP group, nor the exclusion of viral CAP from the definite CAP patients group, made possible the determination of a discriminant cutoff. The combination of CRP and PCT was not more discriminating than each biomarker separately. An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy .", "The combination of CRP and PCT was not more discriminating than each biomarker separately. An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy . According to this strategy, a PCT concentration higher than 0.25 μg/L should prompt administration of antibiotics to patients with suspected LRTI. In our study, this value was associated with poor performance. Additionally, mean PCT levels remained above this threshold both in excluded CAP patients without infectious disorders and in definite CAP presumably related to virus. Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting. This study has some limitations.", "Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting. This study has some limitations. First, the adjudication committee was not blinded to the value of biomarkers measured at bedside in some patients 70 for CRP and 131 for PCT and its CAP classification could thus have been influenced by these results. However, the lack of statistically significant differences in the mean CRP and PCT values in the definite CAP cases, whether or not these biomarkers were available for the adjudication committee, argues against a major impact of these results on adjudication committee classification. Second, another critical point is the prescription of antibiotic therapy 34 % previous to inclusion. We cannot exclude that these previously-treated CAP patients may have altered biomarker performance and reduced the yield of bacterial cultures, although such a population reflects the usual emergency department practice.", "Second, another critical point is the prescription of antibiotic therapy 34 % previous to inclusion. We cannot exclude that these previously-treated CAP patients may have altered biomarker performance and reduced the yield of bacterial cultures, although such a population reflects the usual emergency department practice. Third, multiplex PCR was performed on naso-pharyngeal sampling and not on lower respiratory tract samples, which does not allow definite confirmation of the viral origin of CAP. However, a recent large study on CAP patients which reported a viral etiology of CAP at a comparable rate, did not find upper respiratory tract shedding in a control population without CAP explored during the same year and season . Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty. Given the diversity of the clinical and radiological CAP presentations, CAP diagnosis is often uncertain.", "Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty. Given the diversity of the clinical and radiological CAP presentations, CAP diagnosis is often uncertain. In our population of patients treated in the emergency room with clinical symptoms evoking CAP, neither CRP nor PCT cut-off values carried sufficient weight to confirm or refute CAP diagnosis at bedside; this underlines that these biomarkers are telltales of the host inflammatory response to the intrusion of microorganisms independent of the site of infection. These results, based on a systematic thoracic CT scan evaluation of CAP-suspected patients, do not argue for the use of CRP and PCT in routine care to diagnose CAP with certainty in patients visiting the ED for suspected CAP." ]

[ "nan Text: Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro Jia Liu 1 , Ruiyuan Cao 2 , Mingyue Xu 1,3 , Xi Wang 1 , Huanyu Zhang 1,3 , Hengrui Hu 1,3 , Yufeng Li 1,3 , Zhihong Hu 1 , Wu Zhong 2 and Manli Wang 1 Dear Editor, The outbreak of coronavirus disease 2019 COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 SARS-CoV-2/2019-nCoV poses a serious threat to global public health and local economies. As of March 3, 2020, over 80,000 cases have been confirmed in China, including 2946 deaths as well as over 10,566 confirmed cases in 72 other countries. Such huge numbers of infected and dead people call for an urgent demand of effective, available, and affordable drugs to control and diminish the epidemic. We have recently reported that two drugs, remdesivir GS-5734 and chloroquine CQ phosphate, efficiently inhibited SARS-CoV-2 infection in vitro 1 . Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences USA . A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States 2 , and a phase III clinical trial of remdesivir against SARS-CoV-2 was launched in Wuhan on February 4, 2020.", "Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences USA . A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States 2 , and a phase III clinical trial of remdesivir against SARS-CoV-2 was launched in Wuhan on February 4, 2020. However, as an experimental drug, remdesivir is not expected to be largely available for treating a very large number of patients in a timely manner. Therefore, of the two potential drugs, CQ appears to be the drug of choice for large-scale use due to its availability, proven safety record, and a relatively low cost. In light of the preliminary clinical data, CQ has been added to the list of trial drugs in the Guidelines for the Diagnosis and Treatment of COVID-19 sixth edition published by National Health Commission of the People's Republic of China. CQ N4- 7-Chloro-4-quinolinyl -N1,N1-diethyl-1,4pentanediamine has long been used to treat malaria and amebiasis.", "In light of the preliminary clinical data, CQ has been added to the list of trial drugs in the Guidelines for the Diagnosis and Treatment of COVID-19 sixth edition published by National Health Commission of the People's Republic of China. CQ N4- 7-Chloro-4-quinolinyl -N1,N1-diethyl-1,4pentanediamine has long been used to treat malaria and amebiasis. However, Plasmodium falciparum developed widespread resistance to it, and with the development of new antimalarials, it has become a choice for the prophylaxis of malaria. In addition, an overdose of CQ can cause acute poisoning and death 3 . In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China. Hydroxychloroquine HCQ sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less ~40% toxic than CQ in animals 4 .", "In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China. Hydroxychloroquine HCQ sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less ~40% toxic than CQ in animals 4 . More importantly, HCQ is still widely available to treat autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Since CQ and HCQ share similar chemical structures and mechanisms of acting as a weak base and immunomodulator, it is easy to conjure up the idea that HCQ may be a potent candidate to treat infection by SARS-CoV-2. Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence.", "Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence. To this end, we evaluated the antiviral effect of HCQ against SARS-CoV-2 infection in comparison to CQ in vitro. First, the cytotoxicity of HCQ and CQ in African green monkey kidney VeroE6 cells ATCC-1586 was measured by standard CCK8 assay, and the result showed © The Author s 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author s and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.", "The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit Fig. 1a . To better compare the antiviral activity of CQ versus HCQ, the dose-response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection MOIs by quantification of viral RNA copy numbers in the cell supernatant at 48 h post infection p.i. .", "To better compare the antiviral activity of CQ versus HCQ, the dose-response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection MOIs by quantification of viral RNA copy numbers in the cell supernatant at 48 h post infection p.i. . The data summarized in Fig. 1a and Supplementary Table S1 show that, at all MOIs 0.01, 0.02, 0.2, and 0.8 , the 50% maximal effective concentration EC 50 for CQ 2.71, 3.81, 7.14, and 7.36 μM was lower than that of HCQ 4.51, 4.06, 17.31, and 12.96 μM . The differences in EC 50 values were statistically significant at an MOI of 0.01 P < 0.05 and MOI of 0.2 P < 0.001 Supplementary Table S1 . It is worth noting that the EC 50 values of CQ seemed to be a little higher than that in our previous report 1.13 μM at an MOI of 0.05 1 , which is likely due to the adaptation of the virus in cell culture that significantly increased viral infectivity upon continuous passaging.", "The differences in EC 50 values were statistically significant at an MOI of 0.01 P < 0.05 and MOI of 0.2 P < 0.001 Supplementary Table S1 . It is worth noting that the EC 50 values of CQ seemed to be a little higher than that in our previous report 1.13 μM at an MOI of 0.05 1 , which is likely due to the adaptation of the virus in cell culture that significantly increased viral infectivity upon continuous passaging. Consequently, the selectivity index SI = CC 50 /EC 50 of CQ 100.81, 71.71, 38.26, and 37.12 was higher than that of HCQ 55.32, 61.45, 14.41, 19.25 at MOIs of 0.01, 0.02, 0.2, and 0.8, respectively. These results were corroborated by immunofluorescence microscopy as evidenced by different expression levels of virus nucleoprotein NP at the indicated drug concentrations at 48 h p.i. Supplementary Fig. S1 .", "These results were corroborated by immunofluorescence microscopy as evidenced by different expression levels of virus nucleoprotein NP at the indicated drug concentrations at 48 h p.i. Supplementary Fig. S1 . Taken together, the data suggest that the anti-SARS-CoV-2 activity of HCQ seems to be less potent compared to CQ, at least at certain MOIs. Both CQ and HCQ are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion 5 . In addition, CQ could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein 6 .", "Both CQ and HCQ are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion 5 . In addition, CQ could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein 6 . Time-of-addition experiment confirmed that HCQ effectively inhibited the entry step, as well as the post-entry stages of SARS-CoV-2, which was also found upon CQ treatment Supplementary Fig. S2 . To further explore the detailed mechanism of action of CQ and HCQ in inhibiting virus entry, co-localization of virions with early endosomes EEs or endolysosomes ELs was analyzed by immunofluorescence analysis IFA and confocal microscopy. Quantification analysis showed that, at 90 min p.i.", "To further explore the detailed mechanism of action of CQ and HCQ in inhibiting virus entry, co-localization of virions with early endosomes EEs or endolysosomes ELs was analyzed by immunofluorescence analysis IFA and confocal microscopy. Quantification analysis showed that, at 90 min p.i. in untreated cells, 16.2% of internalized virions anti-NP, red were observed in early endosome antigen 1 EEA1 -positive EEs green , while more virions 34.3% were transported into the late endosomal-lysosomal protein LAMP1 + ELs green n > 30 cells for each group . By contrast, in the presence of CQ or HCQ, significantly more virions 35.3% for CQ and 29.2% for HCQ; P < 0.001 were detected in the EEs, while only very few virions 2.4% for CQ and 0.03% for HCQ; P < 0.001 were found to be co-localized with LAMP1 + ELs n > 30 cells Fig. 1b, c . This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV 7 .", "1b, c . This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV 7 . Interestingly, we found that CQ and HCQ treatment caused noticeable changes in the number and size/morphology of EEs and ELs Fig. 1c . In the untreated cells, most EEs were much smaller than ELs Fig. 1c . In CQand HCQ-treated cells, abnormally enlarged EE vesicles were observed Fig. 1c , arrows in the upper panels , many of which are even larger than ELs in the untreated cells.", "In CQand HCQ-treated cells, abnormally enlarged EE vesicles were observed Fig. 1c , arrows in the upper panels , many of which are even larger than ELs in the untreated cells. This is in agreement with previous report that treatment with CQ induced the formation of expanded cytoplasmic vesicles 8 . Within the EE vesicles, virions red were localized around the membrane green of the vesicle. CQ treatment did not cause obvious changes in the number and size of ELs; however, the regular vesicle structure seemed to be disrupted, at least partially. By contrast, in HCQ-treated cells, the size and number of ELs increased significantly Fig. 1c , arrows in the lower panels .", "By contrast, in HCQ-treated cells, the size and number of ELs increased significantly Fig. 1c , arrows in the lower panels . Since acidification is crucial for endosome maturation and function, we surmise that endosome maturation might be blocked at intermediate stages of endocytosis, resulting in failure of further transport of virions to the ultimate releasing site. CQ was reported to elevate the pH see figure on previous page Fig. 1 Comparative antiviral efficacy and mechanism of action of CQ and HCQ against SARS-CoV-2 infection in vitro. a Cytotoxicity and antiviral activities of CQ and HCQ. The cytotoxicity of the two drugs in Vero E6 cells was determined by CCK-8 assays.", "a Cytotoxicity and antiviral activities of CQ and HCQ. The cytotoxicity of the two drugs in Vero E6 cells was determined by CCK-8 assays. Vero E6 cells were treated with different doses of either compound or with PBS in the controls for 1 h and then infected with SARS-CoV-2 at MOIs of 0.01, 0.02, 0.2, and 0.8. The virus yield in the cell supernatant was quantified by qRT-PCR at 48 h p.i. Y-axis represents the mean of percent inhibition normalized to the PBS group. The experiments were repeated twice. b, c Mechanism of CQ and HCQ in inhibiting virus entry.", "Y-axis represents the mean of percent inhibition normalized to the PBS group. The experiments were repeated twice. b, c Mechanism of CQ and HCQ in inhibiting virus entry. Vero E6 cells were treated with CQ or HCQ 50 μM for 1 h, followed by virus binding MOI = 10 at 4°C for 1 h. Then the unbound virions were removed, and the cells were further supplemented with fresh drug-containing medium at 37°C for 90 min before being fixed and stained with IFA using anti-NP antibody for virions red and antibodies against EEA1 for EEs green or LAMP1 for ELs green . The nuclei blue were stained with Hoechst dye. The portion of virions that co-localized with EEs or ELs in each group n > 30 cells was quantified and is shown in b.", "The nuclei blue were stained with Hoechst dye. The portion of virions that co-localized with EEs or ELs in each group n > 30 cells was quantified and is shown in b. Representative confocal microscopic images of viral particles red , EEA1 + EEs green , or LAMP1 + ELs green in each group are displayed in c. The enlarged images in the boxes indicate a single vesicle-containing virion. The arrows indicated the abnormally enlarged vesicles. Bars, 5 μm. Statistical analysis was performed using a one-way analysis of variance ANOVA with GraphPad Prism F = 102.8, df = 5,182, ***P < 0.001 . of lysosome from about 4.5 to 6.5 at 100 μM 9 .", "Statistical analysis was performed using a one-way analysis of variance ANOVA with GraphPad Prism F = 102.8, df = 5,182, ***P < 0.001 . of lysosome from about 4.5 to 6.5 at 100 μM 9 . To our knowledge, there is a lack of studies on the impact of HCQ on the morphology and pH values of endosomes/ lysosomes. Our observations suggested that the mode of actions of CQ and HCQ appear to be distinct in certain aspects. It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200-700 times higher than those in the plasma 10 .", "It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200-700 times higher than those in the plasma 10 . It was reported that safe dosage 6-6.5 mg/kg per day of HCQ sulfate could generate serum levels of 1.4-1.5 μM in humans 11 . Therefore, with a safe dosage, HCQ concentration in the above tissues is likely to be achieved to inhibit SARS-CoV-2 infection. Clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with SARS-CoV-2, suggesting that cytokine storm was associated with disease severity 12 . Other than its direct antiviral activity, HCQ is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors.", "Clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with SARS-CoV-2, suggesting that cytokine storm was associated with disease severity 12 . Other than its direct antiviral activity, HCQ is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors. Therefore, in COVID-19 patients, HCQ may also contribute to attenuating the inflammatory response. In conclusion, our results show that HCQ can efficiently inhibit SARS-CoV-2 infection in vitro. In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials.", "In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials. We need to point out, although HCQ is less toxic than CQ, prolonged and overdose usage can still cause poisoning. And the relatively low SI of HCQ requires careful designing and conducting of clinical trials to achieve efficient and safe control of the SARS-CoV-2 infection." ]

[ "nan Text: Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro Jia Liu 1 , Ruiyuan Cao 2 , Mingyue Xu 1,3 , Xi Wang 1 , Huanyu Zhang 1,3 , Hengrui Hu 1,3 , Yufeng Li 1,3 , Zhihong Hu 1 , Wu Zhong 2 and Manli Wang 1 Dear Editor, The outbreak of coronavirus disease 2019 COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 SARS-CoV-2/2019-nCoV poses a serious threat to global public health and local economies. As of March 3, 2020, over 80,000 cases have been confirmed in China, including 2946 deaths as well as over 10,566 confirmed cases in 72 other countries. Such huge numbers of infected and dead people call for an urgent demand of effective, available, and affordable drugs to control and diminish the epidemic. We have recently reported that two drugs, remdesivir GS-5734 and chloroquine CQ phosphate, efficiently inhibited SARS-CoV-2 infection in vitro 1 . Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences USA . A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States 2 , and a phase III clinical trial of remdesivir against SARS-CoV-2 was launched in Wuhan on February 4, 2020.", "Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences USA . A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States 2 , and a phase III clinical trial of remdesivir against SARS-CoV-2 was launched in Wuhan on February 4, 2020. However, as an experimental drug, remdesivir is not expected to be largely available for treating a very large number of patients in a timely manner. Therefore, of the two potential drugs, CQ appears to be the drug of choice for large-scale use due to its availability, proven safety record, and a relatively low cost. In light of the preliminary clinical data, CQ has been added to the list of trial drugs in the Guidelines for the Diagnosis and Treatment of COVID-19 sixth edition published by National Health Commission of the People's Republic of China. CQ N4- 7-Chloro-4-quinolinyl -N1,N1-diethyl-1,4pentanediamine has long been used to treat malaria and amebiasis.", "In light of the preliminary clinical data, CQ has been added to the list of trial drugs in the Guidelines for the Diagnosis and Treatment of COVID-19 sixth edition published by National Health Commission of the People's Republic of China. CQ N4- 7-Chloro-4-quinolinyl -N1,N1-diethyl-1,4pentanediamine has long been used to treat malaria and amebiasis. However, Plasmodium falciparum developed widespread resistance to it, and with the development of new antimalarials, it has become a choice for the prophylaxis of malaria. In addition, an overdose of CQ can cause acute poisoning and death 3 . In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China. Hydroxychloroquine HCQ sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less ~40% toxic than CQ in animals 4 .", "In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China. Hydroxychloroquine HCQ sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less ~40% toxic than CQ in animals 4 . More importantly, HCQ is still widely available to treat autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Since CQ and HCQ share similar chemical structures and mechanisms of acting as a weak base and immunomodulator, it is easy to conjure up the idea that HCQ may be a potent candidate to treat infection by SARS-CoV-2. Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence.", "Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence. To this end, we evaluated the antiviral effect of HCQ against SARS-CoV-2 infection in comparison to CQ in vitro. First, the cytotoxicity of HCQ and CQ in African green monkey kidney VeroE6 cells ATCC-1586 was measured by standard CCK8 assay, and the result showed © The Author s 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author s and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.", "The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit Fig. 1a . To better compare the antiviral activity of CQ versus HCQ, the dose-response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection MOIs by quantification of viral RNA copy numbers in the cell supernatant at 48 h post infection p.i. .", "To better compare the antiviral activity of CQ versus HCQ, the dose-response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection MOIs by quantification of viral RNA copy numbers in the cell supernatant at 48 h post infection p.i. . The data summarized in Fig. 1a and Supplementary Table S1 show that, at all MOIs 0.01, 0.02, 0.2, and 0.8 , the 50% maximal effective concentration EC 50 for CQ 2.71, 3.81, 7.14, and 7.36 μM was lower than that of HCQ 4.51, 4.06, 17.31, and 12.96 μM . The differences in EC 50 values were statistically significant at an MOI of 0.01 P < 0.05 and MOI of 0.2 P < 0.001 Supplementary Table S1 . It is worth noting that the EC 50 values of CQ seemed to be a little higher than that in our previous report 1.13 μM at an MOI of 0.05 1 , which is likely due to the adaptation of the virus in cell culture that significantly increased viral infectivity upon continuous passaging.", "The differences in EC 50 values were statistically significant at an MOI of 0.01 P < 0.05 and MOI of 0.2 P < 0.001 Supplementary Table S1 . It is worth noting that the EC 50 values of CQ seemed to be a little higher than that in our previous report 1.13 μM at an MOI of 0.05 1 , which is likely due to the adaptation of the virus in cell culture that significantly increased viral infectivity upon continuous passaging. Consequently, the selectivity index SI = CC 50 /EC 50 of CQ 100.81, 71.71, 38.26, and 37.12 was higher than that of HCQ 55.32, 61.45, 14.41, 19.25 at MOIs of 0.01, 0.02, 0.2, and 0.8, respectively. These results were corroborated by immunofluorescence microscopy as evidenced by different expression levels of virus nucleoprotein NP at the indicated drug concentrations at 48 h p.i. Supplementary Fig. S1 .", "These results were corroborated by immunofluorescence microscopy as evidenced by different expression levels of virus nucleoprotein NP at the indicated drug concentrations at 48 h p.i. Supplementary Fig. S1 . Taken together, the data suggest that the anti-SARS-CoV-2 activity of HCQ seems to be less potent compared to CQ, at least at certain MOIs. Both CQ and HCQ are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion 5 . In addition, CQ could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein 6 .", "Both CQ and HCQ are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion 5 . In addition, CQ could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein 6 . Time-of-addition experiment confirmed that HCQ effectively inhibited the entry step, as well as the post-entry stages of SARS-CoV-2, which was also found upon CQ treatment Supplementary Fig. S2 . To further explore the detailed mechanism of action of CQ and HCQ in inhibiting virus entry, co-localization of virions with early endosomes EEs or endolysosomes ELs was analyzed by immunofluorescence analysis IFA and confocal microscopy. Quantification analysis showed that, at 90 min p.i.", "To further explore the detailed mechanism of action of CQ and HCQ in inhibiting virus entry, co-localization of virions with early endosomes EEs or endolysosomes ELs was analyzed by immunofluorescence analysis IFA and confocal microscopy. Quantification analysis showed that, at 90 min p.i. in untreated cells, 16.2% of internalized virions anti-NP, red were observed in early endosome antigen 1 EEA1 -positive EEs green , while more virions 34.3% were transported into the late endosomal-lysosomal protein LAMP1 + ELs green n > 30 cells for each group . By contrast, in the presence of CQ or HCQ, significantly more virions 35.3% for CQ and 29.2% for HCQ; P < 0.001 were detected in the EEs, while only very few virions 2.4% for CQ and 0.03% for HCQ; P < 0.001 were found to be co-localized with LAMP1 + ELs n > 30 cells Fig. 1b, c . This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV 7 .", "1b, c . This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV 7 . Interestingly, we found that CQ and HCQ treatment caused noticeable changes in the number and size/morphology of EEs and ELs Fig. 1c . In the untreated cells, most EEs were much smaller than ELs Fig. 1c . In CQand HCQ-treated cells, abnormally enlarged EE vesicles were observed Fig. 1c , arrows in the upper panels , many of which are even larger than ELs in the untreated cells.", "In CQand HCQ-treated cells, abnormally enlarged EE vesicles were observed Fig. 1c , arrows in the upper panels , many of which are even larger than ELs in the untreated cells. This is in agreement with previous report that treatment with CQ induced the formation of expanded cytoplasmic vesicles 8 . Within the EE vesicles, virions red were localized around the membrane green of the vesicle. CQ treatment did not cause obvious changes in the number and size of ELs; however, the regular vesicle structure seemed to be disrupted, at least partially. By contrast, in HCQ-treated cells, the size and number of ELs increased significantly Fig. 1c , arrows in the lower panels .", "By contrast, in HCQ-treated cells, the size and number of ELs increased significantly Fig. 1c , arrows in the lower panels . Since acidification is crucial for endosome maturation and function, we surmise that endosome maturation might be blocked at intermediate stages of endocytosis, resulting in failure of further transport of virions to the ultimate releasing site. CQ was reported to elevate the pH see figure on previous page Fig. 1 Comparative antiviral efficacy and mechanism of action of CQ and HCQ against SARS-CoV-2 infection in vitro. a Cytotoxicity and antiviral activities of CQ and HCQ. The cytotoxicity of the two drugs in Vero E6 cells was determined by CCK-8 assays.", "a Cytotoxicity and antiviral activities of CQ and HCQ. The cytotoxicity of the two drugs in Vero E6 cells was determined by CCK-8 assays. Vero E6 cells were treated with different doses of either compound or with PBS in the controls for 1 h and then infected with SARS-CoV-2 at MOIs of 0.01, 0.02, 0.2, and 0.8. The virus yield in the cell supernatant was quantified by qRT-PCR at 48 h p.i. Y-axis represents the mean of percent inhibition normalized to the PBS group. The experiments were repeated twice. b, c Mechanism of CQ and HCQ in inhibiting virus entry.", "Y-axis represents the mean of percent inhibition normalized to the PBS group. The experiments were repeated twice. b, c Mechanism of CQ and HCQ in inhibiting virus entry. Vero E6 cells were treated with CQ or HCQ 50 μM for 1 h, followed by virus binding MOI = 10 at 4°C for 1 h. Then the unbound virions were removed, and the cells were further supplemented with fresh drug-containing medium at 37°C for 90 min before being fixed and stained with IFA using anti-NP antibody for virions red and antibodies against EEA1 for EEs green or LAMP1 for ELs green . The nuclei blue were stained with Hoechst dye. The portion of virions that co-localized with EEs or ELs in each group n > 30 cells was quantified and is shown in b.", "The nuclei blue were stained with Hoechst dye. The portion of virions that co-localized with EEs or ELs in each group n > 30 cells was quantified and is shown in b. Representative confocal microscopic images of viral particles red , EEA1 + EEs green , or LAMP1 + ELs green in each group are displayed in c. The enlarged images in the boxes indicate a single vesicle-containing virion. The arrows indicated the abnormally enlarged vesicles. Bars, 5 μm. Statistical analysis was performed using a one-way analysis of variance ANOVA with GraphPad Prism F = 102.8, df = 5,182, ***P < 0.001 . of lysosome from about 4.5 to 6.5 at 100 μM 9 .", "Statistical analysis was performed using a one-way analysis of variance ANOVA with GraphPad Prism F = 102.8, df = 5,182, ***P < 0.001 . of lysosome from about 4.5 to 6.5 at 100 μM 9 . To our knowledge, there is a lack of studies on the impact of HCQ on the morphology and pH values of endosomes/ lysosomes. Our observations suggested that the mode of actions of CQ and HCQ appear to be distinct in certain aspects. It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200-700 times higher than those in the plasma 10 .", "It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200-700 times higher than those in the plasma 10 . It was reported that safe dosage 6-6.5 mg/kg per day of HCQ sulfate could generate serum levels of 1.4-1.5 μM in humans 11 . Therefore, with a safe dosage, HCQ concentration in the above tissues is likely to be achieved to inhibit SARS-CoV-2 infection. Clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with SARS-CoV-2, suggesting that cytokine storm was associated with disease severity 12 . Other than its direct antiviral activity, HCQ is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors.", "Clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with SARS-CoV-2, suggesting that cytokine storm was associated with disease severity 12 . Other than its direct antiviral activity, HCQ is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors. Therefore, in COVID-19 patients, HCQ may also contribute to attenuating the inflammatory response. In conclusion, our results show that HCQ can efficiently inhibit SARS-CoV-2 infection in vitro. In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials.", "In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials. We need to point out, although HCQ is less toxic than CQ, prolonged and overdose usage can still cause poisoning. And the relatively low SI of HCQ requires careful designing and conducting of clinical trials to achieve efficient and safe control of the SARS-CoV-2 infection." ]

[ "nan Text: Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro Jia Liu 1 , Ruiyuan Cao 2 , Mingyue Xu 1,3 , Xi Wang 1 , Huanyu Zhang 1,3 , Hengrui Hu 1,3 , Yufeng Li 1,3 , Zhihong Hu 1 , Wu Zhong 2 and Manli Wang 1 Dear Editor, The outbreak of coronavirus disease 2019 COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 SARS-CoV-2/2019-nCoV poses a serious threat to global public health and local economies. As of March 3, 2020, over 80,000 cases have been confirmed in China, including 2946 deaths as well as over 10,566 confirmed cases in 72 other countries. Such huge numbers of infected and dead people call for an urgent demand of effective, available, and affordable drugs to control and diminish the epidemic. We have recently reported that two drugs, remdesivir GS-5734 and chloroquine CQ phosphate, efficiently inhibited SARS-CoV-2 infection in vitro 1 . Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences USA . A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States 2 , and a phase III clinical trial of remdesivir against SARS-CoV-2 was launched in Wuhan on February 4, 2020.", "Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences USA . A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States 2 , and a phase III clinical trial of remdesivir against SARS-CoV-2 was launched in Wuhan on February 4, 2020. However, as an experimental drug, remdesivir is not expected to be largely available for treating a very large number of patients in a timely manner. Therefore, of the two potential drugs, CQ appears to be the drug of choice for large-scale use due to its availability, proven safety record, and a relatively low cost. In light of the preliminary clinical data, CQ has been added to the list of trial drugs in the Guidelines for the Diagnosis and Treatment of COVID-19 sixth edition published by National Health Commission of the People's Republic of China. CQ N4- 7-Chloro-4-quinolinyl -N1,N1-diethyl-1,4pentanediamine has long been used to treat malaria and amebiasis.", "In light of the preliminary clinical data, CQ has been added to the list of trial drugs in the Guidelines for the Diagnosis and Treatment of COVID-19 sixth edition published by National Health Commission of the People's Republic of China. CQ N4- 7-Chloro-4-quinolinyl -N1,N1-diethyl-1,4pentanediamine has long been used to treat malaria and amebiasis. However, Plasmodium falciparum developed widespread resistance to it, and with the development of new antimalarials, it has become a choice for the prophylaxis of malaria. In addition, an overdose of CQ can cause acute poisoning and death 3 . In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China. Hydroxychloroquine HCQ sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less ~40% toxic than CQ in animals 4 .", "In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China. Hydroxychloroquine HCQ sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less ~40% toxic than CQ in animals 4 . More importantly, HCQ is still widely available to treat autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Since CQ and HCQ share similar chemical structures and mechanisms of acting as a weak base and immunomodulator, it is easy to conjure up the idea that HCQ may be a potent candidate to treat infection by SARS-CoV-2. Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence.", "Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence. To this end, we evaluated the antiviral effect of HCQ against SARS-CoV-2 infection in comparison to CQ in vitro. First, the cytotoxicity of HCQ and CQ in African green monkey kidney VeroE6 cells ATCC-1586 was measured by standard CCK8 assay, and the result showed © The Author s 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author s and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.", "The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit Fig. 1a . To better compare the antiviral activity of CQ versus HCQ, the dose-response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection MOIs by quantification of viral RNA copy numbers in the cell supernatant at 48 h post infection p.i. .", "To better compare the antiviral activity of CQ versus HCQ, the dose-response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection MOIs by quantification of viral RNA copy numbers in the cell supernatant at 48 h post infection p.i. . The data summarized in Fig. 1a and Supplementary Table S1 show that, at all MOIs 0.01, 0.02, 0.2, and 0.8 , the 50% maximal effective concentration EC 50 for CQ 2.71, 3.81, 7.14, and 7.36 μM was lower than that of HCQ 4.51, 4.06, 17.31, and 12.96 μM . The differences in EC 50 values were statistically significant at an MOI of 0.01 P < 0.05 and MOI of 0.2 P < 0.001 Supplementary Table S1 . It is worth noting that the EC 50 values of CQ seemed to be a little higher than that in our previous report 1.13 μM at an MOI of 0.05 1 , which is likely due to the adaptation of the virus in cell culture that significantly increased viral infectivity upon continuous passaging.", "The differences in EC 50 values were statistically significant at an MOI of 0.01 P < 0.05 and MOI of 0.2 P < 0.001 Supplementary Table S1 . It is worth noting that the EC 50 values of CQ seemed to be a little higher than that in our previous report 1.13 μM at an MOI of 0.05 1 , which is likely due to the adaptation of the virus in cell culture that significantly increased viral infectivity upon continuous passaging. Consequently, the selectivity index SI = CC 50 /EC 50 of CQ 100.81, 71.71, 38.26, and 37.12 was higher than that of HCQ 55.32, 61.45, 14.41, 19.25 at MOIs of 0.01, 0.02, 0.2, and 0.8, respectively. These results were corroborated by immunofluorescence microscopy as evidenced by different expression levels of virus nucleoprotein NP at the indicated drug concentrations at 48 h p.i. Supplementary Fig. S1 .", "These results were corroborated by immunofluorescence microscopy as evidenced by different expression levels of virus nucleoprotein NP at the indicated drug concentrations at 48 h p.i. Supplementary Fig. S1 . Taken together, the data suggest that the anti-SARS-CoV-2 activity of HCQ seems to be less potent compared to CQ, at least at certain MOIs. Both CQ and HCQ are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion 5 . In addition, CQ could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein 6 .", "Both CQ and HCQ are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion 5 . In addition, CQ could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein 6 . Time-of-addition experiment confirmed that HCQ effectively inhibited the entry step, as well as the post-entry stages of SARS-CoV-2, which was also found upon CQ treatment Supplementary Fig. S2 . To further explore the detailed mechanism of action of CQ and HCQ in inhibiting virus entry, co-localization of virions with early endosomes EEs or endolysosomes ELs was analyzed by immunofluorescence analysis IFA and confocal microscopy. Quantification analysis showed that, at 90 min p.i.", "To further explore the detailed mechanism of action of CQ and HCQ in inhibiting virus entry, co-localization of virions with early endosomes EEs or endolysosomes ELs was analyzed by immunofluorescence analysis IFA and confocal microscopy. Quantification analysis showed that, at 90 min p.i. in untreated cells, 16.2% of internalized virions anti-NP, red were observed in early endosome antigen 1 EEA1 -positive EEs green , while more virions 34.3% were transported into the late endosomal-lysosomal protein LAMP1 + ELs green n > 30 cells for each group . By contrast, in the presence of CQ or HCQ, significantly more virions 35.3% for CQ and 29.2% for HCQ; P < 0.001 were detected in the EEs, while only very few virions 2.4% for CQ and 0.03% for HCQ; P < 0.001 were found to be co-localized with LAMP1 + ELs n > 30 cells Fig. 1b, c . This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV 7 .", "1b, c . This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV 7 . Interestingly, we found that CQ and HCQ treatment caused noticeable changes in the number and size/morphology of EEs and ELs Fig. 1c . In the untreated cells, most EEs were much smaller than ELs Fig. 1c . In CQand HCQ-treated cells, abnormally enlarged EE vesicles were observed Fig. 1c , arrows in the upper panels , many of which are even larger than ELs in the untreated cells.", "In CQand HCQ-treated cells, abnormally enlarged EE vesicles were observed Fig. 1c , arrows in the upper panels , many of which are even larger than ELs in the untreated cells. This is in agreement with previous report that treatment with CQ induced the formation of expanded cytoplasmic vesicles 8 . Within the EE vesicles, virions red were localized around the membrane green of the vesicle. CQ treatment did not cause obvious changes in the number and size of ELs; however, the regular vesicle structure seemed to be disrupted, at least partially. By contrast, in HCQ-treated cells, the size and number of ELs increased significantly Fig. 1c , arrows in the lower panels .", "By contrast, in HCQ-treated cells, the size and number of ELs increased significantly Fig. 1c , arrows in the lower panels . Since acidification is crucial for endosome maturation and function, we surmise that endosome maturation might be blocked at intermediate stages of endocytosis, resulting in failure of further transport of virions to the ultimate releasing site. CQ was reported to elevate the pH see figure on previous page Fig. 1 Comparative antiviral efficacy and mechanism of action of CQ and HCQ against SARS-CoV-2 infection in vitro. a Cytotoxicity and antiviral activities of CQ and HCQ. The cytotoxicity of the two drugs in Vero E6 cells was determined by CCK-8 assays.", "a Cytotoxicity and antiviral activities of CQ and HCQ. The cytotoxicity of the two drugs in Vero E6 cells was determined by CCK-8 assays. Vero E6 cells were treated with different doses of either compound or with PBS in the controls for 1 h and then infected with SARS-CoV-2 at MOIs of 0.01, 0.02, 0.2, and 0.8. The virus yield in the cell supernatant was quantified by qRT-PCR at 48 h p.i. Y-axis represents the mean of percent inhibition normalized to the PBS group. The experiments were repeated twice. b, c Mechanism of CQ and HCQ in inhibiting virus entry.", "Y-axis represents the mean of percent inhibition normalized to the PBS group. The experiments were repeated twice. b, c Mechanism of CQ and HCQ in inhibiting virus entry. Vero E6 cells were treated with CQ or HCQ 50 μM for 1 h, followed by virus binding MOI = 10 at 4°C for 1 h. Then the unbound virions were removed, and the cells were further supplemented with fresh drug-containing medium at 37°C for 90 min before being fixed and stained with IFA using anti-NP antibody for virions red and antibodies against EEA1 for EEs green or LAMP1 for ELs green . The nuclei blue were stained with Hoechst dye. The portion of virions that co-localized with EEs or ELs in each group n > 30 cells was quantified and is shown in b.", "The nuclei blue were stained with Hoechst dye. The portion of virions that co-localized with EEs or ELs in each group n > 30 cells was quantified and is shown in b. Representative confocal microscopic images of viral particles red , EEA1 + EEs green , or LAMP1 + ELs green in each group are displayed in c. The enlarged images in the boxes indicate a single vesicle-containing virion. The arrows indicated the abnormally enlarged vesicles. Bars, 5 μm. Statistical analysis was performed using a one-way analysis of variance ANOVA with GraphPad Prism F = 102.8, df = 5,182, ***P < 0.001 . of lysosome from about 4.5 to 6.5 at 100 μM 9 .", "Statistical analysis was performed using a one-way analysis of variance ANOVA with GraphPad Prism F = 102.8, df = 5,182, ***P < 0.001 . of lysosome from about 4.5 to 6.5 at 100 μM 9 . To our knowledge, there is a lack of studies on the impact of HCQ on the morphology and pH values of endosomes/ lysosomes. Our observations suggested that the mode of actions of CQ and HCQ appear to be distinct in certain aspects. It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200-700 times higher than those in the plasma 10 .", "It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200-700 times higher than those in the plasma 10 . It was reported that safe dosage 6-6.5 mg/kg per day of HCQ sulfate could generate serum levels of 1.4-1.5 μM in humans 11 . Therefore, with a safe dosage, HCQ concentration in the above tissues is likely to be achieved to inhibit SARS-CoV-2 infection. Clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with SARS-CoV-2, suggesting that cytokine storm was associated with disease severity 12 . Other than its direct antiviral activity, HCQ is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors.", "Clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with SARS-CoV-2, suggesting that cytokine storm was associated with disease severity 12 . Other than its direct antiviral activity, HCQ is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors. Therefore, in COVID-19 patients, HCQ may also contribute to attenuating the inflammatory response. In conclusion, our results show that HCQ can efficiently inhibit SARS-CoV-2 infection in vitro. In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials.", "In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials. We need to point out, although HCQ is less toxic than CQ, prolonged and overdose usage can still cause poisoning. And the relatively low SI of HCQ requires careful designing and conducting of clinical trials to achieve efficient and safe control of the SARS-CoV-2 infection." ]

[ "nan Text: Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro Jia Liu 1 , Ruiyuan Cao 2 , Mingyue Xu 1,3 , Xi Wang 1 , Huanyu Zhang 1,3 , Hengrui Hu 1,3 , Yufeng Li 1,3 , Zhihong Hu 1 , Wu Zhong 2 and Manli Wang 1 Dear Editor, The outbreak of coronavirus disease 2019 COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 SARS-CoV-2/2019-nCoV poses a serious threat to global public health and local economies. As of March 3, 2020, over 80,000 cases have been confirmed in China, including 2946 deaths as well as over 10,566 confirmed cases in 72 other countries. Such huge numbers of infected and dead people call for an urgent demand of effective, available, and affordable drugs to control and diminish the epidemic. We have recently reported that two drugs, remdesivir GS-5734 and chloroquine CQ phosphate, efficiently inhibited SARS-CoV-2 infection in vitro 1 . Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences USA . A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States 2 , and a phase III clinical trial of remdesivir against SARS-CoV-2 was launched in Wuhan on February 4, 2020.", "Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences USA . A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States 2 , and a phase III clinical trial of remdesivir against SARS-CoV-2 was launched in Wuhan on February 4, 2020. However, as an experimental drug, remdesivir is not expected to be largely available for treating a very large number of patients in a timely manner. Therefore, of the two potential drugs, CQ appears to be the drug of choice for large-scale use due to its availability, proven safety record, and a relatively low cost. In light of the preliminary clinical data, CQ has been added to the list of trial drugs in the Guidelines for the Diagnosis and Treatment of COVID-19 sixth edition published by National Health Commission of the People's Republic of China. CQ N4- 7-Chloro-4-quinolinyl -N1,N1-diethyl-1,4pentanediamine has long been used to treat malaria and amebiasis.", "In light of the preliminary clinical data, CQ has been added to the list of trial drugs in the Guidelines for the Diagnosis and Treatment of COVID-19 sixth edition published by National Health Commission of the People's Republic of China. CQ N4- 7-Chloro-4-quinolinyl -N1,N1-diethyl-1,4pentanediamine has long been used to treat malaria and amebiasis. However, Plasmodium falciparum developed widespread resistance to it, and with the development of new antimalarials, it has become a choice for the prophylaxis of malaria. In addition, an overdose of CQ can cause acute poisoning and death 3 . In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China. Hydroxychloroquine HCQ sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less ~40% toxic than CQ in animals 4 .", "In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China. Hydroxychloroquine HCQ sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less ~40% toxic than CQ in animals 4 . More importantly, HCQ is still widely available to treat autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Since CQ and HCQ share similar chemical structures and mechanisms of acting as a weak base and immunomodulator, it is easy to conjure up the idea that HCQ may be a potent candidate to treat infection by SARS-CoV-2. Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence.", "Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence. To this end, we evaluated the antiviral effect of HCQ against SARS-CoV-2 infection in comparison to CQ in vitro. First, the cytotoxicity of HCQ and CQ in African green monkey kidney VeroE6 cells ATCC-1586 was measured by standard CCK8 assay, and the result showed © The Author s 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author s and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.", "The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit Fig. 1a . To better compare the antiviral activity of CQ versus HCQ, the dose-response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection MOIs by quantification of viral RNA copy numbers in the cell supernatant at 48 h post infection p.i. .", "To better compare the antiviral activity of CQ versus HCQ, the dose-response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection MOIs by quantification of viral RNA copy numbers in the cell supernatant at 48 h post infection p.i. . The data summarized in Fig. 1a and Supplementary Table S1 show that, at all MOIs 0.01, 0.02, 0.2, and 0.8 , the 50% maximal effective concentration EC 50 for CQ 2.71, 3.81, 7.14, and 7.36 μM was lower than that of HCQ 4.51, 4.06, 17.31, and 12.96 μM . The differences in EC 50 values were statistically significant at an MOI of 0.01 P < 0.05 and MOI of 0.2 P < 0.001 Supplementary Table S1 . It is worth noting that the EC 50 values of CQ seemed to be a little higher than that in our previous report 1.13 μM at an MOI of 0.05 1 , which is likely due to the adaptation of the virus in cell culture that significantly increased viral infectivity upon continuous passaging.", "The differences in EC 50 values were statistically significant at an MOI of 0.01 P < 0.05 and MOI of 0.2 P < 0.001 Supplementary Table S1 . It is worth noting that the EC 50 values of CQ seemed to be a little higher than that in our previous report 1.13 μM at an MOI of 0.05 1 , which is likely due to the adaptation of the virus in cell culture that significantly increased viral infectivity upon continuous passaging. Consequently, the selectivity index SI = CC 50 /EC 50 of CQ 100.81, 71.71, 38.26, and 37.12 was higher than that of HCQ 55.32, 61.45, 14.41, 19.25 at MOIs of 0.01, 0.02, 0.2, and 0.8, respectively. These results were corroborated by immunofluorescence microscopy as evidenced by different expression levels of virus nucleoprotein NP at the indicated drug concentrations at 48 h p.i. Supplementary Fig. S1 .", "These results were corroborated by immunofluorescence microscopy as evidenced by different expression levels of virus nucleoprotein NP at the indicated drug concentrations at 48 h p.i. Supplementary Fig. S1 . Taken together, the data suggest that the anti-SARS-CoV-2 activity of HCQ seems to be less potent compared to CQ, at least at certain MOIs. Both CQ and HCQ are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion 5 . In addition, CQ could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein 6 .", "Both CQ and HCQ are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion 5 . In addition, CQ could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein 6 . Time-of-addition experiment confirmed that HCQ effectively inhibited the entry step, as well as the post-entry stages of SARS-CoV-2, which was also found upon CQ treatment Supplementary Fig. S2 . To further explore the detailed mechanism of action of CQ and HCQ in inhibiting virus entry, co-localization of virions with early endosomes EEs or endolysosomes ELs was analyzed by immunofluorescence analysis IFA and confocal microscopy. Quantification analysis showed that, at 90 min p.i.", "To further explore the detailed mechanism of action of CQ and HCQ in inhibiting virus entry, co-localization of virions with early endosomes EEs or endolysosomes ELs was analyzed by immunofluorescence analysis IFA and confocal microscopy. Quantification analysis showed that, at 90 min p.i. in untreated cells, 16.2% of internalized virions anti-NP, red were observed in early endosome antigen 1 EEA1 -positive EEs green , while more virions 34.3% were transported into the late endosomal-lysosomal protein LAMP1 + ELs green n > 30 cells for each group . By contrast, in the presence of CQ or HCQ, significantly more virions 35.3% for CQ and 29.2% for HCQ; P < 0.001 were detected in the EEs, while only very few virions 2.4% for CQ and 0.03% for HCQ; P < 0.001 were found to be co-localized with LAMP1 + ELs n > 30 cells Fig. 1b, c . This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV 7 .", "1b, c . This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV 7 . Interestingly, we found that CQ and HCQ treatment caused noticeable changes in the number and size/morphology of EEs and ELs Fig. 1c . In the untreated cells, most EEs were much smaller than ELs Fig. 1c . In CQand HCQ-treated cells, abnormally enlarged EE vesicles were observed Fig. 1c , arrows in the upper panels , many of which are even larger than ELs in the untreated cells.", "In CQand HCQ-treated cells, abnormally enlarged EE vesicles were observed Fig. 1c , arrows in the upper panels , many of which are even larger than ELs in the untreated cells. This is in agreement with previous report that treatment with CQ induced the formation of expanded cytoplasmic vesicles 8 . Within the EE vesicles, virions red were localized around the membrane green of the vesicle. CQ treatment did not cause obvious changes in the number and size of ELs; however, the regular vesicle structure seemed to be disrupted, at least partially. By contrast, in HCQ-treated cells, the size and number of ELs increased significantly Fig. 1c , arrows in the lower panels .", "By contrast, in HCQ-treated cells, the size and number of ELs increased significantly Fig. 1c , arrows in the lower panels . Since acidification is crucial for endosome maturation and function, we surmise that endosome maturation might be blocked at intermediate stages of endocytosis, resulting in failure of further transport of virions to the ultimate releasing site. CQ was reported to elevate the pH see figure on previous page Fig. 1 Comparative antiviral efficacy and mechanism of action of CQ and HCQ against SARS-CoV-2 infection in vitro. a Cytotoxicity and antiviral activities of CQ and HCQ. The cytotoxicity of the two drugs in Vero E6 cells was determined by CCK-8 assays.", "a Cytotoxicity and antiviral activities of CQ and HCQ. The cytotoxicity of the two drugs in Vero E6 cells was determined by CCK-8 assays. Vero E6 cells were treated with different doses of either compound or with PBS in the controls for 1 h and then infected with SARS-CoV-2 at MOIs of 0.01, 0.02, 0.2, and 0.8. The virus yield in the cell supernatant was quantified by qRT-PCR at 48 h p.i. Y-axis represents the mean of percent inhibition normalized to the PBS group. The experiments were repeated twice. b, c Mechanism of CQ and HCQ in inhibiting virus entry.", "Y-axis represents the mean of percent inhibition normalized to the PBS group. The experiments were repeated twice. b, c Mechanism of CQ and HCQ in inhibiting virus entry. Vero E6 cells were treated with CQ or HCQ 50 μM for 1 h, followed by virus binding MOI = 10 at 4°C for 1 h. Then the unbound virions were removed, and the cells were further supplemented with fresh drug-containing medium at 37°C for 90 min before being fixed and stained with IFA using anti-NP antibody for virions red and antibodies against EEA1 for EEs green or LAMP1 for ELs green . The nuclei blue were stained with Hoechst dye. The portion of virions that co-localized with EEs or ELs in each group n > 30 cells was quantified and is shown in b.", "The nuclei blue were stained with Hoechst dye. The portion of virions that co-localized with EEs or ELs in each group n > 30 cells was quantified and is shown in b. Representative confocal microscopic images of viral particles red , EEA1 + EEs green , or LAMP1 + ELs green in each group are displayed in c. The enlarged images in the boxes indicate a single vesicle-containing virion. The arrows indicated the abnormally enlarged vesicles. Bars, 5 μm. Statistical analysis was performed using a one-way analysis of variance ANOVA with GraphPad Prism F = 102.8, df = 5,182, ***P < 0.001 . of lysosome from about 4.5 to 6.5 at 100 μM 9 .", "Statistical analysis was performed using a one-way analysis of variance ANOVA with GraphPad Prism F = 102.8, df = 5,182, ***P < 0.001 . of lysosome from about 4.5 to 6.5 at 100 μM 9 . To our knowledge, there is a lack of studies on the impact of HCQ on the morphology and pH values of endosomes/ lysosomes. Our observations suggested that the mode of actions of CQ and HCQ appear to be distinct in certain aspects. It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200-700 times higher than those in the plasma 10 .", "It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200-700 times higher than those in the plasma 10 . It was reported that safe dosage 6-6.5 mg/kg per day of HCQ sulfate could generate serum levels of 1.4-1.5 μM in humans 11 . Therefore, with a safe dosage, HCQ concentration in the above tissues is likely to be achieved to inhibit SARS-CoV-2 infection. Clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with SARS-CoV-2, suggesting that cytokine storm was associated with disease severity 12 . Other than its direct antiviral activity, HCQ is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors.", "Clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with SARS-CoV-2, suggesting that cytokine storm was associated with disease severity 12 . Other than its direct antiviral activity, HCQ is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors. Therefore, in COVID-19 patients, HCQ may also contribute to attenuating the inflammatory response. In conclusion, our results show that HCQ can efficiently inhibit SARS-CoV-2 infection in vitro. In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials.", "In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials. We need to point out, although HCQ is less toxic than CQ, prolonged and overdose usage can still cause poisoning. And the relatively low SI of HCQ requires careful designing and conducting of clinical trials to achieve efficient and safe control of the SARS-CoV-2 infection." ]

[ "nan Text: Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro Jia Liu 1 , Ruiyuan Cao 2 , Mingyue Xu 1,3 , Xi Wang 1 , Huanyu Zhang 1,3 , Hengrui Hu 1,3 , Yufeng Li 1,3 , Zhihong Hu 1 , Wu Zhong 2 and Manli Wang 1 Dear Editor, The outbreak of coronavirus disease 2019 COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 SARS-CoV-2/2019-nCoV poses a serious threat to global public health and local economies. As of March 3, 2020, over 80,000 cases have been confirmed in China, including 2946 deaths as well as over 10,566 confirmed cases in 72 other countries. Such huge numbers of infected and dead people call for an urgent demand of effective, available, and affordable drugs to control and diminish the epidemic. We have recently reported that two drugs, remdesivir GS-5734 and chloroquine CQ phosphate, efficiently inhibited SARS-CoV-2 infection in vitro 1 . Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences USA . A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States 2 , and a phase III clinical trial of remdesivir against SARS-CoV-2 was launched in Wuhan on February 4, 2020.", "Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences USA . A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States 2 , and a phase III clinical trial of remdesivir against SARS-CoV-2 was launched in Wuhan on February 4, 2020. However, as an experimental drug, remdesivir is not expected to be largely available for treating a very large number of patients in a timely manner. Therefore, of the two potential drugs, CQ appears to be the drug of choice for large-scale use due to its availability, proven safety record, and a relatively low cost. In light of the preliminary clinical data, CQ has been added to the list of trial drugs in the Guidelines for the Diagnosis and Treatment of COVID-19 sixth edition published by National Health Commission of the People's Republic of China. CQ N4- 7-Chloro-4-quinolinyl -N1,N1-diethyl-1,4pentanediamine has long been used to treat malaria and amebiasis.", "In light of the preliminary clinical data, CQ has been added to the list of trial drugs in the Guidelines for the Diagnosis and Treatment of COVID-19 sixth edition published by National Health Commission of the People's Republic of China. CQ N4- 7-Chloro-4-quinolinyl -N1,N1-diethyl-1,4pentanediamine has long been used to treat malaria and amebiasis. However, Plasmodium falciparum developed widespread resistance to it, and with the development of new antimalarials, it has become a choice for the prophylaxis of malaria. In addition, an overdose of CQ can cause acute poisoning and death 3 . In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China. Hydroxychloroquine HCQ sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less ~40% toxic than CQ in animals 4 .", "In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China. Hydroxychloroquine HCQ sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less ~40% toxic than CQ in animals 4 . More importantly, HCQ is still widely available to treat autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Since CQ and HCQ share similar chemical structures and mechanisms of acting as a weak base and immunomodulator, it is easy to conjure up the idea that HCQ may be a potent candidate to treat infection by SARS-CoV-2. Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence.", "Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence. To this end, we evaluated the antiviral effect of HCQ against SARS-CoV-2 infection in comparison to CQ in vitro. First, the cytotoxicity of HCQ and CQ in African green monkey kidney VeroE6 cells ATCC-1586 was measured by standard CCK8 assay, and the result showed © The Author s 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author s and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.", "The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit Fig. 1a . To better compare the antiviral activity of CQ versus HCQ, the dose-response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection MOIs by quantification of viral RNA copy numbers in the cell supernatant at 48 h post infection p.i. .", "To better compare the antiviral activity of CQ versus HCQ, the dose-response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection MOIs by quantification of viral RNA copy numbers in the cell supernatant at 48 h post infection p.i. . The data summarized in Fig. 1a and Supplementary Table S1 show that, at all MOIs 0.01, 0.02, 0.2, and 0.8 , the 50% maximal effective concentration EC 50 for CQ 2.71, 3.81, 7.14, and 7.36 μM was lower than that of HCQ 4.51, 4.06, 17.31, and 12.96 μM . The differences in EC 50 values were statistically significant at an MOI of 0.01 P < 0.05 and MOI of 0.2 P < 0.001 Supplementary Table S1 . It is worth noting that the EC 50 values of CQ seemed to be a little higher than that in our previous report 1.13 μM at an MOI of 0.05 1 , which is likely due to the adaptation of the virus in cell culture that significantly increased viral infectivity upon continuous passaging.", "The differences in EC 50 values were statistically significant at an MOI of 0.01 P < 0.05 and MOI of 0.2 P < 0.001 Supplementary Table S1 . It is worth noting that the EC 50 values of CQ seemed to be a little higher than that in our previous report 1.13 μM at an MOI of 0.05 1 , which is likely due to the adaptation of the virus in cell culture that significantly increased viral infectivity upon continuous passaging. Consequently, the selectivity index SI = CC 50 /EC 50 of CQ 100.81, 71.71, 38.26, and 37.12 was higher than that of HCQ 55.32, 61.45, 14.41, 19.25 at MOIs of 0.01, 0.02, 0.2, and 0.8, respectively. These results were corroborated by immunofluorescence microscopy as evidenced by different expression levels of virus nucleoprotein NP at the indicated drug concentrations at 48 h p.i. Supplementary Fig. S1 .", "These results were corroborated by immunofluorescence microscopy as evidenced by different expression levels of virus nucleoprotein NP at the indicated drug concentrations at 48 h p.i. Supplementary Fig. S1 . Taken together, the data suggest that the anti-SARS-CoV-2 activity of HCQ seems to be less potent compared to CQ, at least at certain MOIs. Both CQ and HCQ are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion 5 . In addition, CQ could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein 6 .", "Both CQ and HCQ are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion 5 . In addition, CQ could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein 6 . Time-of-addition experiment confirmed that HCQ effectively inhibited the entry step, as well as the post-entry stages of SARS-CoV-2, which was also found upon CQ treatment Supplementary Fig. S2 . To further explore the detailed mechanism of action of CQ and HCQ in inhibiting virus entry, co-localization of virions with early endosomes EEs or endolysosomes ELs was analyzed by immunofluorescence analysis IFA and confocal microscopy. Quantification analysis showed that, at 90 min p.i.", "To further explore the detailed mechanism of action of CQ and HCQ in inhibiting virus entry, co-localization of virions with early endosomes EEs or endolysosomes ELs was analyzed by immunofluorescence analysis IFA and confocal microscopy. Quantification analysis showed that, at 90 min p.i. in untreated cells, 16.2% of internalized virions anti-NP, red were observed in early endosome antigen 1 EEA1 -positive EEs green , while more virions 34.3% were transported into the late endosomal-lysosomal protein LAMP1 + ELs green n > 30 cells for each group . By contrast, in the presence of CQ or HCQ, significantly more virions 35.3% for CQ and 29.2% for HCQ; P < 0.001 were detected in the EEs, while only very few virions 2.4% for CQ and 0.03% for HCQ; P < 0.001 were found to be co-localized with LAMP1 + ELs n > 30 cells Fig. 1b, c . This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV 7 .", "1b, c . This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV 7 . Interestingly, we found that CQ and HCQ treatment caused noticeable changes in the number and size/morphology of EEs and ELs Fig. 1c . In the untreated cells, most EEs were much smaller than ELs Fig. 1c . In CQand HCQ-treated cells, abnormally enlarged EE vesicles were observed Fig. 1c , arrows in the upper panels , many of which are even larger than ELs in the untreated cells.", "In CQand HCQ-treated cells, abnormally enlarged EE vesicles were observed Fig. 1c , arrows in the upper panels , many of which are even larger than ELs in the untreated cells. This is in agreement with previous report that treatment with CQ induced the formation of expanded cytoplasmic vesicles 8 . Within the EE vesicles, virions red were localized around the membrane green of the vesicle. CQ treatment did not cause obvious changes in the number and size of ELs; however, the regular vesicle structure seemed to be disrupted, at least partially. By contrast, in HCQ-treated cells, the size and number of ELs increased significantly Fig. 1c , arrows in the lower panels .", "By contrast, in HCQ-treated cells, the size and number of ELs increased significantly Fig. 1c , arrows in the lower panels . Since acidification is crucial for endosome maturation and function, we surmise that endosome maturation might be blocked at intermediate stages of endocytosis, resulting in failure of further transport of virions to the ultimate releasing site. CQ was reported to elevate the pH see figure on previous page Fig. 1 Comparative antiviral efficacy and mechanism of action of CQ and HCQ against SARS-CoV-2 infection in vitro. a Cytotoxicity and antiviral activities of CQ and HCQ. The cytotoxicity of the two drugs in Vero E6 cells was determined by CCK-8 assays.", "a Cytotoxicity and antiviral activities of CQ and HCQ. The cytotoxicity of the two drugs in Vero E6 cells was determined by CCK-8 assays. Vero E6 cells were treated with different doses of either compound or with PBS in the controls for 1 h and then infected with SARS-CoV-2 at MOIs of 0.01, 0.02, 0.2, and 0.8. The virus yield in the cell supernatant was quantified by qRT-PCR at 48 h p.i. Y-axis represents the mean of percent inhibition normalized to the PBS group. The experiments were repeated twice. b, c Mechanism of CQ and HCQ in inhibiting virus entry.", "Y-axis represents the mean of percent inhibition normalized to the PBS group. The experiments were repeated twice. b, c Mechanism of CQ and HCQ in inhibiting virus entry. Vero E6 cells were treated with CQ or HCQ 50 μM for 1 h, followed by virus binding MOI = 10 at 4°C for 1 h. Then the unbound virions were removed, and the cells were further supplemented with fresh drug-containing medium at 37°C for 90 min before being fixed and stained with IFA using anti-NP antibody for virions red and antibodies against EEA1 for EEs green or LAMP1 for ELs green . The nuclei blue were stained with Hoechst dye. The portion of virions that co-localized with EEs or ELs in each group n > 30 cells was quantified and is shown in b.", "The nuclei blue were stained with Hoechst dye. The portion of virions that co-localized with EEs or ELs in each group n > 30 cells was quantified and is shown in b. Representative confocal microscopic images of viral particles red , EEA1 + EEs green , or LAMP1 + ELs green in each group are displayed in c. The enlarged images in the boxes indicate a single vesicle-containing virion. The arrows indicated the abnormally enlarged vesicles. Bars, 5 μm. Statistical analysis was performed using a one-way analysis of variance ANOVA with GraphPad Prism F = 102.8, df = 5,182, ***P < 0.001 . of lysosome from about 4.5 to 6.5 at 100 μM 9 .", "Statistical analysis was performed using a one-way analysis of variance ANOVA with GraphPad Prism F = 102.8, df = 5,182, ***P < 0.001 . of lysosome from about 4.5 to 6.5 at 100 μM 9 . To our knowledge, there is a lack of studies on the impact of HCQ on the morphology and pH values of endosomes/ lysosomes. Our observations suggested that the mode of actions of CQ and HCQ appear to be distinct in certain aspects. It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200-700 times higher than those in the plasma 10 .", "It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200-700 times higher than those in the plasma 10 . It was reported that safe dosage 6-6.5 mg/kg per day of HCQ sulfate could generate serum levels of 1.4-1.5 μM in humans 11 . Therefore, with a safe dosage, HCQ concentration in the above tissues is likely to be achieved to inhibit SARS-CoV-2 infection. Clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with SARS-CoV-2, suggesting that cytokine storm was associated with disease severity 12 . Other than its direct antiviral activity, HCQ is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors.", "Clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with SARS-CoV-2, suggesting that cytokine storm was associated with disease severity 12 . Other than its direct antiviral activity, HCQ is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors. Therefore, in COVID-19 patients, HCQ may also contribute to attenuating the inflammatory response. In conclusion, our results show that HCQ can efficiently inhibit SARS-CoV-2 infection in vitro. In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials.", "In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials. We need to point out, although HCQ is less toxic than CQ, prolonged and overdose usage can still cause poisoning. And the relatively low SI of HCQ requires careful designing and conducting of clinical trials to achieve efficient and safe control of the SARS-CoV-2 infection." ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 COVID-19 could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 64.7% were aged 26 to 40 years, and 964 76.7% were women. Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% .", "Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% . Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers eg, median IQR Patient Health Questionnaire scores among physicians vs nurses: 4.0 1.0-7.0 vs 5.0 2.0-8.0 ; P = .007; median interquartile range {IQR} Generalized Anxiety Disorder scale scores among men vs women: 2.0 0-6.0 vs 4.0 1.0-7.0 ; P < .001; median IQR Insomnia Severity Index scores among frontline vs second-line workers: 6.0 2.0-11.0 vs 4.0 1.0-8.0 ; P < .001; median IQR Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 8.5-34.5 vs 18.0 6.0-28.0 in Hubei outside Wuhan and 15.0 4.0-26.0 outside Hubei; P < .001 . Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan odds ratio OR , 0.62; 95% CI, 0.43-0.88; P = .008 . Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19.", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median IQR 6.0 2.0, 10.0 6.0 2.0, 9.0 .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 COVID-19 could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 64.7% were aged 26 to 40 years, and 964 76.7% were women. Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% .", "Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% . Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers eg, median IQR Patient Health Questionnaire scores among physicians vs nurses: 4.0 1.0-7.0 vs 5.0 2.0-8.0 ; P = .007; median interquartile range {IQR} Generalized Anxiety Disorder scale scores among men vs women: 2.0 0-6.0 vs 4.0 1.0-7.0 ; P < .001; median IQR Insomnia Severity Index scores among frontline vs second-line workers: 6.0 2.0-11.0 vs 4.0 1.0-8.0 ; P < .001; median IQR Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 8.5-34.5 vs 18.0 6.0-28.0 in Hubei outside Wuhan and 15.0 4.0-26.0 outside Hubei; P < .001 . Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan odds ratio OR , 0.62; 95% CI, 0.43-0.88; P = .008 . Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19.", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median IQR 6.0 2.0, 10.0 6.0 2.0, 9.0 .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 COVID-19 could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 64.7% were aged 26 to 40 years, and 964 76.7% were women. Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% .", "Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% . Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers eg, median IQR Patient Health Questionnaire scores among physicians vs nurses: 4.0 1.0-7.0 vs 5.0 2.0-8.0 ; P = .007; median interquartile range {IQR} Generalized Anxiety Disorder scale scores among men vs women: 2.0 0-6.0 vs 4.0 1.0-7.0 ; P < .001; median IQR Insomnia Severity Index scores among frontline vs second-line workers: 6.0 2.0-11.0 vs 4.0 1.0-8.0 ; P < .001; median IQR Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 8.5-34.5 vs 18.0 6.0-28.0 in Hubei outside Wuhan and 15.0 4.0-26.0 outside Hubei; P < .001 . Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan odds ratio OR , 0.62; 95% CI, 0.43-0.88; P = .008 . Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19.", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median IQR 6.0 2.0, 10.0 6.0 2.0, 9.0 .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 COVID-19 could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 64.7% were aged 26 to 40 years, and 964 76.7% were women. Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% .", "Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% . Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers eg, median IQR Patient Health Questionnaire scores among physicians vs nurses: 4.0 1.0-7.0 vs 5.0 2.0-8.0 ; P = .007; median interquartile range {IQR} Generalized Anxiety Disorder scale scores among men vs women: 2.0 0-6.0 vs 4.0 1.0-7.0 ; P < .001; median IQR Insomnia Severity Index scores among frontline vs second-line workers: 6.0 2.0-11.0 vs 4.0 1.0-8.0 ; P < .001; median IQR Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 8.5-34.5 vs 18.0 6.0-28.0 in Hubei outside Wuhan and 15.0 4.0-26.0 outside Hubei; P < .001 . Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan odds ratio OR , 0.62; 95% CI, 0.43-0.88; P = .008 . Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19.", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median IQR 6.0 2.0, 10.0 6.0 2.0, 9.0 .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 COVID-19 could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 64.7% were aged 26 to 40 years, and 964 76.7% were women. Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% .", "Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% . Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers eg, median IQR Patient Health Questionnaire scores among physicians vs nurses: 4.0 1.0-7.0 vs 5.0 2.0-8.0 ; P = .007; median interquartile range {IQR} Generalized Anxiety Disorder scale scores among men vs women: 2.0 0-6.0 vs 4.0 1.0-7.0 ; P < .001; median IQR Insomnia Severity Index scores among frontline vs second-line workers: 6.0 2.0-11.0 vs 4.0 1.0-8.0 ; P < .001; median IQR Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 8.5-34.5 vs 18.0 6.0-28.0 in Hubei outside Wuhan and 15.0 4.0-26.0 outside Hubei; P < .001 . Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan odds ratio OR , 0.62; 95% CI, 0.43-0.88; P = .008 . Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19.", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median IQR 6.0 2.0, 10.0 6.0 2.0, 9.0 .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 COVID-19 could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 64.7% were aged 26 to 40 years, and 964 76.7% were women. Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% .", "Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% . Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers eg, median IQR Patient Health Questionnaire scores among physicians vs nurses: 4.0 1.0-7.0 vs 5.0 2.0-8.0 ; P = .007; median interquartile range {IQR} Generalized Anxiety Disorder scale scores among men vs women: 2.0 0-6.0 vs 4.0 1.0-7.0 ; P < .001; median IQR Insomnia Severity Index scores among frontline vs second-line workers: 6.0 2.0-11.0 vs 4.0 1.0-8.0 ; P < .001; median IQR Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 8.5-34.5 vs 18.0 6.0-28.0 in Hubei outside Wuhan and 15.0 4.0-26.0 outside Hubei; P < .001 . Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan odds ratio OR , 0.62; 95% CI, 0.43-0.88; P = .008 . Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19.", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median IQR 6.0 2.0, 10.0 6.0 2.0, 9.0 .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 COVID-19 could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 64.7% were aged 26 to 40 years, and 964 76.7% were women. Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% .", "Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% . Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers eg, median IQR Patient Health Questionnaire scores among physicians vs nurses: 4.0 1.0-7.0 vs 5.0 2.0-8.0 ; P = .007; median interquartile range {IQR} Generalized Anxiety Disorder scale scores among men vs women: 2.0 0-6.0 vs 4.0 1.0-7.0 ; P < .001; median IQR Insomnia Severity Index scores among frontline vs second-line workers: 6.0 2.0-11.0 vs 4.0 1.0-8.0 ; P < .001; median IQR Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 8.5-34.5 vs 18.0 6.0-28.0 in Hubei outside Wuhan and 15.0 4.0-26.0 outside Hubei; P < .001 . Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan odds ratio OR , 0.62; 95% CI, 0.43-0.88; P = .008 . Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19.", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median IQR 6.0 2.0, 10.0 6.0 2.0, 9.0 .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 COVID-19 could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 64.7% were aged 26 to 40 years, and 964 76.7% were women. Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% .", "Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% . Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers eg, median IQR Patient Health Questionnaire scores among physicians vs nurses: 4.0 1.0-7.0 vs 5.0 2.0-8.0 ; P = .007; median interquartile range {IQR} Generalized Anxiety Disorder scale scores among men vs women: 2.0 0-6.0 vs 4.0 1.0-7.0 ; P < .001; median IQR Insomnia Severity Index scores among frontline vs second-line workers: 6.0 2.0-11.0 vs 4.0 1.0-8.0 ; P < .001; median IQR Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 8.5-34.5 vs 18.0 6.0-28.0 in Hubei outside Wuhan and 15.0 4.0-26.0 outside Hubei; P < .001 . Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan odds ratio OR , 0.62; 95% CI, 0.43-0.88; P = .008 . Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19.", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median IQR 6.0 2.0, 10.0 6.0 2.0, 9.0 .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 COVID-19 could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 64.7% were aged 26 to 40 years, and 964 76.7% were women. Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% .", "Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% . Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers eg, median IQR Patient Health Questionnaire scores among physicians vs nurses: 4.0 1.0-7.0 vs 5.0 2.0-8.0 ; P = .007; median interquartile range {IQR} Generalized Anxiety Disorder scale scores among men vs women: 2.0 0-6.0 vs 4.0 1.0-7.0 ; P < .001; median IQR Insomnia Severity Index scores among frontline vs second-line workers: 6.0 2.0-11.0 vs 4.0 1.0-8.0 ; P < .001; median IQR Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 8.5-34.5 vs 18.0 6.0-28.0 in Hubei outside Wuhan and 15.0 4.0-26.0 outside Hubei; P < .001 . Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan odds ratio OR , 0.62; 95% CI, 0.43-0.88; P = .008 . Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19.", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median IQR 6.0 2.0, 10.0 6.0 2.0, 9.0 .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 COVID-19 could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 64.7% were aged 26 to 40 years, and 964 76.7% were women. Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% .", "Of all participants, 764 60.8% were nurses, and 493 39.2% were physicians; 760 60.5% worked in hospitals in Wuhan, and 522 41.5% were frontline health care workers. A considerable proportion of participants reported symptoms of depression 634 50.4% , anxiety 560 44.6% , insomnia 427 34.0% , and distress 899 71.5% . Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers eg, median IQR Patient Health Questionnaire scores among physicians vs nurses: 4.0 1.0-7.0 vs 5.0 2.0-8.0 ; P = .007; median interquartile range {IQR} Generalized Anxiety Disorder scale scores among men vs women: 2.0 0-6.0 vs 4.0 1.0-7.0 ; P < .001; median IQR Insomnia Severity Index scores among frontline vs second-line workers: 6.0 2.0-11.0 vs 4.0 1.0-8.0 ; P < .001; median IQR Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 8.5-34.5 vs 18.0 6.0-28.0 in Hubei outside Wuhan and 15.0 4.0-26.0 outside Hubei; P < .001 . Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan odds ratio OR , 0.62; 95% CI, 0.43-0.88; P = .008 . Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19.", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression OR, 1.52; 95% CI, 1.11-2.09; P = .01 , anxiety OR, 1.57; 95% CI, 1.22-2.02; P < .001 , insomnia OR, 2.97; 95% CI, 1.92-4.60; P < .001 , and distress OR, 1.60; 95% CI, 1.25-2.04; P < .001 . CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median IQR 6.0 2.0, 10.0 6.0 2.0, 9.0 .29" ]

[ "Microtubule severing enzymes implement a diverse range of tissue-specific molecular functions throughout development and into adulthood. Although microtubule severing is fundamental to many dynamic neural processes, little is known regarding the role of the family member Katanin p60 subunit A-like 1, KATNAL1, in central nervous system CNS function. Recent studies reporting that microdeletions incorporating the KATNAL1 locus in humans result in intellectual disability and microcephaly suggest that KATNAL1 may play a prominent role in the CNS; however, such associations lack the functional data required to highlight potential mechanisms which link the gene to disease symptoms. Here we identify and characterise a mouse line carrying a loss of function allele in Katnal1. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology.", "We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. Furthermore we demonstrate defects in the motile cilia of the ventricular ependymal cells of mutants, suggesting a role for Katnal1 in the development of ciliary function. We believe the data we present here are the first to associate KATNAL1 with such phenotypes, demonstrating that the protein plays keys roles in a number of processes integral to the development of neuronal function and behaviour. Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development.", "Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, and mutations in Fign cause a range of phenotypes in mice. 7 Currently the microtubule severing enzyme KATNAL1 is poorly characterised and it is not yet understood how the enzyme functions in the nervous system. Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus.", "Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus. 11, 12 Although such association studies strongly suggest that KATNAL1 plays a fundamental role in the central nervous system CNS , additional studies using cellular or animals models are required to understand how the gene may be causative for disease. Here we present the first study describing neural and behavioural deficits associated with a loss of function allele of Katnal1 in the mouse. This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits.", "This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits. Underlying these behavioural phenotypes, we identified histopathological abnormalities in the brains of Katnal1 1H/1H mutants, including disordered cellular layers in the hippocampus and cortex and substantially larger ventricles. Further investigations demonstrated that Katnal1 1H/1H mice show neuronal migration and ciliary function deficits suggesting KATNAL1 plays an essential role in these processes. These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background.", "These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background. Circadian wheel running was performed as previously described. 14 Sleep assessment by electroencephalography and electromyography Electroencephalography and electromyography was performed as previously described. 15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice.", "15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice. The mouse was confined in the goal arm for 30 s, before being allowed a second free choice of goal arm. An alternation was recorded if the second choice differed from that of the first. One trial was performed per day for 10 days. Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min.", "Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min. Animals were monitored by EthoVision XT analysis software Noldus, Wageningen, Netherlands . Video tracking in the home cage. Activity in the home cage was recorded by video tracking as previously described. 16 Morris water maze and ultrasonic vocalisation. These tests were performed as previously described. 17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols.", "17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols. In vivo neuronal migration assessment was performed as previously described 18 using embryos at either E13 or E15 three mothers per age group and pups at P9. Cell counts were performed using ImageJ NIH, Bethesda, MD, USA . In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm.", "In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm. Segmentation, volume calculation and 3D modelling was performed using ITK-SNAP version 3.0.0 ref. 20 and 3DSlicer. 21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ.", "21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ. Brains from P2 mice were dissected, and the dorsal cerebral half was sectioned 250 μm through the floor of the lateral and 3rd ventricle using a vibratome. Ciliary beat frequency and pattern was analysed as previously described. 22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies .", "22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies . and visualised using a JEOL LSM-6010 scanning electron microscope Jeol, Herts, UK . Transmission electron microscopy was performed as previously described. 22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05.", "22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05. All graphs are presented showing mean ± s.e.m. Additional and more detailed methods can be found in supplementary information. Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened .", "Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened . An outcross using an affected female produced no affected animals 33 animals screened . In subsequent intercross screens 15.5% of animals were affected 53 out of 342 animals screened , suggesting that the pedigree carries a mutation causing a recessive circadian phenotype which is 60% penetrant. We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree.", "We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree. 23 Genome-wide SNP linkage analysis mapped the circadian and sterility phenotypes to the same region on chromosome 5 and subsequent sequencing identified the causative mutation as a T to G single point mutation within exon seven of the Katnal1 gene. For full details of mapping and identification of the mutation see reference 23. This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele.", "This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele. 23 3D modelling of the protein suggests that this loss of function is due to hydrophobic changes in the AAA domain of the enzyme Supplementary Figure S1 . Genotyping confirmed that the mutation was homozygous in affected circadian animals and wild type or heterozygous in unaffected animals, confirming that Katnal1 1H was causative for the circadian phenotype. Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 .", "Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 . Bioluminescence recordings performed using PER2::LUCIFERASE reporter mice carrying the Katnal1 1H mutation revealed that these circadian changes were not due to changes to the core molecular clock of the suprachiasmatic nucleus the site of the master circadian clock in the brain; Supplementary Figure S2 . Circadian disruptions are often associated with deficits in sleep homeostasis. Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1.", "Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1. Compared to wildtype littermates, the non-REM delta power of Katnal1 1H/1H mice was higher in the dark phase of baseline sleep mixed ANOVA, interaction factors 'genotype X time, F 1,88 = 8.91, P = 0.0175 Figure 1f and in both the light and dark phases of recovery sleep mixed ANOVA, interaction factors 'genotype X time', F 1,136 = 11.93, P = 0.0086; Figure 1g . All other sleep parameters were unaffected in Katnal1 1H/1H animals. Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests.", "Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests. Data and cohort details are given in Supplementary Table S2 . Both working memory and spatial memory were significantly poorer in Katnal1 1H/1H mice, as evidenced by reduced spontaneous alternations in a T-maze Figure 2a and in the Morris water maze where mutants take longer to find the platform in acquisition trials Figure 2b Compared to wild-type littermates, Katnal1 1H/1H animals have a shorter period c , are more active in the light phase of the light/dark cycle d and show an earlier onset of activity in light/dark transitions and in the transition from light/dark cycles to constant darkness e . In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001.", "In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001. EEG, electroencephalography; DD, constant darkness; LD, light/dark cycle. type = 164 ± 12 m, Katnal1 1H/1H = 243 ± 20 m, P = 0.02; distance travelled in periphery of open field: wild type = 4.3 ± 0.2 m, Katnal1 1H/1H = 6 ± 0.3 m, P = 0.004 . Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs .", "Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs . To test whether Katnal1 1H/1H animals vocalised differently to wild types, we separated pups at postnatal days 7-8 the age at which mice show peak of USV emission 24 and recorded their USVs. In these tests, compared to wild types, Katnal1 1H/1H pups produced fewer Figure 2g and shorter Figure 2h vocalisations, containing fewer phrases Figure 2i . Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 .", "Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 . Analysis of hematoxylin and eosin stained brain sections revealed that, compared to wildtype littermates, Katnal1 1H/1H animals had less tightly packed pyramidal cell layers in the hippocampus Figures 3a and b and a narrower cortical layer 1 and wider cortical layer 6 Figures 3c-e . To confirm these cortical layer differences, immunofluorescence was performed using the Figures 3l and m . Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n .", "Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n . Finally, three dimensional models of the ventricular system were constructed from brain micro-computed tomography scans Figures 3o and p . Volumetric analysis revealed that Katnal1 1H/1H mice had substantially larger ventricles than wild types Figure 3q . Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 .", "Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 . At both ages Katnal1 1H/1H animals had greater numbers of labelled neurons in bins close to the cortical surface neurons positioned closer to the cortical surface compared to wild type. To confirm these results we used a Boyden chamber 19 and performed in vitro neuronal migration analysis in E13.5 primary cortical neuronal cultures. Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration.", "Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration. Given its role in cytoskeletal organisation, we also hypothesised that neuronal morphology is modulated by Katnal1. Analysis of golgi stained neurons from layers 2-3 of the cortex Figures 4g and i demonstrated that, compared to wild-type littermates, Katnal1 1H/1H neurons had larger soma Figure 4k , and shorter and thinner axons Figures 4l and m data and cohort details are given in Supplementary Table S3 . Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia.", "Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia. 5 Since such ciliary defects could underlie the phenotypes described above we studied the motile cilia of the ependymal lining of the lateral ventricle in sections of postnatal day 2 mouse brains from both Katnal1 1H/1H n = 4 and wild-type animals n = 3 . We found that the ciliary beat frequency CBF of Katnal1 1H/1H animals was significantly attenuated compared to wild-type CBF: wildtype = 22.39 ± 0.94 Hz, Katnal1 1H/1H = 14.25 ± 0.92 Hz, P = 0.0001; Figure 5a , Supplementary Movies S1 . This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles.", "This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles. These abnormalities were observed in approximately 25% of Katnal1 1H/1H brain slices. The abnormal cilia always showed a dyskinetic beat pattern and lower beat frequency. To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 .", "To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 . However in Katnal1 1H/1H samples we noted the presence of both long and short cilia Figures 5e and f ; defined as two standard deviations longer or shorter than the average cilia length that were not present in wild-type samples. In addition, inspection of Katnal1 1H/1H cilia identified ciliary abnormalities including bifurcated cilia Figure 5g , abnormal kinks and bends in the cilia Figure 5h and swellings along the length of the cilia Figure 5i . Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia.", "Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia. Microtubule severing enzymes play diverse roles in the nervous system. 1, 2 However, at present the microtubule severing enzyme Katnal1 is poorly defined in the context of CNS development and function. Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes.", "Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes. Finally we also demonstrate that Katnal1 1H causes a range of defects in the motile cilia of ventricular ependymal cells. The data we present here are the first to associate KATNAL1 with such dysfunctions with important implications for clinical association studies. The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation.", "The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation. Phenotypes in circadian running wheel rhythms that are not associated with changes to the core clock mechanism have also been reported in mouse models of schizophrenia. 25 Here it has been suggested that the wheel running changes observed are the result in defects in output pathways from the SCN circadian clock. Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed.", "Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed. The behavioural consequences of microtubule severing enzyme dysfunction in mouse models have been poorly characterised. Currently the phenotypes described are limited to motor dysfunction in mice lacking the Spg4 gene 27 and head shaking and circling in the Fign mutant. 7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1.", "7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1. 8, 9 While it is also worth noting that mutant mice spend more time the centre of the open field than wild types implying that Katnal1 1H/1H animals show reduced anxiety , we suggest that this result is confounded by the hyperactivity in novel environments phenotype we also describe in mutant mice. This observation is backed up by the fact that mutant animals showed increased activity in all regions of the open field rather than just the anxiolytic periphery. Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions.", "Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions. For example, defects in neuronal migration, synaptic spines and neuronal morphology such as those we have demonstrated here, have been suggested to underpin ID in conditions such as lissencephaly, 18 Down's syndrome 31 and Rett syndrome. 32 While we are not suggesting that Katnal1 is causative for these conditions, similarities in symptoms and neuronal phenotypes between these conditions and those linked to Katnal1 dysfunction should be appreciated. Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice.", "Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice. Thus the range of phenotypes associated with defects in the function of Katnal1 strongly suggests that the gene should be considered in the pathology of disorders such as ID and schizophrenia. We do note one key genetic difference between the human patients and Katnal1 1H/1H animals. While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1.", "While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1. 17 While heterozygous loss of function mutations in CTNNB1 are causative for intellectual disability in humans, conditional knock outs for CTNNB1 have no reported behavioural or craniofacial phenotypes. 36, 37 These differences demonstrate that while mouse models of intellectual disability are of great use in our understanding of the causative mechanisms which underlie the condition, there are still genetic and neurodevelopmental differences between species which also must be taken into account. We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons.", "We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons. The data we present here also demonstrate defects in motile cilia in Katnal1 1H/1H mice. Ciliary disruptions in humans ciliopathies include Bardet-Biedl and Joubert syndrome. 38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations.", "38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations. Furthermore in Bardet-Biedl syndrome mouse models ciliary defects such as reduced CBF 45 and structural defects such as abnormal lengthening and swellings along their length 41 have been described, that are similar to those we describe in Katnal1 1H/1H mice. There is strong evidence that ciliopathy associated genes play a number of roles in neuronal development by affecting processes such as progenitor proliferation or maintenance of the radial glia scaffold. 43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice.", "43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice. Further investigations are required to clarify the impacts of these two processes. However it is notable that while defects in cilia structure may contribute to the phenotypes we describe in Katnal1 1H/1H mice, they are far less prominent in Katnal1 1H/1H mice than in other mouse ciliopathy models, 41 suggesting that the ciliary component of KATNAL1 dysfunction may be mild compared to other ciliopathies. Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function.", "Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function. The downstream effect of these defects leads in turn to a number of behavioural changes including in learning and memory, reaction to anxiogenic situations and circadian rhythms. These data therefore highlight how perturbations in KATNAL1 may play a role in neuronal dysfunction and demonstrates that the enzyme is a novel candidate in the study of behavioural and neurodevelopmental disorders. The authors declare no conflict of interest." ]

[ "Microtubule severing enzymes implement a diverse range of tissue-specific molecular functions throughout development and into adulthood. Although microtubule severing is fundamental to many dynamic neural processes, little is known regarding the role of the family member Katanin p60 subunit A-like 1, KATNAL1, in central nervous system CNS function. Recent studies reporting that microdeletions incorporating the KATNAL1 locus in humans result in intellectual disability and microcephaly suggest that KATNAL1 may play a prominent role in the CNS; however, such associations lack the functional data required to highlight potential mechanisms which link the gene to disease symptoms. Here we identify and characterise a mouse line carrying a loss of function allele in Katnal1. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology.", "We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. Furthermore we demonstrate defects in the motile cilia of the ventricular ependymal cells of mutants, suggesting a role for Katnal1 in the development of ciliary function. We believe the data we present here are the first to associate KATNAL1 with such phenotypes, demonstrating that the protein plays keys roles in a number of processes integral to the development of neuronal function and behaviour. Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development.", "Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, and mutations in Fign cause a range of phenotypes in mice. 7 Currently the microtubule severing enzyme KATNAL1 is poorly characterised and it is not yet understood how the enzyme functions in the nervous system. Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus.", "Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus. 11, 12 Although such association studies strongly suggest that KATNAL1 plays a fundamental role in the central nervous system CNS , additional studies using cellular or animals models are required to understand how the gene may be causative for disease. Here we present the first study describing neural and behavioural deficits associated with a loss of function allele of Katnal1 in the mouse. This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits.", "This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits. Underlying these behavioural phenotypes, we identified histopathological abnormalities in the brains of Katnal1 1H/1H mutants, including disordered cellular layers in the hippocampus and cortex and substantially larger ventricles. Further investigations demonstrated that Katnal1 1H/1H mice show neuronal migration and ciliary function deficits suggesting KATNAL1 plays an essential role in these processes. These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background.", "These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background. Circadian wheel running was performed as previously described. 14 Sleep assessment by electroencephalography and electromyography Electroencephalography and electromyography was performed as previously described. 15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice.", "15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice. The mouse was confined in the goal arm for 30 s, before being allowed a second free choice of goal arm. An alternation was recorded if the second choice differed from that of the first. One trial was performed per day for 10 days. Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min.", "Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min. Animals were monitored by EthoVision XT analysis software Noldus, Wageningen, Netherlands . Video tracking in the home cage. Activity in the home cage was recorded by video tracking as previously described. 16 Morris water maze and ultrasonic vocalisation. These tests were performed as previously described. 17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols.", "17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols. In vivo neuronal migration assessment was performed as previously described 18 using embryos at either E13 or E15 three mothers per age group and pups at P9. Cell counts were performed using ImageJ NIH, Bethesda, MD, USA . In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm.", "In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm. Segmentation, volume calculation and 3D modelling was performed using ITK-SNAP version 3.0.0 ref. 20 and 3DSlicer. 21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ.", "21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ. Brains from P2 mice were dissected, and the dorsal cerebral half was sectioned 250 μm through the floor of the lateral and 3rd ventricle using a vibratome. Ciliary beat frequency and pattern was analysed as previously described. 22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies .", "22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies . and visualised using a JEOL LSM-6010 scanning electron microscope Jeol, Herts, UK . Transmission electron microscopy was performed as previously described. 22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05.", "22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05. All graphs are presented showing mean ± s.e.m. Additional and more detailed methods can be found in supplementary information. Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened .", "Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened . An outcross using an affected female produced no affected animals 33 animals screened . In subsequent intercross screens 15.5% of animals were affected 53 out of 342 animals screened , suggesting that the pedigree carries a mutation causing a recessive circadian phenotype which is 60% penetrant. We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree.", "We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree. 23 Genome-wide SNP linkage analysis mapped the circadian and sterility phenotypes to the same region on chromosome 5 and subsequent sequencing identified the causative mutation as a T to G single point mutation within exon seven of the Katnal1 gene. For full details of mapping and identification of the mutation see reference 23. This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele.", "This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele. 23 3D modelling of the protein suggests that this loss of function is due to hydrophobic changes in the AAA domain of the enzyme Supplementary Figure S1 . Genotyping confirmed that the mutation was homozygous in affected circadian animals and wild type or heterozygous in unaffected animals, confirming that Katnal1 1H was causative for the circadian phenotype. Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 .", "Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 . Bioluminescence recordings performed using PER2::LUCIFERASE reporter mice carrying the Katnal1 1H mutation revealed that these circadian changes were not due to changes to the core molecular clock of the suprachiasmatic nucleus the site of the master circadian clock in the brain; Supplementary Figure S2 . Circadian disruptions are often associated with deficits in sleep homeostasis. Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1.", "Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1. Compared to wildtype littermates, the non-REM delta power of Katnal1 1H/1H mice was higher in the dark phase of baseline sleep mixed ANOVA, interaction factors 'genotype X time, F 1,88 = 8.91, P = 0.0175 Figure 1f and in both the light and dark phases of recovery sleep mixed ANOVA, interaction factors 'genotype X time', F 1,136 = 11.93, P = 0.0086; Figure 1g . All other sleep parameters were unaffected in Katnal1 1H/1H animals. Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests.", "Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests. Data and cohort details are given in Supplementary Table S2 . Both working memory and spatial memory were significantly poorer in Katnal1 1H/1H mice, as evidenced by reduced spontaneous alternations in a T-maze Figure 2a and in the Morris water maze where mutants take longer to find the platform in acquisition trials Figure 2b Compared to wild-type littermates, Katnal1 1H/1H animals have a shorter period c , are more active in the light phase of the light/dark cycle d and show an earlier onset of activity in light/dark transitions and in the transition from light/dark cycles to constant darkness e . In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001.", "In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001. EEG, electroencephalography; DD, constant darkness; LD, light/dark cycle. type = 164 ± 12 m, Katnal1 1H/1H = 243 ± 20 m, P = 0.02; distance travelled in periphery of open field: wild type = 4.3 ± 0.2 m, Katnal1 1H/1H = 6 ± 0.3 m, P = 0.004 . Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs .", "Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs . To test whether Katnal1 1H/1H animals vocalised differently to wild types, we separated pups at postnatal days 7-8 the age at which mice show peak of USV emission 24 and recorded their USVs. In these tests, compared to wild types, Katnal1 1H/1H pups produced fewer Figure 2g and shorter Figure 2h vocalisations, containing fewer phrases Figure 2i . Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 .", "Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 . Analysis of hematoxylin and eosin stained brain sections revealed that, compared to wildtype littermates, Katnal1 1H/1H animals had less tightly packed pyramidal cell layers in the hippocampus Figures 3a and b and a narrower cortical layer 1 and wider cortical layer 6 Figures 3c-e . To confirm these cortical layer differences, immunofluorescence was performed using the Figures 3l and m . Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n .", "Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n . Finally, three dimensional models of the ventricular system were constructed from brain micro-computed tomography scans Figures 3o and p . Volumetric analysis revealed that Katnal1 1H/1H mice had substantially larger ventricles than wild types Figure 3q . Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 .", "Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 . At both ages Katnal1 1H/1H animals had greater numbers of labelled neurons in bins close to the cortical surface neurons positioned closer to the cortical surface compared to wild type. To confirm these results we used a Boyden chamber 19 and performed in vitro neuronal migration analysis in E13.5 primary cortical neuronal cultures. Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration.", "Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration. Given its role in cytoskeletal organisation, we also hypothesised that neuronal morphology is modulated by Katnal1. Analysis of golgi stained neurons from layers 2-3 of the cortex Figures 4g and i demonstrated that, compared to wild-type littermates, Katnal1 1H/1H neurons had larger soma Figure 4k , and shorter and thinner axons Figures 4l and m data and cohort details are given in Supplementary Table S3 . Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia.", "Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia. 5 Since such ciliary defects could underlie the phenotypes described above we studied the motile cilia of the ependymal lining of the lateral ventricle in sections of postnatal day 2 mouse brains from both Katnal1 1H/1H n = 4 and wild-type animals n = 3 . We found that the ciliary beat frequency CBF of Katnal1 1H/1H animals was significantly attenuated compared to wild-type CBF: wildtype = 22.39 ± 0.94 Hz, Katnal1 1H/1H = 14.25 ± 0.92 Hz, P = 0.0001; Figure 5a , Supplementary Movies S1 . This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles.", "This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles. These abnormalities were observed in approximately 25% of Katnal1 1H/1H brain slices. The abnormal cilia always showed a dyskinetic beat pattern and lower beat frequency. To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 .", "To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 . However in Katnal1 1H/1H samples we noted the presence of both long and short cilia Figures 5e and f ; defined as two standard deviations longer or shorter than the average cilia length that were not present in wild-type samples. In addition, inspection of Katnal1 1H/1H cilia identified ciliary abnormalities including bifurcated cilia Figure 5g , abnormal kinks and bends in the cilia Figure 5h and swellings along the length of the cilia Figure 5i . Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia.", "Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia. Microtubule severing enzymes play diverse roles in the nervous system. 1, 2 However, at present the microtubule severing enzyme Katnal1 is poorly defined in the context of CNS development and function. Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes.", "Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes. Finally we also demonstrate that Katnal1 1H causes a range of defects in the motile cilia of ventricular ependymal cells. The data we present here are the first to associate KATNAL1 with such dysfunctions with important implications for clinical association studies. The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation.", "The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation. Phenotypes in circadian running wheel rhythms that are not associated with changes to the core clock mechanism have also been reported in mouse models of schizophrenia. 25 Here it has been suggested that the wheel running changes observed are the result in defects in output pathways from the SCN circadian clock. Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed.", "Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed. The behavioural consequences of microtubule severing enzyme dysfunction in mouse models have been poorly characterised. Currently the phenotypes described are limited to motor dysfunction in mice lacking the Spg4 gene 27 and head shaking and circling in the Fign mutant. 7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1.", "7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1. 8, 9 While it is also worth noting that mutant mice spend more time the centre of the open field than wild types implying that Katnal1 1H/1H animals show reduced anxiety , we suggest that this result is confounded by the hyperactivity in novel environments phenotype we also describe in mutant mice. This observation is backed up by the fact that mutant animals showed increased activity in all regions of the open field rather than just the anxiolytic periphery. Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions.", "Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions. For example, defects in neuronal migration, synaptic spines and neuronal morphology such as those we have demonstrated here, have been suggested to underpin ID in conditions such as lissencephaly, 18 Down's syndrome 31 and Rett syndrome. 32 While we are not suggesting that Katnal1 is causative for these conditions, similarities in symptoms and neuronal phenotypes between these conditions and those linked to Katnal1 dysfunction should be appreciated. Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice.", "Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice. Thus the range of phenotypes associated with defects in the function of Katnal1 strongly suggests that the gene should be considered in the pathology of disorders such as ID and schizophrenia. We do note one key genetic difference between the human patients and Katnal1 1H/1H animals. While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1.", "While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1. 17 While heterozygous loss of function mutations in CTNNB1 are causative for intellectual disability in humans, conditional knock outs for CTNNB1 have no reported behavioural or craniofacial phenotypes. 36, 37 These differences demonstrate that while mouse models of intellectual disability are of great use in our understanding of the causative mechanisms which underlie the condition, there are still genetic and neurodevelopmental differences between species which also must be taken into account. We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons.", "We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons. The data we present here also demonstrate defects in motile cilia in Katnal1 1H/1H mice. Ciliary disruptions in humans ciliopathies include Bardet-Biedl and Joubert syndrome. 38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations.", "38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations. Furthermore in Bardet-Biedl syndrome mouse models ciliary defects such as reduced CBF 45 and structural defects such as abnormal lengthening and swellings along their length 41 have been described, that are similar to those we describe in Katnal1 1H/1H mice. There is strong evidence that ciliopathy associated genes play a number of roles in neuronal development by affecting processes such as progenitor proliferation or maintenance of the radial glia scaffold. 43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice.", "43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice. Further investigations are required to clarify the impacts of these two processes. However it is notable that while defects in cilia structure may contribute to the phenotypes we describe in Katnal1 1H/1H mice, they are far less prominent in Katnal1 1H/1H mice than in other mouse ciliopathy models, 41 suggesting that the ciliary component of KATNAL1 dysfunction may be mild compared to other ciliopathies. Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function.", "Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function. The downstream effect of these defects leads in turn to a number of behavioural changes including in learning and memory, reaction to anxiogenic situations and circadian rhythms. These data therefore highlight how perturbations in KATNAL1 may play a role in neuronal dysfunction and demonstrates that the enzyme is a novel candidate in the study of behavioural and neurodevelopmental disorders. The authors declare no conflict of interest." ]

[ "Microtubule severing enzymes implement a diverse range of tissue-specific molecular functions throughout development and into adulthood. Although microtubule severing is fundamental to many dynamic neural processes, little is known regarding the role of the family member Katanin p60 subunit A-like 1, KATNAL1, in central nervous system CNS function. Recent studies reporting that microdeletions incorporating the KATNAL1 locus in humans result in intellectual disability and microcephaly suggest that KATNAL1 may play a prominent role in the CNS; however, such associations lack the functional data required to highlight potential mechanisms which link the gene to disease symptoms. Here we identify and characterise a mouse line carrying a loss of function allele in Katnal1. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology.", "We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. Furthermore we demonstrate defects in the motile cilia of the ventricular ependymal cells of mutants, suggesting a role for Katnal1 in the development of ciliary function. We believe the data we present here are the first to associate KATNAL1 with such phenotypes, demonstrating that the protein plays keys roles in a number of processes integral to the development of neuronal function and behaviour. Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development.", "Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, and mutations in Fign cause a range of phenotypes in mice. 7 Currently the microtubule severing enzyme KATNAL1 is poorly characterised and it is not yet understood how the enzyme functions in the nervous system. Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus.", "Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus. 11, 12 Although such association studies strongly suggest that KATNAL1 plays a fundamental role in the central nervous system CNS , additional studies using cellular or animals models are required to understand how the gene may be causative for disease. Here we present the first study describing neural and behavioural deficits associated with a loss of function allele of Katnal1 in the mouse. This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits.", "This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits. Underlying these behavioural phenotypes, we identified histopathological abnormalities in the brains of Katnal1 1H/1H mutants, including disordered cellular layers in the hippocampus and cortex and substantially larger ventricles. Further investigations demonstrated that Katnal1 1H/1H mice show neuronal migration and ciliary function deficits suggesting KATNAL1 plays an essential role in these processes. These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background.", "These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background. Circadian wheel running was performed as previously described. 14 Sleep assessment by electroencephalography and electromyography Electroencephalography and electromyography was performed as previously described. 15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice.", "15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice. The mouse was confined in the goal arm for 30 s, before being allowed a second free choice of goal arm. An alternation was recorded if the second choice differed from that of the first. One trial was performed per day for 10 days. Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min.", "Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min. Animals were monitored by EthoVision XT analysis software Noldus, Wageningen, Netherlands . Video tracking in the home cage. Activity in the home cage was recorded by video tracking as previously described. 16 Morris water maze and ultrasonic vocalisation. These tests were performed as previously described. 17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols.", "17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols. In vivo neuronal migration assessment was performed as previously described 18 using embryos at either E13 or E15 three mothers per age group and pups at P9. Cell counts were performed using ImageJ NIH, Bethesda, MD, USA . In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm.", "In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm. Segmentation, volume calculation and 3D modelling was performed using ITK-SNAP version 3.0.0 ref. 20 and 3DSlicer. 21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ.", "21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ. Brains from P2 mice were dissected, and the dorsal cerebral half was sectioned 250 μm through the floor of the lateral and 3rd ventricle using a vibratome. Ciliary beat frequency and pattern was analysed as previously described. 22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies .", "22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies . and visualised using a JEOL LSM-6010 scanning electron microscope Jeol, Herts, UK . Transmission electron microscopy was performed as previously described. 22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05.", "22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05. All graphs are presented showing mean ± s.e.m. Additional and more detailed methods can be found in supplementary information. Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened .", "Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened . An outcross using an affected female produced no affected animals 33 animals screened . In subsequent intercross screens 15.5% of animals were affected 53 out of 342 animals screened , suggesting that the pedigree carries a mutation causing a recessive circadian phenotype which is 60% penetrant. We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree.", "We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree. 23 Genome-wide SNP linkage analysis mapped the circadian and sterility phenotypes to the same region on chromosome 5 and subsequent sequencing identified the causative mutation as a T to G single point mutation within exon seven of the Katnal1 gene. For full details of mapping and identification of the mutation see reference 23. This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele.", "This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele. 23 3D modelling of the protein suggests that this loss of function is due to hydrophobic changes in the AAA domain of the enzyme Supplementary Figure S1 . Genotyping confirmed that the mutation was homozygous in affected circadian animals and wild type or heterozygous in unaffected animals, confirming that Katnal1 1H was causative for the circadian phenotype. Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 .", "Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 . Bioluminescence recordings performed using PER2::LUCIFERASE reporter mice carrying the Katnal1 1H mutation revealed that these circadian changes were not due to changes to the core molecular clock of the suprachiasmatic nucleus the site of the master circadian clock in the brain; Supplementary Figure S2 . Circadian disruptions are often associated with deficits in sleep homeostasis. Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1.", "Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1. Compared to wildtype littermates, the non-REM delta power of Katnal1 1H/1H mice was higher in the dark phase of baseline sleep mixed ANOVA, interaction factors 'genotype X time, F 1,88 = 8.91, P = 0.0175 Figure 1f and in both the light and dark phases of recovery sleep mixed ANOVA, interaction factors 'genotype X time', F 1,136 = 11.93, P = 0.0086; Figure 1g . All other sleep parameters were unaffected in Katnal1 1H/1H animals. Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests.", "Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests. Data and cohort details are given in Supplementary Table S2 . Both working memory and spatial memory were significantly poorer in Katnal1 1H/1H mice, as evidenced by reduced spontaneous alternations in a T-maze Figure 2a and in the Morris water maze where mutants take longer to find the platform in acquisition trials Figure 2b Compared to wild-type littermates, Katnal1 1H/1H animals have a shorter period c , are more active in the light phase of the light/dark cycle d and show an earlier onset of activity in light/dark transitions and in the transition from light/dark cycles to constant darkness e . In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001.", "In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001. EEG, electroencephalography; DD, constant darkness; LD, light/dark cycle. type = 164 ± 12 m, Katnal1 1H/1H = 243 ± 20 m, P = 0.02; distance travelled in periphery of open field: wild type = 4.3 ± 0.2 m, Katnal1 1H/1H = 6 ± 0.3 m, P = 0.004 . Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs .", "Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs . To test whether Katnal1 1H/1H animals vocalised differently to wild types, we separated pups at postnatal days 7-8 the age at which mice show peak of USV emission 24 and recorded their USVs. In these tests, compared to wild types, Katnal1 1H/1H pups produced fewer Figure 2g and shorter Figure 2h vocalisations, containing fewer phrases Figure 2i . Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 .", "Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 . Analysis of hematoxylin and eosin stained brain sections revealed that, compared to wildtype littermates, Katnal1 1H/1H animals had less tightly packed pyramidal cell layers in the hippocampus Figures 3a and b and a narrower cortical layer 1 and wider cortical layer 6 Figures 3c-e . To confirm these cortical layer differences, immunofluorescence was performed using the Figures 3l and m . Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n .", "Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n . Finally, three dimensional models of the ventricular system were constructed from brain micro-computed tomography scans Figures 3o and p . Volumetric analysis revealed that Katnal1 1H/1H mice had substantially larger ventricles than wild types Figure 3q . Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 .", "Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 . At both ages Katnal1 1H/1H animals had greater numbers of labelled neurons in bins close to the cortical surface neurons positioned closer to the cortical surface compared to wild type. To confirm these results we used a Boyden chamber 19 and performed in vitro neuronal migration analysis in E13.5 primary cortical neuronal cultures. Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration.", "Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration. Given its role in cytoskeletal organisation, we also hypothesised that neuronal morphology is modulated by Katnal1. Analysis of golgi stained neurons from layers 2-3 of the cortex Figures 4g and i demonstrated that, compared to wild-type littermates, Katnal1 1H/1H neurons had larger soma Figure 4k , and shorter and thinner axons Figures 4l and m data and cohort details are given in Supplementary Table S3 . Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia.", "Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia. 5 Since such ciliary defects could underlie the phenotypes described above we studied the motile cilia of the ependymal lining of the lateral ventricle in sections of postnatal day 2 mouse brains from both Katnal1 1H/1H n = 4 and wild-type animals n = 3 . We found that the ciliary beat frequency CBF of Katnal1 1H/1H animals was significantly attenuated compared to wild-type CBF: wildtype = 22.39 ± 0.94 Hz, Katnal1 1H/1H = 14.25 ± 0.92 Hz, P = 0.0001; Figure 5a , Supplementary Movies S1 . This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles.", "This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles. These abnormalities were observed in approximately 25% of Katnal1 1H/1H brain slices. The abnormal cilia always showed a dyskinetic beat pattern and lower beat frequency. To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 .", "To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 . However in Katnal1 1H/1H samples we noted the presence of both long and short cilia Figures 5e and f ; defined as two standard deviations longer or shorter than the average cilia length that were not present in wild-type samples. In addition, inspection of Katnal1 1H/1H cilia identified ciliary abnormalities including bifurcated cilia Figure 5g , abnormal kinks and bends in the cilia Figure 5h and swellings along the length of the cilia Figure 5i . Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia.", "Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia. Microtubule severing enzymes play diverse roles in the nervous system. 1, 2 However, at present the microtubule severing enzyme Katnal1 is poorly defined in the context of CNS development and function. Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes.", "Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes. Finally we also demonstrate that Katnal1 1H causes a range of defects in the motile cilia of ventricular ependymal cells. The data we present here are the first to associate KATNAL1 with such dysfunctions with important implications for clinical association studies. The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation.", "The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation. Phenotypes in circadian running wheel rhythms that are not associated with changes to the core clock mechanism have also been reported in mouse models of schizophrenia. 25 Here it has been suggested that the wheel running changes observed are the result in defects in output pathways from the SCN circadian clock. Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed.", "Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed. The behavioural consequences of microtubule severing enzyme dysfunction in mouse models have been poorly characterised. Currently the phenotypes described are limited to motor dysfunction in mice lacking the Spg4 gene 27 and head shaking and circling in the Fign mutant. 7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1.", "7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1. 8, 9 While it is also worth noting that mutant mice spend more time the centre of the open field than wild types implying that Katnal1 1H/1H animals show reduced anxiety , we suggest that this result is confounded by the hyperactivity in novel environments phenotype we also describe in mutant mice. This observation is backed up by the fact that mutant animals showed increased activity in all regions of the open field rather than just the anxiolytic periphery. Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions.", "Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions. For example, defects in neuronal migration, synaptic spines and neuronal morphology such as those we have demonstrated here, have been suggested to underpin ID in conditions such as lissencephaly, 18 Down's syndrome 31 and Rett syndrome. 32 While we are not suggesting that Katnal1 is causative for these conditions, similarities in symptoms and neuronal phenotypes between these conditions and those linked to Katnal1 dysfunction should be appreciated. Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice.", "Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice. Thus the range of phenotypes associated with defects in the function of Katnal1 strongly suggests that the gene should be considered in the pathology of disorders such as ID and schizophrenia. We do note one key genetic difference between the human patients and Katnal1 1H/1H animals. While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1.", "While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1. 17 While heterozygous loss of function mutations in CTNNB1 are causative for intellectual disability in humans, conditional knock outs for CTNNB1 have no reported behavioural or craniofacial phenotypes. 36, 37 These differences demonstrate that while mouse models of intellectual disability are of great use in our understanding of the causative mechanisms which underlie the condition, there are still genetic and neurodevelopmental differences between species which also must be taken into account. We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons.", "We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons. The data we present here also demonstrate defects in motile cilia in Katnal1 1H/1H mice. Ciliary disruptions in humans ciliopathies include Bardet-Biedl and Joubert syndrome. 38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations.", "38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations. Furthermore in Bardet-Biedl syndrome mouse models ciliary defects such as reduced CBF 45 and structural defects such as abnormal lengthening and swellings along their length 41 have been described, that are similar to those we describe in Katnal1 1H/1H mice. There is strong evidence that ciliopathy associated genes play a number of roles in neuronal development by affecting processes such as progenitor proliferation or maintenance of the radial glia scaffold. 43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice.", "43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice. Further investigations are required to clarify the impacts of these two processes. However it is notable that while defects in cilia structure may contribute to the phenotypes we describe in Katnal1 1H/1H mice, they are far less prominent in Katnal1 1H/1H mice than in other mouse ciliopathy models, 41 suggesting that the ciliary component of KATNAL1 dysfunction may be mild compared to other ciliopathies. Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function.", "Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function. The downstream effect of these defects leads in turn to a number of behavioural changes including in learning and memory, reaction to anxiogenic situations and circadian rhythms. These data therefore highlight how perturbations in KATNAL1 may play a role in neuronal dysfunction and demonstrates that the enzyme is a novel candidate in the study of behavioural and neurodevelopmental disorders. The authors declare no conflict of interest." ]

[ "Microtubule severing enzymes implement a diverse range of tissue-specific molecular functions throughout development and into adulthood. Although microtubule severing is fundamental to many dynamic neural processes, little is known regarding the role of the family member Katanin p60 subunit A-like 1, KATNAL1, in central nervous system CNS function. Recent studies reporting that microdeletions incorporating the KATNAL1 locus in humans result in intellectual disability and microcephaly suggest that KATNAL1 may play a prominent role in the CNS; however, such associations lack the functional data required to highlight potential mechanisms which link the gene to disease symptoms. Here we identify and characterise a mouse line carrying a loss of function allele in Katnal1. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology.", "We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. Furthermore we demonstrate defects in the motile cilia of the ventricular ependymal cells of mutants, suggesting a role for Katnal1 in the development of ciliary function. We believe the data we present here are the first to associate KATNAL1 with such phenotypes, demonstrating that the protein plays keys roles in a number of processes integral to the development of neuronal function and behaviour. Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development.", "Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, and mutations in Fign cause a range of phenotypes in mice. 7 Currently the microtubule severing enzyme KATNAL1 is poorly characterised and it is not yet understood how the enzyme functions in the nervous system. Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus.", "Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus. 11, 12 Although such association studies strongly suggest that KATNAL1 plays a fundamental role in the central nervous system CNS , additional studies using cellular or animals models are required to understand how the gene may be causative for disease. Here we present the first study describing neural and behavioural deficits associated with a loss of function allele of Katnal1 in the mouse. This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits.", "This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits. Underlying these behavioural phenotypes, we identified histopathological abnormalities in the brains of Katnal1 1H/1H mutants, including disordered cellular layers in the hippocampus and cortex and substantially larger ventricles. Further investigations demonstrated that Katnal1 1H/1H mice show neuronal migration and ciliary function deficits suggesting KATNAL1 plays an essential role in these processes. These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background.", "These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background. Circadian wheel running was performed as previously described. 14 Sleep assessment by electroencephalography and electromyography Electroencephalography and electromyography was performed as previously described. 15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice.", "15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice. The mouse was confined in the goal arm for 30 s, before being allowed a second free choice of goal arm. An alternation was recorded if the second choice differed from that of the first. One trial was performed per day for 10 days. Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min.", "Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min. Animals were monitored by EthoVision XT analysis software Noldus, Wageningen, Netherlands . Video tracking in the home cage. Activity in the home cage was recorded by video tracking as previously described. 16 Morris water maze and ultrasonic vocalisation. These tests were performed as previously described. 17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols.", "17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols. In vivo neuronal migration assessment was performed as previously described 18 using embryos at either E13 or E15 three mothers per age group and pups at P9. Cell counts were performed using ImageJ NIH, Bethesda, MD, USA . In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm.", "In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm. Segmentation, volume calculation and 3D modelling was performed using ITK-SNAP version 3.0.0 ref. 20 and 3DSlicer. 21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ.", "21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ. Brains from P2 mice were dissected, and the dorsal cerebral half was sectioned 250 μm through the floor of the lateral and 3rd ventricle using a vibratome. Ciliary beat frequency and pattern was analysed as previously described. 22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies .", "22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies . and visualised using a JEOL LSM-6010 scanning electron microscope Jeol, Herts, UK . Transmission electron microscopy was performed as previously described. 22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05.", "22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05. All graphs are presented showing mean ± s.e.m. Additional and more detailed methods can be found in supplementary information. Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened .", "Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened . An outcross using an affected female produced no affected animals 33 animals screened . In subsequent intercross screens 15.5% of animals were affected 53 out of 342 animals screened , suggesting that the pedigree carries a mutation causing a recessive circadian phenotype which is 60% penetrant. We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree.", "We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree. 23 Genome-wide SNP linkage analysis mapped the circadian and sterility phenotypes to the same region on chromosome 5 and subsequent sequencing identified the causative mutation as a T to G single point mutation within exon seven of the Katnal1 gene. For full details of mapping and identification of the mutation see reference 23. This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele.", "This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele. 23 3D modelling of the protein suggests that this loss of function is due to hydrophobic changes in the AAA domain of the enzyme Supplementary Figure S1 . Genotyping confirmed that the mutation was homozygous in affected circadian animals and wild type or heterozygous in unaffected animals, confirming that Katnal1 1H was causative for the circadian phenotype. Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 .", "Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 . Bioluminescence recordings performed using PER2::LUCIFERASE reporter mice carrying the Katnal1 1H mutation revealed that these circadian changes were not due to changes to the core molecular clock of the suprachiasmatic nucleus the site of the master circadian clock in the brain; Supplementary Figure S2 . Circadian disruptions are often associated with deficits in sleep homeostasis. Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1.", "Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1. Compared to wildtype littermates, the non-REM delta power of Katnal1 1H/1H mice was higher in the dark phase of baseline sleep mixed ANOVA, interaction factors 'genotype X time, F 1,88 = 8.91, P = 0.0175 Figure 1f and in both the light and dark phases of recovery sleep mixed ANOVA, interaction factors 'genotype X time', F 1,136 = 11.93, P = 0.0086; Figure 1g . All other sleep parameters were unaffected in Katnal1 1H/1H animals. Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests.", "Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests. Data and cohort details are given in Supplementary Table S2 . Both working memory and spatial memory were significantly poorer in Katnal1 1H/1H mice, as evidenced by reduced spontaneous alternations in a T-maze Figure 2a and in the Morris water maze where mutants take longer to find the platform in acquisition trials Figure 2b Compared to wild-type littermates, Katnal1 1H/1H animals have a shorter period c , are more active in the light phase of the light/dark cycle d and show an earlier onset of activity in light/dark transitions and in the transition from light/dark cycles to constant darkness e . In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001.", "In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001. EEG, electroencephalography; DD, constant darkness; LD, light/dark cycle. type = 164 ± 12 m, Katnal1 1H/1H = 243 ± 20 m, P = 0.02; distance travelled in periphery of open field: wild type = 4.3 ± 0.2 m, Katnal1 1H/1H = 6 ± 0.3 m, P = 0.004 . Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs .", "Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs . To test whether Katnal1 1H/1H animals vocalised differently to wild types, we separated pups at postnatal days 7-8 the age at which mice show peak of USV emission 24 and recorded their USVs. In these tests, compared to wild types, Katnal1 1H/1H pups produced fewer Figure 2g and shorter Figure 2h vocalisations, containing fewer phrases Figure 2i . Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 .", "Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 . Analysis of hematoxylin and eosin stained brain sections revealed that, compared to wildtype littermates, Katnal1 1H/1H animals had less tightly packed pyramidal cell layers in the hippocampus Figures 3a and b and a narrower cortical layer 1 and wider cortical layer 6 Figures 3c-e . To confirm these cortical layer differences, immunofluorescence was performed using the Figures 3l and m . Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n .", "Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n . Finally, three dimensional models of the ventricular system were constructed from brain micro-computed tomography scans Figures 3o and p . Volumetric analysis revealed that Katnal1 1H/1H mice had substantially larger ventricles than wild types Figure 3q . Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 .", "Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 . At both ages Katnal1 1H/1H animals had greater numbers of labelled neurons in bins close to the cortical surface neurons positioned closer to the cortical surface compared to wild type. To confirm these results we used a Boyden chamber 19 and performed in vitro neuronal migration analysis in E13.5 primary cortical neuronal cultures. Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration.", "Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration. Given its role in cytoskeletal organisation, we also hypothesised that neuronal morphology is modulated by Katnal1. Analysis of golgi stained neurons from layers 2-3 of the cortex Figures 4g and i demonstrated that, compared to wild-type littermates, Katnal1 1H/1H neurons had larger soma Figure 4k , and shorter and thinner axons Figures 4l and m data and cohort details are given in Supplementary Table S3 . Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia.", "Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia. 5 Since such ciliary defects could underlie the phenotypes described above we studied the motile cilia of the ependymal lining of the lateral ventricle in sections of postnatal day 2 mouse brains from both Katnal1 1H/1H n = 4 and wild-type animals n = 3 . We found that the ciliary beat frequency CBF of Katnal1 1H/1H animals was significantly attenuated compared to wild-type CBF: wildtype = 22.39 ± 0.94 Hz, Katnal1 1H/1H = 14.25 ± 0.92 Hz, P = 0.0001; Figure 5a , Supplementary Movies S1 . This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles.", "This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles. These abnormalities were observed in approximately 25% of Katnal1 1H/1H brain slices. The abnormal cilia always showed a dyskinetic beat pattern and lower beat frequency. To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 .", "To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 . However in Katnal1 1H/1H samples we noted the presence of both long and short cilia Figures 5e and f ; defined as two standard deviations longer or shorter than the average cilia length that were not present in wild-type samples. In addition, inspection of Katnal1 1H/1H cilia identified ciliary abnormalities including bifurcated cilia Figure 5g , abnormal kinks and bends in the cilia Figure 5h and swellings along the length of the cilia Figure 5i . Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia.", "Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia. Microtubule severing enzymes play diverse roles in the nervous system. 1, 2 However, at present the microtubule severing enzyme Katnal1 is poorly defined in the context of CNS development and function. Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes.", "Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes. Finally we also demonstrate that Katnal1 1H causes a range of defects in the motile cilia of ventricular ependymal cells. The data we present here are the first to associate KATNAL1 with such dysfunctions with important implications for clinical association studies. The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation.", "The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation. Phenotypes in circadian running wheel rhythms that are not associated with changes to the core clock mechanism have also been reported in mouse models of schizophrenia. 25 Here it has been suggested that the wheel running changes observed are the result in defects in output pathways from the SCN circadian clock. Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed.", "Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed. The behavioural consequences of microtubule severing enzyme dysfunction in mouse models have been poorly characterised. Currently the phenotypes described are limited to motor dysfunction in mice lacking the Spg4 gene 27 and head shaking and circling in the Fign mutant. 7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1.", "7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1. 8, 9 While it is also worth noting that mutant mice spend more time the centre of the open field than wild types implying that Katnal1 1H/1H animals show reduced anxiety , we suggest that this result is confounded by the hyperactivity in novel environments phenotype we also describe in mutant mice. This observation is backed up by the fact that mutant animals showed increased activity in all regions of the open field rather than just the anxiolytic periphery. Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions.", "Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions. For example, defects in neuronal migration, synaptic spines and neuronal morphology such as those we have demonstrated here, have been suggested to underpin ID in conditions such as lissencephaly, 18 Down's syndrome 31 and Rett syndrome. 32 While we are not suggesting that Katnal1 is causative for these conditions, similarities in symptoms and neuronal phenotypes between these conditions and those linked to Katnal1 dysfunction should be appreciated. Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice.", "Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice. Thus the range of phenotypes associated with defects in the function of Katnal1 strongly suggests that the gene should be considered in the pathology of disorders such as ID and schizophrenia. We do note one key genetic difference between the human patients and Katnal1 1H/1H animals. While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1.", "While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1. 17 While heterozygous loss of function mutations in CTNNB1 are causative for intellectual disability in humans, conditional knock outs for CTNNB1 have no reported behavioural or craniofacial phenotypes. 36, 37 These differences demonstrate that while mouse models of intellectual disability are of great use in our understanding of the causative mechanisms which underlie the condition, there are still genetic and neurodevelopmental differences between species which also must be taken into account. We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons.", "We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons. The data we present here also demonstrate defects in motile cilia in Katnal1 1H/1H mice. Ciliary disruptions in humans ciliopathies include Bardet-Biedl and Joubert syndrome. 38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations.", "38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations. Furthermore in Bardet-Biedl syndrome mouse models ciliary defects such as reduced CBF 45 and structural defects such as abnormal lengthening and swellings along their length 41 have been described, that are similar to those we describe in Katnal1 1H/1H mice. There is strong evidence that ciliopathy associated genes play a number of roles in neuronal development by affecting processes such as progenitor proliferation or maintenance of the radial glia scaffold. 43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice.", "43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice. Further investigations are required to clarify the impacts of these two processes. However it is notable that while defects in cilia structure may contribute to the phenotypes we describe in Katnal1 1H/1H mice, they are far less prominent in Katnal1 1H/1H mice than in other mouse ciliopathy models, 41 suggesting that the ciliary component of KATNAL1 dysfunction may be mild compared to other ciliopathies. Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function.", "Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function. The downstream effect of these defects leads in turn to a number of behavioural changes including in learning and memory, reaction to anxiogenic situations and circadian rhythms. These data therefore highlight how perturbations in KATNAL1 may play a role in neuronal dysfunction and demonstrates that the enzyme is a novel candidate in the study of behavioural and neurodevelopmental disorders. The authors declare no conflict of interest." ]

[ "Microtubule severing enzymes implement a diverse range of tissue-specific molecular functions throughout development and into adulthood. Although microtubule severing is fundamental to many dynamic neural processes, little is known regarding the role of the family member Katanin p60 subunit A-like 1, KATNAL1, in central nervous system CNS function. Recent studies reporting that microdeletions incorporating the KATNAL1 locus in humans result in intellectual disability and microcephaly suggest that KATNAL1 may play a prominent role in the CNS; however, such associations lack the functional data required to highlight potential mechanisms which link the gene to disease symptoms. Here we identify and characterise a mouse line carrying a loss of function allele in Katnal1. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology.", "We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. Furthermore we demonstrate defects in the motile cilia of the ventricular ependymal cells of mutants, suggesting a role for Katnal1 in the development of ciliary function. We believe the data we present here are the first to associate KATNAL1 with such phenotypes, demonstrating that the protein plays keys roles in a number of processes integral to the development of neuronal function and behaviour. Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development.", "Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, and mutations in Fign cause a range of phenotypes in mice. 7 Currently the microtubule severing enzyme KATNAL1 is poorly characterised and it is not yet understood how the enzyme functions in the nervous system. Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus.", "Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus. 11, 12 Although such association studies strongly suggest that KATNAL1 plays a fundamental role in the central nervous system CNS , additional studies using cellular or animals models are required to understand how the gene may be causative for disease. Here we present the first study describing neural and behavioural deficits associated with a loss of function allele of Katnal1 in the mouse. This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits.", "This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits. Underlying these behavioural phenotypes, we identified histopathological abnormalities in the brains of Katnal1 1H/1H mutants, including disordered cellular layers in the hippocampus and cortex and substantially larger ventricles. Further investigations demonstrated that Katnal1 1H/1H mice show neuronal migration and ciliary function deficits suggesting KATNAL1 plays an essential role in these processes. These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background.", "These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background. Circadian wheel running was performed as previously described. 14 Sleep assessment by electroencephalography and electromyography Electroencephalography and electromyography was performed as previously described. 15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice.", "15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice. The mouse was confined in the goal arm for 30 s, before being allowed a second free choice of goal arm. An alternation was recorded if the second choice differed from that of the first. One trial was performed per day for 10 days. Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min.", "Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min. Animals were monitored by EthoVision XT analysis software Noldus, Wageningen, Netherlands . Video tracking in the home cage. Activity in the home cage was recorded by video tracking as previously described. 16 Morris water maze and ultrasonic vocalisation. These tests were performed as previously described. 17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols.", "17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols. In vivo neuronal migration assessment was performed as previously described 18 using embryos at either E13 or E15 three mothers per age group and pups at P9. Cell counts were performed using ImageJ NIH, Bethesda, MD, USA . In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm.", "In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm. Segmentation, volume calculation and 3D modelling was performed using ITK-SNAP version 3.0.0 ref. 20 and 3DSlicer. 21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ.", "21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ. Brains from P2 mice were dissected, and the dorsal cerebral half was sectioned 250 μm through the floor of the lateral and 3rd ventricle using a vibratome. Ciliary beat frequency and pattern was analysed as previously described. 22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies .", "22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies . and visualised using a JEOL LSM-6010 scanning electron microscope Jeol, Herts, UK . Transmission electron microscopy was performed as previously described. 22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05.", "22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05. All graphs are presented showing mean ± s.e.m. Additional and more detailed methods can be found in supplementary information. Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened .", "Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened . An outcross using an affected female produced no affected animals 33 animals screened . In subsequent intercross screens 15.5% of animals were affected 53 out of 342 animals screened , suggesting that the pedigree carries a mutation causing a recessive circadian phenotype which is 60% penetrant. We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree.", "We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree. 23 Genome-wide SNP linkage analysis mapped the circadian and sterility phenotypes to the same region on chromosome 5 and subsequent sequencing identified the causative mutation as a T to G single point mutation within exon seven of the Katnal1 gene. For full details of mapping and identification of the mutation see reference 23. This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele.", "This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele. 23 3D modelling of the protein suggests that this loss of function is due to hydrophobic changes in the AAA domain of the enzyme Supplementary Figure S1 . Genotyping confirmed that the mutation was homozygous in affected circadian animals and wild type or heterozygous in unaffected animals, confirming that Katnal1 1H was causative for the circadian phenotype. Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 .", "Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 . Bioluminescence recordings performed using PER2::LUCIFERASE reporter mice carrying the Katnal1 1H mutation revealed that these circadian changes were not due to changes to the core molecular clock of the suprachiasmatic nucleus the site of the master circadian clock in the brain; Supplementary Figure S2 . Circadian disruptions are often associated with deficits in sleep homeostasis. Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1.", "Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1. Compared to wildtype littermates, the non-REM delta power of Katnal1 1H/1H mice was higher in the dark phase of baseline sleep mixed ANOVA, interaction factors 'genotype X time, F 1,88 = 8.91, P = 0.0175 Figure 1f and in both the light and dark phases of recovery sleep mixed ANOVA, interaction factors 'genotype X time', F 1,136 = 11.93, P = 0.0086; Figure 1g . All other sleep parameters were unaffected in Katnal1 1H/1H animals. Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests.", "Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests. Data and cohort details are given in Supplementary Table S2 . Both working memory and spatial memory were significantly poorer in Katnal1 1H/1H mice, as evidenced by reduced spontaneous alternations in a T-maze Figure 2a and in the Morris water maze where mutants take longer to find the platform in acquisition trials Figure 2b Compared to wild-type littermates, Katnal1 1H/1H animals have a shorter period c , are more active in the light phase of the light/dark cycle d and show an earlier onset of activity in light/dark transitions and in the transition from light/dark cycles to constant darkness e . In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001.", "In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001. EEG, electroencephalography; DD, constant darkness; LD, light/dark cycle. type = 164 ± 12 m, Katnal1 1H/1H = 243 ± 20 m, P = 0.02; distance travelled in periphery of open field: wild type = 4.3 ± 0.2 m, Katnal1 1H/1H = 6 ± 0.3 m, P = 0.004 . Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs .", "Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs . To test whether Katnal1 1H/1H animals vocalised differently to wild types, we separated pups at postnatal days 7-8 the age at which mice show peak of USV emission 24 and recorded their USVs. In these tests, compared to wild types, Katnal1 1H/1H pups produced fewer Figure 2g and shorter Figure 2h vocalisations, containing fewer phrases Figure 2i . Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 .", "Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 . Analysis of hematoxylin and eosin stained brain sections revealed that, compared to wildtype littermates, Katnal1 1H/1H animals had less tightly packed pyramidal cell layers in the hippocampus Figures 3a and b and a narrower cortical layer 1 and wider cortical layer 6 Figures 3c-e . To confirm these cortical layer differences, immunofluorescence was performed using the Figures 3l and m . Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n .", "Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n . Finally, three dimensional models of the ventricular system were constructed from brain micro-computed tomography scans Figures 3o and p . Volumetric analysis revealed that Katnal1 1H/1H mice had substantially larger ventricles than wild types Figure 3q . Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 .", "Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 . At both ages Katnal1 1H/1H animals had greater numbers of labelled neurons in bins close to the cortical surface neurons positioned closer to the cortical surface compared to wild type. To confirm these results we used a Boyden chamber 19 and performed in vitro neuronal migration analysis in E13.5 primary cortical neuronal cultures. Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration.", "Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration. Given its role in cytoskeletal organisation, we also hypothesised that neuronal morphology is modulated by Katnal1. Analysis of golgi stained neurons from layers 2-3 of the cortex Figures 4g and i demonstrated that, compared to wild-type littermates, Katnal1 1H/1H neurons had larger soma Figure 4k , and shorter and thinner axons Figures 4l and m data and cohort details are given in Supplementary Table S3 . Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia.", "Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia. 5 Since such ciliary defects could underlie the phenotypes described above we studied the motile cilia of the ependymal lining of the lateral ventricle in sections of postnatal day 2 mouse brains from both Katnal1 1H/1H n = 4 and wild-type animals n = 3 . We found that the ciliary beat frequency CBF of Katnal1 1H/1H animals was significantly attenuated compared to wild-type CBF: wildtype = 22.39 ± 0.94 Hz, Katnal1 1H/1H = 14.25 ± 0.92 Hz, P = 0.0001; Figure 5a , Supplementary Movies S1 . This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles.", "This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles. These abnormalities were observed in approximately 25% of Katnal1 1H/1H brain slices. The abnormal cilia always showed a dyskinetic beat pattern and lower beat frequency. To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 .", "To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 . However in Katnal1 1H/1H samples we noted the presence of both long and short cilia Figures 5e and f ; defined as two standard deviations longer or shorter than the average cilia length that were not present in wild-type samples. In addition, inspection of Katnal1 1H/1H cilia identified ciliary abnormalities including bifurcated cilia Figure 5g , abnormal kinks and bends in the cilia Figure 5h and swellings along the length of the cilia Figure 5i . Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia.", "Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia. Microtubule severing enzymes play diverse roles in the nervous system. 1, 2 However, at present the microtubule severing enzyme Katnal1 is poorly defined in the context of CNS development and function. Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes.", "Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes. Finally we also demonstrate that Katnal1 1H causes a range of defects in the motile cilia of ventricular ependymal cells. The data we present here are the first to associate KATNAL1 with such dysfunctions with important implications for clinical association studies. The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation.", "The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation. Phenotypes in circadian running wheel rhythms that are not associated with changes to the core clock mechanism have also been reported in mouse models of schizophrenia. 25 Here it has been suggested that the wheel running changes observed are the result in defects in output pathways from the SCN circadian clock. Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed.", "Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed. The behavioural consequences of microtubule severing enzyme dysfunction in mouse models have been poorly characterised. Currently the phenotypes described are limited to motor dysfunction in mice lacking the Spg4 gene 27 and head shaking and circling in the Fign mutant. 7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1.", "7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1. 8, 9 While it is also worth noting that mutant mice spend more time the centre of the open field than wild types implying that Katnal1 1H/1H animals show reduced anxiety , we suggest that this result is confounded by the hyperactivity in novel environments phenotype we also describe in mutant mice. This observation is backed up by the fact that mutant animals showed increased activity in all regions of the open field rather than just the anxiolytic periphery. Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions.", "Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions. For example, defects in neuronal migration, synaptic spines and neuronal morphology such as those we have demonstrated here, have been suggested to underpin ID in conditions such as lissencephaly, 18 Down's syndrome 31 and Rett syndrome. 32 While we are not suggesting that Katnal1 is causative for these conditions, similarities in symptoms and neuronal phenotypes between these conditions and those linked to Katnal1 dysfunction should be appreciated. Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice.", "Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice. Thus the range of phenotypes associated with defects in the function of Katnal1 strongly suggests that the gene should be considered in the pathology of disorders such as ID and schizophrenia. We do note one key genetic difference between the human patients and Katnal1 1H/1H animals. While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1.", "While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1. 17 While heterozygous loss of function mutations in CTNNB1 are causative for intellectual disability in humans, conditional knock outs for CTNNB1 have no reported behavioural or craniofacial phenotypes. 36, 37 These differences demonstrate that while mouse models of intellectual disability are of great use in our understanding of the causative mechanisms which underlie the condition, there are still genetic and neurodevelopmental differences between species which also must be taken into account. We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons.", "We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons. The data we present here also demonstrate defects in motile cilia in Katnal1 1H/1H mice. Ciliary disruptions in humans ciliopathies include Bardet-Biedl and Joubert syndrome. 38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations.", "38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations. Furthermore in Bardet-Biedl syndrome mouse models ciliary defects such as reduced CBF 45 and structural defects such as abnormal lengthening and swellings along their length 41 have been described, that are similar to those we describe in Katnal1 1H/1H mice. There is strong evidence that ciliopathy associated genes play a number of roles in neuronal development by affecting processes such as progenitor proliferation or maintenance of the radial glia scaffold. 43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice.", "43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice. Further investigations are required to clarify the impacts of these two processes. However it is notable that while defects in cilia structure may contribute to the phenotypes we describe in Katnal1 1H/1H mice, they are far less prominent in Katnal1 1H/1H mice than in other mouse ciliopathy models, 41 suggesting that the ciliary component of KATNAL1 dysfunction may be mild compared to other ciliopathies. Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function.", "Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function. The downstream effect of these defects leads in turn to a number of behavioural changes including in learning and memory, reaction to anxiogenic situations and circadian rhythms. These data therefore highlight how perturbations in KATNAL1 may play a role in neuronal dysfunction and demonstrates that the enzyme is a novel candidate in the study of behavioural and neurodevelopmental disorders. The authors declare no conflict of interest." ]

[ "Microtubule severing enzymes implement a diverse range of tissue-specific molecular functions throughout development and into adulthood. Although microtubule severing is fundamental to many dynamic neural processes, little is known regarding the role of the family member Katanin p60 subunit A-like 1, KATNAL1, in central nervous system CNS function. Recent studies reporting that microdeletions incorporating the KATNAL1 locus in humans result in intellectual disability and microcephaly suggest that KATNAL1 may play a prominent role in the CNS; however, such associations lack the functional data required to highlight potential mechanisms which link the gene to disease symptoms. Here we identify and characterise a mouse line carrying a loss of function allele in Katnal1. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology.", "We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. Furthermore we demonstrate defects in the motile cilia of the ventricular ependymal cells of mutants, suggesting a role for Katnal1 in the development of ciliary function. We believe the data we present here are the first to associate KATNAL1 with such phenotypes, demonstrating that the protein plays keys roles in a number of processes integral to the development of neuronal function and behaviour. Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development.", "Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, and mutations in Fign cause a range of phenotypes in mice. 7 Currently the microtubule severing enzyme KATNAL1 is poorly characterised and it is not yet understood how the enzyme functions in the nervous system. Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus.", "Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus. 11, 12 Although such association studies strongly suggest that KATNAL1 plays a fundamental role in the central nervous system CNS , additional studies using cellular or animals models are required to understand how the gene may be causative for disease. Here we present the first study describing neural and behavioural deficits associated with a loss of function allele of Katnal1 in the mouse. This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits.", "This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits. Underlying these behavioural phenotypes, we identified histopathological abnormalities in the brains of Katnal1 1H/1H mutants, including disordered cellular layers in the hippocampus and cortex and substantially larger ventricles. Further investigations demonstrated that Katnal1 1H/1H mice show neuronal migration and ciliary function deficits suggesting KATNAL1 plays an essential role in these processes. These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background.", "These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background. Circadian wheel running was performed as previously described. 14 Sleep assessment by electroencephalography and electromyography Electroencephalography and electromyography was performed as previously described. 15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice.", "15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice. The mouse was confined in the goal arm for 30 s, before being allowed a second free choice of goal arm. An alternation was recorded if the second choice differed from that of the first. One trial was performed per day for 10 days. Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min.", "Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min. Animals were monitored by EthoVision XT analysis software Noldus, Wageningen, Netherlands . Video tracking in the home cage. Activity in the home cage was recorded by video tracking as previously described. 16 Morris water maze and ultrasonic vocalisation. These tests were performed as previously described. 17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols.", "17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols. In vivo neuronal migration assessment was performed as previously described 18 using embryos at either E13 or E15 three mothers per age group and pups at P9. Cell counts were performed using ImageJ NIH, Bethesda, MD, USA . In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm.", "In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm. Segmentation, volume calculation and 3D modelling was performed using ITK-SNAP version 3.0.0 ref. 20 and 3DSlicer. 21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ.", "21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ. Brains from P2 mice were dissected, and the dorsal cerebral half was sectioned 250 μm through the floor of the lateral and 3rd ventricle using a vibratome. Ciliary beat frequency and pattern was analysed as previously described. 22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies .", "22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies . and visualised using a JEOL LSM-6010 scanning electron microscope Jeol, Herts, UK . Transmission electron microscopy was performed as previously described. 22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05.", "22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05. All graphs are presented showing mean ± s.e.m. Additional and more detailed methods can be found in supplementary information. Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened .", "Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened . An outcross using an affected female produced no affected animals 33 animals screened . In subsequent intercross screens 15.5% of animals were affected 53 out of 342 animals screened , suggesting that the pedigree carries a mutation causing a recessive circadian phenotype which is 60% penetrant. We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree.", "We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree. 23 Genome-wide SNP linkage analysis mapped the circadian and sterility phenotypes to the same region on chromosome 5 and subsequent sequencing identified the causative mutation as a T to G single point mutation within exon seven of the Katnal1 gene. For full details of mapping and identification of the mutation see reference 23. This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele.", "This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele. 23 3D modelling of the protein suggests that this loss of function is due to hydrophobic changes in the AAA domain of the enzyme Supplementary Figure S1 . Genotyping confirmed that the mutation was homozygous in affected circadian animals and wild type or heterozygous in unaffected animals, confirming that Katnal1 1H was causative for the circadian phenotype. Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 .", "Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 . Bioluminescence recordings performed using PER2::LUCIFERASE reporter mice carrying the Katnal1 1H mutation revealed that these circadian changes were not due to changes to the core molecular clock of the suprachiasmatic nucleus the site of the master circadian clock in the brain; Supplementary Figure S2 . Circadian disruptions are often associated with deficits in sleep homeostasis. Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1.", "Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1. Compared to wildtype littermates, the non-REM delta power of Katnal1 1H/1H mice was higher in the dark phase of baseline sleep mixed ANOVA, interaction factors 'genotype X time, F 1,88 = 8.91, P = 0.0175 Figure 1f and in both the light and dark phases of recovery sleep mixed ANOVA, interaction factors 'genotype X time', F 1,136 = 11.93, P = 0.0086; Figure 1g . All other sleep parameters were unaffected in Katnal1 1H/1H animals. Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests.", "Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests. Data and cohort details are given in Supplementary Table S2 . Both working memory and spatial memory were significantly poorer in Katnal1 1H/1H mice, as evidenced by reduced spontaneous alternations in a T-maze Figure 2a and in the Morris water maze where mutants take longer to find the platform in acquisition trials Figure 2b Compared to wild-type littermates, Katnal1 1H/1H animals have a shorter period c , are more active in the light phase of the light/dark cycle d and show an earlier onset of activity in light/dark transitions and in the transition from light/dark cycles to constant darkness e . In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001.", "In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001. EEG, electroencephalography; DD, constant darkness; LD, light/dark cycle. type = 164 ± 12 m, Katnal1 1H/1H = 243 ± 20 m, P = 0.02; distance travelled in periphery of open field: wild type = 4.3 ± 0.2 m, Katnal1 1H/1H = 6 ± 0.3 m, P = 0.004 . Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs .", "Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs . To test whether Katnal1 1H/1H animals vocalised differently to wild types, we separated pups at postnatal days 7-8 the age at which mice show peak of USV emission 24 and recorded their USVs. In these tests, compared to wild types, Katnal1 1H/1H pups produced fewer Figure 2g and shorter Figure 2h vocalisations, containing fewer phrases Figure 2i . Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 .", "Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 . Analysis of hematoxylin and eosin stained brain sections revealed that, compared to wildtype littermates, Katnal1 1H/1H animals had less tightly packed pyramidal cell layers in the hippocampus Figures 3a and b and a narrower cortical layer 1 and wider cortical layer 6 Figures 3c-e . To confirm these cortical layer differences, immunofluorescence was performed using the Figures 3l and m . Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n .", "Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n . Finally, three dimensional models of the ventricular system were constructed from brain micro-computed tomography scans Figures 3o and p . Volumetric analysis revealed that Katnal1 1H/1H mice had substantially larger ventricles than wild types Figure 3q . Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 .", "Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 . At both ages Katnal1 1H/1H animals had greater numbers of labelled neurons in bins close to the cortical surface neurons positioned closer to the cortical surface compared to wild type. To confirm these results we used a Boyden chamber 19 and performed in vitro neuronal migration analysis in E13.5 primary cortical neuronal cultures. Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration.", "Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration. Given its role in cytoskeletal organisation, we also hypothesised that neuronal morphology is modulated by Katnal1. Analysis of golgi stained neurons from layers 2-3 of the cortex Figures 4g and i demonstrated that, compared to wild-type littermates, Katnal1 1H/1H neurons had larger soma Figure 4k , and shorter and thinner axons Figures 4l and m data and cohort details are given in Supplementary Table S3 . Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia.", "Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia. 5 Since such ciliary defects could underlie the phenotypes described above we studied the motile cilia of the ependymal lining of the lateral ventricle in sections of postnatal day 2 mouse brains from both Katnal1 1H/1H n = 4 and wild-type animals n = 3 . We found that the ciliary beat frequency CBF of Katnal1 1H/1H animals was significantly attenuated compared to wild-type CBF: wildtype = 22.39 ± 0.94 Hz, Katnal1 1H/1H = 14.25 ± 0.92 Hz, P = 0.0001; Figure 5a , Supplementary Movies S1 . This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles.", "This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles. These abnormalities were observed in approximately 25% of Katnal1 1H/1H brain slices. The abnormal cilia always showed a dyskinetic beat pattern and lower beat frequency. To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 .", "To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 . However in Katnal1 1H/1H samples we noted the presence of both long and short cilia Figures 5e and f ; defined as two standard deviations longer or shorter than the average cilia length that were not present in wild-type samples. In addition, inspection of Katnal1 1H/1H cilia identified ciliary abnormalities including bifurcated cilia Figure 5g , abnormal kinks and bends in the cilia Figure 5h and swellings along the length of the cilia Figure 5i . Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia.", "Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia. Microtubule severing enzymes play diverse roles in the nervous system. 1, 2 However, at present the microtubule severing enzyme Katnal1 is poorly defined in the context of CNS development and function. Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes.", "Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes. Finally we also demonstrate that Katnal1 1H causes a range of defects in the motile cilia of ventricular ependymal cells. The data we present here are the first to associate KATNAL1 with such dysfunctions with important implications for clinical association studies. The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation.", "The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation. Phenotypes in circadian running wheel rhythms that are not associated with changes to the core clock mechanism have also been reported in mouse models of schizophrenia. 25 Here it has been suggested that the wheel running changes observed are the result in defects in output pathways from the SCN circadian clock. Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed.", "Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed. The behavioural consequences of microtubule severing enzyme dysfunction in mouse models have been poorly characterised. Currently the phenotypes described are limited to motor dysfunction in mice lacking the Spg4 gene 27 and head shaking and circling in the Fign mutant. 7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1.", "7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1. 8, 9 While it is also worth noting that mutant mice spend more time the centre of the open field than wild types implying that Katnal1 1H/1H animals show reduced anxiety , we suggest that this result is confounded by the hyperactivity in novel environments phenotype we also describe in mutant mice. This observation is backed up by the fact that mutant animals showed increased activity in all regions of the open field rather than just the anxiolytic periphery. Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions.", "Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions. For example, defects in neuronal migration, synaptic spines and neuronal morphology such as those we have demonstrated here, have been suggested to underpin ID in conditions such as lissencephaly, 18 Down's syndrome 31 and Rett syndrome. 32 While we are not suggesting that Katnal1 is causative for these conditions, similarities in symptoms and neuronal phenotypes between these conditions and those linked to Katnal1 dysfunction should be appreciated. Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice.", "Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice. Thus the range of phenotypes associated with defects in the function of Katnal1 strongly suggests that the gene should be considered in the pathology of disorders such as ID and schizophrenia. We do note one key genetic difference between the human patients and Katnal1 1H/1H animals. While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1.", "While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1. 17 While heterozygous loss of function mutations in CTNNB1 are causative for intellectual disability in humans, conditional knock outs for CTNNB1 have no reported behavioural or craniofacial phenotypes. 36, 37 These differences demonstrate that while mouse models of intellectual disability are of great use in our understanding of the causative mechanisms which underlie the condition, there are still genetic and neurodevelopmental differences between species which also must be taken into account. We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons.", "We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons. The data we present here also demonstrate defects in motile cilia in Katnal1 1H/1H mice. Ciliary disruptions in humans ciliopathies include Bardet-Biedl and Joubert syndrome. 38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations.", "38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations. Furthermore in Bardet-Biedl syndrome mouse models ciliary defects such as reduced CBF 45 and structural defects such as abnormal lengthening and swellings along their length 41 have been described, that are similar to those we describe in Katnal1 1H/1H mice. There is strong evidence that ciliopathy associated genes play a number of roles in neuronal development by affecting processes such as progenitor proliferation or maintenance of the radial glia scaffold. 43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice.", "43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice. Further investigations are required to clarify the impacts of these two processes. However it is notable that while defects in cilia structure may contribute to the phenotypes we describe in Katnal1 1H/1H mice, they are far less prominent in Katnal1 1H/1H mice than in other mouse ciliopathy models, 41 suggesting that the ciliary component of KATNAL1 dysfunction may be mild compared to other ciliopathies. Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function.", "Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function. The downstream effect of these defects leads in turn to a number of behavioural changes including in learning and memory, reaction to anxiogenic situations and circadian rhythms. These data therefore highlight how perturbations in KATNAL1 may play a role in neuronal dysfunction and demonstrates that the enzyme is a novel candidate in the study of behavioural and neurodevelopmental disorders. The authors declare no conflict of interest." ]

[ "Microtubule severing enzymes implement a diverse range of tissue-specific molecular functions throughout development and into adulthood. Although microtubule severing is fundamental to many dynamic neural processes, little is known regarding the role of the family member Katanin p60 subunit A-like 1, KATNAL1, in central nervous system CNS function. Recent studies reporting that microdeletions incorporating the KATNAL1 locus in humans result in intellectual disability and microcephaly suggest that KATNAL1 may play a prominent role in the CNS; however, such associations lack the functional data required to highlight potential mechanisms which link the gene to disease symptoms. Here we identify and characterise a mouse line carrying a loss of function allele in Katnal1. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology.", "We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. Furthermore we demonstrate defects in the motile cilia of the ventricular ependymal cells of mutants, suggesting a role for Katnal1 in the development of ciliary function. We believe the data we present here are the first to associate KATNAL1 with such phenotypes, demonstrating that the protein plays keys roles in a number of processes integral to the development of neuronal function and behaviour. Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development.", "Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, and mutations in Fign cause a range of phenotypes in mice. 7 Currently the microtubule severing enzyme KATNAL1 is poorly characterised and it is not yet understood how the enzyme functions in the nervous system. Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus.", "Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus. 11, 12 Although such association studies strongly suggest that KATNAL1 plays a fundamental role in the central nervous system CNS , additional studies using cellular or animals models are required to understand how the gene may be causative for disease. Here we present the first study describing neural and behavioural deficits associated with a loss of function allele of Katnal1 in the mouse. This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits.", "This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits. Underlying these behavioural phenotypes, we identified histopathological abnormalities in the brains of Katnal1 1H/1H mutants, including disordered cellular layers in the hippocampus and cortex and substantially larger ventricles. Further investigations demonstrated that Katnal1 1H/1H mice show neuronal migration and ciliary function deficits suggesting KATNAL1 plays an essential role in these processes. These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background.", "These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background. Circadian wheel running was performed as previously described. 14 Sleep assessment by electroencephalography and electromyography Electroencephalography and electromyography was performed as previously described. 15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice.", "15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice. The mouse was confined in the goal arm for 30 s, before being allowed a second free choice of goal arm. An alternation was recorded if the second choice differed from that of the first. One trial was performed per day for 10 days. Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min.", "Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min. Animals were monitored by EthoVision XT analysis software Noldus, Wageningen, Netherlands . Video tracking in the home cage. Activity in the home cage was recorded by video tracking as previously described. 16 Morris water maze and ultrasonic vocalisation. These tests were performed as previously described. 17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols.", "17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols. In vivo neuronal migration assessment was performed as previously described 18 using embryos at either E13 or E15 three mothers per age group and pups at P9. Cell counts were performed using ImageJ NIH, Bethesda, MD, USA . In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm.", "In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm. Segmentation, volume calculation and 3D modelling was performed using ITK-SNAP version 3.0.0 ref. 20 and 3DSlicer. 21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ.", "21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ. Brains from P2 mice were dissected, and the dorsal cerebral half was sectioned 250 μm through the floor of the lateral and 3rd ventricle using a vibratome. Ciliary beat frequency and pattern was analysed as previously described. 22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies .", "22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies . and visualised using a JEOL LSM-6010 scanning electron microscope Jeol, Herts, UK . Transmission electron microscopy was performed as previously described. 22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05.", "22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05. All graphs are presented showing mean ± s.e.m. Additional and more detailed methods can be found in supplementary information. Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened .", "Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened . An outcross using an affected female produced no affected animals 33 animals screened . In subsequent intercross screens 15.5% of animals were affected 53 out of 342 animals screened , suggesting that the pedigree carries a mutation causing a recessive circadian phenotype which is 60% penetrant. We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree.", "We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree. 23 Genome-wide SNP linkage analysis mapped the circadian and sterility phenotypes to the same region on chromosome 5 and subsequent sequencing identified the causative mutation as a T to G single point mutation within exon seven of the Katnal1 gene. For full details of mapping and identification of the mutation see reference 23. This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele.", "This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele. 23 3D modelling of the protein suggests that this loss of function is due to hydrophobic changes in the AAA domain of the enzyme Supplementary Figure S1 . Genotyping confirmed that the mutation was homozygous in affected circadian animals and wild type or heterozygous in unaffected animals, confirming that Katnal1 1H was causative for the circadian phenotype. Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 .", "Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 . Bioluminescence recordings performed using PER2::LUCIFERASE reporter mice carrying the Katnal1 1H mutation revealed that these circadian changes were not due to changes to the core molecular clock of the suprachiasmatic nucleus the site of the master circadian clock in the brain; Supplementary Figure S2 . Circadian disruptions are often associated with deficits in sleep homeostasis. Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1.", "Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1. Compared to wildtype littermates, the non-REM delta power of Katnal1 1H/1H mice was higher in the dark phase of baseline sleep mixed ANOVA, interaction factors 'genotype X time, F 1,88 = 8.91, P = 0.0175 Figure 1f and in both the light and dark phases of recovery sleep mixed ANOVA, interaction factors 'genotype X time', F 1,136 = 11.93, P = 0.0086; Figure 1g . All other sleep parameters were unaffected in Katnal1 1H/1H animals. Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests.", "Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests. Data and cohort details are given in Supplementary Table S2 . Both working memory and spatial memory were significantly poorer in Katnal1 1H/1H mice, as evidenced by reduced spontaneous alternations in a T-maze Figure 2a and in the Morris water maze where mutants take longer to find the platform in acquisition trials Figure 2b Compared to wild-type littermates, Katnal1 1H/1H animals have a shorter period c , are more active in the light phase of the light/dark cycle d and show an earlier onset of activity in light/dark transitions and in the transition from light/dark cycles to constant darkness e . In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001.", "In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001. EEG, electroencephalography; DD, constant darkness; LD, light/dark cycle. type = 164 ± 12 m, Katnal1 1H/1H = 243 ± 20 m, P = 0.02; distance travelled in periphery of open field: wild type = 4.3 ± 0.2 m, Katnal1 1H/1H = 6 ± 0.3 m, P = 0.004 . Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs .", "Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs . To test whether Katnal1 1H/1H animals vocalised differently to wild types, we separated pups at postnatal days 7-8 the age at which mice show peak of USV emission 24 and recorded their USVs. In these tests, compared to wild types, Katnal1 1H/1H pups produced fewer Figure 2g and shorter Figure 2h vocalisations, containing fewer phrases Figure 2i . Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 .", "Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 . Analysis of hematoxylin and eosin stained brain sections revealed that, compared to wildtype littermates, Katnal1 1H/1H animals had less tightly packed pyramidal cell layers in the hippocampus Figures 3a and b and a narrower cortical layer 1 and wider cortical layer 6 Figures 3c-e . To confirm these cortical layer differences, immunofluorescence was performed using the Figures 3l and m . Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n .", "Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n . Finally, three dimensional models of the ventricular system were constructed from brain micro-computed tomography scans Figures 3o and p . Volumetric analysis revealed that Katnal1 1H/1H mice had substantially larger ventricles than wild types Figure 3q . Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 .", "Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 . At both ages Katnal1 1H/1H animals had greater numbers of labelled neurons in bins close to the cortical surface neurons positioned closer to the cortical surface compared to wild type. To confirm these results we used a Boyden chamber 19 and performed in vitro neuronal migration analysis in E13.5 primary cortical neuronal cultures. Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration.", "Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration. Given its role in cytoskeletal organisation, we also hypothesised that neuronal morphology is modulated by Katnal1. Analysis of golgi stained neurons from layers 2-3 of the cortex Figures 4g and i demonstrated that, compared to wild-type littermates, Katnal1 1H/1H neurons had larger soma Figure 4k , and shorter and thinner axons Figures 4l and m data and cohort details are given in Supplementary Table S3 . Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia.", "Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia. 5 Since such ciliary defects could underlie the phenotypes described above we studied the motile cilia of the ependymal lining of the lateral ventricle in sections of postnatal day 2 mouse brains from both Katnal1 1H/1H n = 4 and wild-type animals n = 3 . We found that the ciliary beat frequency CBF of Katnal1 1H/1H animals was significantly attenuated compared to wild-type CBF: wildtype = 22.39 ± 0.94 Hz, Katnal1 1H/1H = 14.25 ± 0.92 Hz, P = 0.0001; Figure 5a , Supplementary Movies S1 . This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles.", "This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles. These abnormalities were observed in approximately 25% of Katnal1 1H/1H brain slices. The abnormal cilia always showed a dyskinetic beat pattern and lower beat frequency. To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 .", "To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 . However in Katnal1 1H/1H samples we noted the presence of both long and short cilia Figures 5e and f ; defined as two standard deviations longer or shorter than the average cilia length that were not present in wild-type samples. In addition, inspection of Katnal1 1H/1H cilia identified ciliary abnormalities including bifurcated cilia Figure 5g , abnormal kinks and bends in the cilia Figure 5h and swellings along the length of the cilia Figure 5i . Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia.", "Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia. Microtubule severing enzymes play diverse roles in the nervous system. 1, 2 However, at present the microtubule severing enzyme Katnal1 is poorly defined in the context of CNS development and function. Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes.", "Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes. Finally we also demonstrate that Katnal1 1H causes a range of defects in the motile cilia of ventricular ependymal cells. The data we present here are the first to associate KATNAL1 with such dysfunctions with important implications for clinical association studies. The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation.", "The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation. Phenotypes in circadian running wheel rhythms that are not associated with changes to the core clock mechanism have also been reported in mouse models of schizophrenia. 25 Here it has been suggested that the wheel running changes observed are the result in defects in output pathways from the SCN circadian clock. Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed.", "Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed. The behavioural consequences of microtubule severing enzyme dysfunction in mouse models have been poorly characterised. Currently the phenotypes described are limited to motor dysfunction in mice lacking the Spg4 gene 27 and head shaking and circling in the Fign mutant. 7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1.", "7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1. 8, 9 While it is also worth noting that mutant mice spend more time the centre of the open field than wild types implying that Katnal1 1H/1H animals show reduced anxiety , we suggest that this result is confounded by the hyperactivity in novel environments phenotype we also describe in mutant mice. This observation is backed up by the fact that mutant animals showed increased activity in all regions of the open field rather than just the anxiolytic periphery. Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions.", "Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions. For example, defects in neuronal migration, synaptic spines and neuronal morphology such as those we have demonstrated here, have been suggested to underpin ID in conditions such as lissencephaly, 18 Down's syndrome 31 and Rett syndrome. 32 While we are not suggesting that Katnal1 is causative for these conditions, similarities in symptoms and neuronal phenotypes between these conditions and those linked to Katnal1 dysfunction should be appreciated. Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice.", "Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice. Thus the range of phenotypes associated with defects in the function of Katnal1 strongly suggests that the gene should be considered in the pathology of disorders such as ID and schizophrenia. We do note one key genetic difference between the human patients and Katnal1 1H/1H animals. While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1.", "While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1. 17 While heterozygous loss of function mutations in CTNNB1 are causative for intellectual disability in humans, conditional knock outs for CTNNB1 have no reported behavioural or craniofacial phenotypes. 36, 37 These differences demonstrate that while mouse models of intellectual disability are of great use in our understanding of the causative mechanisms which underlie the condition, there are still genetic and neurodevelopmental differences between species which also must be taken into account. We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons.", "We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons. The data we present here also demonstrate defects in motile cilia in Katnal1 1H/1H mice. Ciliary disruptions in humans ciliopathies include Bardet-Biedl and Joubert syndrome. 38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations.", "38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations. Furthermore in Bardet-Biedl syndrome mouse models ciliary defects such as reduced CBF 45 and structural defects such as abnormal lengthening and swellings along their length 41 have been described, that are similar to those we describe in Katnal1 1H/1H mice. There is strong evidence that ciliopathy associated genes play a number of roles in neuronal development by affecting processes such as progenitor proliferation or maintenance of the radial glia scaffold. 43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice.", "43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice. Further investigations are required to clarify the impacts of these two processes. However it is notable that while defects in cilia structure may contribute to the phenotypes we describe in Katnal1 1H/1H mice, they are far less prominent in Katnal1 1H/1H mice than in other mouse ciliopathy models, 41 suggesting that the ciliary component of KATNAL1 dysfunction may be mild compared to other ciliopathies. Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function.", "Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function. The downstream effect of these defects leads in turn to a number of behavioural changes including in learning and memory, reaction to anxiogenic situations and circadian rhythms. These data therefore highlight how perturbations in KATNAL1 may play a role in neuronal dysfunction and demonstrates that the enzyme is a novel candidate in the study of behavioural and neurodevelopmental disorders. The authors declare no conflict of interest." ]

[ "Microtubule severing enzymes implement a diverse range of tissue-specific molecular functions throughout development and into adulthood. Although microtubule severing is fundamental to many dynamic neural processes, little is known regarding the role of the family member Katanin p60 subunit A-like 1, KATNAL1, in central nervous system CNS function. Recent studies reporting that microdeletions incorporating the KATNAL1 locus in humans result in intellectual disability and microcephaly suggest that KATNAL1 may play a prominent role in the CNS; however, such associations lack the functional data required to highlight potential mechanisms which link the gene to disease symptoms. Here we identify and characterise a mouse line carrying a loss of function allele in Katnal1. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology.", "We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. Furthermore we demonstrate defects in the motile cilia of the ventricular ependymal cells of mutants, suggesting a role for Katnal1 in the development of ciliary function. We believe the data we present here are the first to associate KATNAL1 with such phenotypes, demonstrating that the protein plays keys roles in a number of processes integral to the development of neuronal function and behaviour. Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development.", "Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, and mutations in Fign cause a range of phenotypes in mice. 7 Currently the microtubule severing enzyme KATNAL1 is poorly characterised and it is not yet understood how the enzyme functions in the nervous system. Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus.", "Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus. 11, 12 Although such association studies strongly suggest that KATNAL1 plays a fundamental role in the central nervous system CNS , additional studies using cellular or animals models are required to understand how the gene may be causative for disease. Here we present the first study describing neural and behavioural deficits associated with a loss of function allele of Katnal1 in the mouse. This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits.", "This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits. Underlying these behavioural phenotypes, we identified histopathological abnormalities in the brains of Katnal1 1H/1H mutants, including disordered cellular layers in the hippocampus and cortex and substantially larger ventricles. Further investigations demonstrated that Katnal1 1H/1H mice show neuronal migration and ciliary function deficits suggesting KATNAL1 plays an essential role in these processes. These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background.", "These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background. Circadian wheel running was performed as previously described. 14 Sleep assessment by electroencephalography and electromyography Electroencephalography and electromyography was performed as previously described. 15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice.", "15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice. The mouse was confined in the goal arm for 30 s, before being allowed a second free choice of goal arm. An alternation was recorded if the second choice differed from that of the first. One trial was performed per day for 10 days. Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min.", "Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min. Animals were monitored by EthoVision XT analysis software Noldus, Wageningen, Netherlands . Video tracking in the home cage. Activity in the home cage was recorded by video tracking as previously described. 16 Morris water maze and ultrasonic vocalisation. These tests were performed as previously described. 17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols.", "17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols. In vivo neuronal migration assessment was performed as previously described 18 using embryos at either E13 or E15 three mothers per age group and pups at P9. Cell counts were performed using ImageJ NIH, Bethesda, MD, USA . In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm.", "In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm. Segmentation, volume calculation and 3D modelling was performed using ITK-SNAP version 3.0.0 ref. 20 and 3DSlicer. 21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ.", "21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ. Brains from P2 mice were dissected, and the dorsal cerebral half was sectioned 250 μm through the floor of the lateral and 3rd ventricle using a vibratome. Ciliary beat frequency and pattern was analysed as previously described. 22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies .", "22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies . and visualised using a JEOL LSM-6010 scanning electron microscope Jeol, Herts, UK . Transmission electron microscopy was performed as previously described. 22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05.", "22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05. All graphs are presented showing mean ± s.e.m. Additional and more detailed methods can be found in supplementary information. Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened .", "Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened . An outcross using an affected female produced no affected animals 33 animals screened . In subsequent intercross screens 15.5% of animals were affected 53 out of 342 animals screened , suggesting that the pedigree carries a mutation causing a recessive circadian phenotype which is 60% penetrant. We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree.", "We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree. 23 Genome-wide SNP linkage analysis mapped the circadian and sterility phenotypes to the same region on chromosome 5 and subsequent sequencing identified the causative mutation as a T to G single point mutation within exon seven of the Katnal1 gene. For full details of mapping and identification of the mutation see reference 23. This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele.", "This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele. 23 3D modelling of the protein suggests that this loss of function is due to hydrophobic changes in the AAA domain of the enzyme Supplementary Figure S1 . Genotyping confirmed that the mutation was homozygous in affected circadian animals and wild type or heterozygous in unaffected animals, confirming that Katnal1 1H was causative for the circadian phenotype. Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 .", "Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 . Bioluminescence recordings performed using PER2::LUCIFERASE reporter mice carrying the Katnal1 1H mutation revealed that these circadian changes were not due to changes to the core molecular clock of the suprachiasmatic nucleus the site of the master circadian clock in the brain; Supplementary Figure S2 . Circadian disruptions are often associated with deficits in sleep homeostasis. Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1.", "Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1. Compared to wildtype littermates, the non-REM delta power of Katnal1 1H/1H mice was higher in the dark phase of baseline sleep mixed ANOVA, interaction factors 'genotype X time, F 1,88 = 8.91, P = 0.0175 Figure 1f and in both the light and dark phases of recovery sleep mixed ANOVA, interaction factors 'genotype X time', F 1,136 = 11.93, P = 0.0086; Figure 1g . All other sleep parameters were unaffected in Katnal1 1H/1H animals. Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests.", "Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests. Data and cohort details are given in Supplementary Table S2 . Both working memory and spatial memory were significantly poorer in Katnal1 1H/1H mice, as evidenced by reduced spontaneous alternations in a T-maze Figure 2a and in the Morris water maze where mutants take longer to find the platform in acquisition trials Figure 2b Compared to wild-type littermates, Katnal1 1H/1H animals have a shorter period c , are more active in the light phase of the light/dark cycle d and show an earlier onset of activity in light/dark transitions and in the transition from light/dark cycles to constant darkness e . In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001.", "In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001. EEG, electroencephalography; DD, constant darkness; LD, light/dark cycle. type = 164 ± 12 m, Katnal1 1H/1H = 243 ± 20 m, P = 0.02; distance travelled in periphery of open field: wild type = 4.3 ± 0.2 m, Katnal1 1H/1H = 6 ± 0.3 m, P = 0.004 . Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs .", "Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs . To test whether Katnal1 1H/1H animals vocalised differently to wild types, we separated pups at postnatal days 7-8 the age at which mice show peak of USV emission 24 and recorded their USVs. In these tests, compared to wild types, Katnal1 1H/1H pups produced fewer Figure 2g and shorter Figure 2h vocalisations, containing fewer phrases Figure 2i . Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 .", "Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 . Analysis of hematoxylin and eosin stained brain sections revealed that, compared to wildtype littermates, Katnal1 1H/1H animals had less tightly packed pyramidal cell layers in the hippocampus Figures 3a and b and a narrower cortical layer 1 and wider cortical layer 6 Figures 3c-e . To confirm these cortical layer differences, immunofluorescence was performed using the Figures 3l and m . Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n .", "Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n . Finally, three dimensional models of the ventricular system were constructed from brain micro-computed tomography scans Figures 3o and p . Volumetric analysis revealed that Katnal1 1H/1H mice had substantially larger ventricles than wild types Figure 3q . Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 .", "Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 . At both ages Katnal1 1H/1H animals had greater numbers of labelled neurons in bins close to the cortical surface neurons positioned closer to the cortical surface compared to wild type. To confirm these results we used a Boyden chamber 19 and performed in vitro neuronal migration analysis in E13.5 primary cortical neuronal cultures. Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration.", "Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration. Given its role in cytoskeletal organisation, we also hypothesised that neuronal morphology is modulated by Katnal1. Analysis of golgi stained neurons from layers 2-3 of the cortex Figures 4g and i demonstrated that, compared to wild-type littermates, Katnal1 1H/1H neurons had larger soma Figure 4k , and shorter and thinner axons Figures 4l and m data and cohort details are given in Supplementary Table S3 . Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia.", "Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia. 5 Since such ciliary defects could underlie the phenotypes described above we studied the motile cilia of the ependymal lining of the lateral ventricle in sections of postnatal day 2 mouse brains from both Katnal1 1H/1H n = 4 and wild-type animals n = 3 . We found that the ciliary beat frequency CBF of Katnal1 1H/1H animals was significantly attenuated compared to wild-type CBF: wildtype = 22.39 ± 0.94 Hz, Katnal1 1H/1H = 14.25 ± 0.92 Hz, P = 0.0001; Figure 5a , Supplementary Movies S1 . This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles.", "This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles. These abnormalities were observed in approximately 25% of Katnal1 1H/1H brain slices. The abnormal cilia always showed a dyskinetic beat pattern and lower beat frequency. To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 .", "To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 . However in Katnal1 1H/1H samples we noted the presence of both long and short cilia Figures 5e and f ; defined as two standard deviations longer or shorter than the average cilia length that were not present in wild-type samples. In addition, inspection of Katnal1 1H/1H cilia identified ciliary abnormalities including bifurcated cilia Figure 5g , abnormal kinks and bends in the cilia Figure 5h and swellings along the length of the cilia Figure 5i . Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia.", "Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia. Microtubule severing enzymes play diverse roles in the nervous system. 1, 2 However, at present the microtubule severing enzyme Katnal1 is poorly defined in the context of CNS development and function. Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes.", "Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes. Finally we also demonstrate that Katnal1 1H causes a range of defects in the motile cilia of ventricular ependymal cells. The data we present here are the first to associate KATNAL1 with such dysfunctions with important implications for clinical association studies. The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation.", "The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation. Phenotypes in circadian running wheel rhythms that are not associated with changes to the core clock mechanism have also been reported in mouse models of schizophrenia. 25 Here it has been suggested that the wheel running changes observed are the result in defects in output pathways from the SCN circadian clock. Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed.", "Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed. The behavioural consequences of microtubule severing enzyme dysfunction in mouse models have been poorly characterised. Currently the phenotypes described are limited to motor dysfunction in mice lacking the Spg4 gene 27 and head shaking and circling in the Fign mutant. 7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1.", "7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1. 8, 9 While it is also worth noting that mutant mice spend more time the centre of the open field than wild types implying that Katnal1 1H/1H animals show reduced anxiety , we suggest that this result is confounded by the hyperactivity in novel environments phenotype we also describe in mutant mice. This observation is backed up by the fact that mutant animals showed increased activity in all regions of the open field rather than just the anxiolytic periphery. Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions.", "Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions. For example, defects in neuronal migration, synaptic spines and neuronal morphology such as those we have demonstrated here, have been suggested to underpin ID in conditions such as lissencephaly, 18 Down's syndrome 31 and Rett syndrome. 32 While we are not suggesting that Katnal1 is causative for these conditions, similarities in symptoms and neuronal phenotypes between these conditions and those linked to Katnal1 dysfunction should be appreciated. Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice.", "Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice. Thus the range of phenotypes associated with defects in the function of Katnal1 strongly suggests that the gene should be considered in the pathology of disorders such as ID and schizophrenia. We do note one key genetic difference between the human patients and Katnal1 1H/1H animals. While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1.", "While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1. 17 While heterozygous loss of function mutations in CTNNB1 are causative for intellectual disability in humans, conditional knock outs for CTNNB1 have no reported behavioural or craniofacial phenotypes. 36, 37 These differences demonstrate that while mouse models of intellectual disability are of great use in our understanding of the causative mechanisms which underlie the condition, there are still genetic and neurodevelopmental differences between species which also must be taken into account. We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons.", "We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons. The data we present here also demonstrate defects in motile cilia in Katnal1 1H/1H mice. Ciliary disruptions in humans ciliopathies include Bardet-Biedl and Joubert syndrome. 38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations.", "38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations. Furthermore in Bardet-Biedl syndrome mouse models ciliary defects such as reduced CBF 45 and structural defects such as abnormal lengthening and swellings along their length 41 have been described, that are similar to those we describe in Katnal1 1H/1H mice. There is strong evidence that ciliopathy associated genes play a number of roles in neuronal development by affecting processes such as progenitor proliferation or maintenance of the radial glia scaffold. 43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice.", "43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice. Further investigations are required to clarify the impacts of these two processes. However it is notable that while defects in cilia structure may contribute to the phenotypes we describe in Katnal1 1H/1H mice, they are far less prominent in Katnal1 1H/1H mice than in other mouse ciliopathy models, 41 suggesting that the ciliary component of KATNAL1 dysfunction may be mild compared to other ciliopathies. Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function.", "Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function. The downstream effect of these defects leads in turn to a number of behavioural changes including in learning and memory, reaction to anxiogenic situations and circadian rhythms. These data therefore highlight how perturbations in KATNAL1 may play a role in neuronal dysfunction and demonstrates that the enzyme is a novel candidate in the study of behavioural and neurodevelopmental disorders. The authors declare no conflict of interest." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]