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Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/ SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0 Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen Date: 2014-08-12 DOI: 10.1186/s13054-014-0456-6 License: cc-by Abstract: 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. 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(8) copies in three patients and was 1 × 10(4) 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. 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. 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 [1] [2] [3] [4] [5] . 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 [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . 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) [6] . 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). 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: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) 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. 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. 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 [6] . 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. 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. 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). 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. 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. 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). 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. 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. 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. 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. 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. 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) . 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 following are the main findings of this study. (1) 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. (2) 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. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) 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 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . 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 [6] . 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. 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 [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . 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 [18] . 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. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . 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. 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. 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.

How long did it take for patients with positive human adenovirus type 55 (HAdV-55) endotracheal aspirates to develop a measurable viremia?

Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/ SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0 Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen Date: 2014-08-12 DOI: 10.1186/s13054-014-0456-6 License: cc-by Abstract: 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. 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(8) copies in three patients and was 1 × 10(4) 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. 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. 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 [1] [2] [3] [4] [5] . 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 [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . 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) [6] . 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). 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: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) 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. 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. 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 [6] . 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. 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. 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). 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. 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. 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). 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. 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. 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. 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. 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. 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) . 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 following are the main findings of this study. (1) 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. (2) 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. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) 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 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . 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 [6] . 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. 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 [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . 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 [18] . 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. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . 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. 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. 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.

Does blood type play a role in the severity of human adenovirus type 55 (HAdV-55) infection?

Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/ SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0 Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen Date: 2014-08-12 DOI: 10.1186/s13054-014-0456-6 License: cc-by Abstract: 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. 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(8) copies in three patients and was 1 × 10(4) 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. 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. 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 [1] [2] [3] [4] [5] . 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 [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . 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) [6] . 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). 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: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) 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. 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. 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 [6] . 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. 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. 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). 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. 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. 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). 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. 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. 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. 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. 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. 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) . 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 following are the main findings of this study. (1) 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. (2) 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. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) 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 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . 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 [6] . 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. 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 [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . 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 [18] . 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. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . 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. 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. 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.

What are the most common clinical manifestations of severe human adenovirus type 55 (HAdV-55) induced ARDS?

Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/ SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0 Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen Date: 2014-08-12 DOI: 10.1186/s13054-014-0456-6 License: cc-by Abstract: 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. 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(8) copies in three patients and was 1 × 10(4) 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. 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. 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 [1] [2] [3] [4] [5] . 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 [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . 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) [6] . 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). 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: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) 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. 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. 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 [6] . 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. 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. 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). 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. 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. 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). 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. 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. 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. 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. 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. 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) . 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 following are the main findings of this study. (1) 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. (2) 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. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) 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 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . 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 [6] . 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. 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 [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . 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 [18] . 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. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . 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. 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. 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.

What is the mortality rate of severe ARDS from human adenovirus type 55 (HAdV-55)?

Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/ SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0 Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen Date: 2014-08-12 DOI: 10.1186/s13054-014-0456-6 License: cc-by Abstract: 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. 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(8) copies in three patients and was 1 × 10(4) 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. 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. 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 [1] [2] [3] [4] [5] . 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 [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . 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) [6] . 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). 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: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) 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. 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. 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 [6] . 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. 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. 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). 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. 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. 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). 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. 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. 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. 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. 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. 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) . 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 following are the main findings of this study. (1) 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. (2) 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. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) 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 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . 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 [6] . 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. 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 [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . 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 [18] . 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. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . 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. 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. 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.

What role does T-cell count play in severe human adenovirus type 55 (HAdV-55) infection?

Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/ SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0 Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen Date: 2014-08-12 DOI: 10.1186/s13054-014-0456-6 License: cc-by Abstract: 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. 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(8) copies in three patients and was 1 × 10(4) 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. 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. 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 [1] [2] [3] [4] [5] . 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 [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . 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) [6] . 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). 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: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) 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. 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. 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 [6] . 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. 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. 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). 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. 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. 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). 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. 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. 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. 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. 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. 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) . 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 following are the main findings of this study. (1) 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. (2) 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. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) 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 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . 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 [6] . 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. 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 [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . 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 [18] . 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. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . 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. 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. 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.

How successful are the use of invasive mechanical ventilation (IMV) and non-invasive positive pressure ventilation (NPPV) in the treatment of severe ARDS from human adenovirus type 55 infection?

Diagnostic accuracy of C-reactive protein and procalcitonin in suspected community-acquired pneumonia adults visiting emergency department and having a systematic thoracic CT scan https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608327/ SHA: f3d150545162ff3cc253c235011a02a91ee676cb Authors: Le Bel, Josselin; Hausfater, Pierre; Chenevier-Gobeaux, Camille; Blanc, François-Xavier; Benjoar, Mikhael; Ficko, Cécile; Ray, Patrick; Choquet, Christophe; Duval, Xavier; Claessens, Yann-Erick Date: 2015-10-16 DOI: 10.1186/s13054-015-1083-6 License: cc-by Abstract: 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. 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. 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. 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 (doi:10.1186/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 [1] . 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 [2, 3] . CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms [4, 5] , 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 [6, 7] . Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis [8, 9] ; 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 [10] . 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) [11] . 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 [11, 12] . 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 [13] [14] [15] [16] [17] . 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. Setting ESCAPED was a multicenter, prospective, interventional study, entitled "Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department (ESCAPED)" [11] , 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 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. 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 [18] . Consecutive adults ( [19] . 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. 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. 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 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. 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 [15, 20, 21] . Several cut-off points for PCT were chosen at the level of 0.10 μg/L [18] , 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. 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 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 . 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. 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. 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) . 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. 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 [21, 22] . 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 [19] . 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 [23] . 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 [21] . 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 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 [15] . The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections [14] . However, this issue is still unresolved [24] , with conflicting positions [14, 15, 25, 26] . 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) [15, 27, 28] . 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 [14, 26, 29] . 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. 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. 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 [30] . 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.

How many patients were analyzed in the study?

Diagnostic accuracy of C-reactive protein and procalcitonin in suspected community-acquired pneumonia adults visiting emergency department and having a systematic thoracic CT scan https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608327/ SHA: f3d150545162ff3cc253c235011a02a91ee676cb Authors: Le Bel, Josselin; Hausfater, Pierre; Chenevier-Gobeaux, Camille; Blanc, François-Xavier; Benjoar, Mikhael; Ficko, Cécile; Ray, Patrick; Choquet, Christophe; Duval, Xavier; Claessens, Yann-Erick Date: 2015-10-16 DOI: 10.1186/s13054-015-1083-6 License: cc-by Abstract: 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. 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. 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. 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 (doi:10.1186/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 [1] . 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 [2, 3] . CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms [4, 5] , 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 [6, 7] . Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis [8, 9] ; 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 [10] . 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) [11] . 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 [11, 12] . 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 [13] [14] [15] [16] [17] . 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. Setting ESCAPED was a multicenter, prospective, interventional study, entitled "Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department (ESCAPED)" [11] , 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 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. 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 [18] . Consecutive adults ( [19] . 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. 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. 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 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. 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 [15, 20, 21] . Several cut-off points for PCT were chosen at the level of 0.10 μg/L [18] , 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. 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 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 . 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. 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. 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) . 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. 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 [21, 22] . 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 [19] . 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 [23] . 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 [21] . 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 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 [15] . The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections [14] . However, this issue is still unresolved [24] , with conflicting positions [14, 15, 25, 26] . 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) [15, 27, 28] . 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 [14, 26, 29] . 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. 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. 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 [30] . 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.

How many patients with community-acquired pneumonia are hospitalized each year?

Diagnostic accuracy of C-reactive protein and procalcitonin in suspected community-acquired pneumonia adults visiting emergency department and having a systematic thoracic CT scan https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608327/ SHA: f3d150545162ff3cc253c235011a02a91ee676cb Authors: Le Bel, Josselin; Hausfater, Pierre; Chenevier-Gobeaux, Camille; Blanc, François-Xavier; Benjoar, Mikhael; Ficko, Cécile; Ray, Patrick; Choquet, Christophe; Duval, Xavier; Claessens, Yann-Erick Date: 2015-10-16 DOI: 10.1186/s13054-015-1083-6 License: cc-by Abstract: 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. 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. 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. 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 (doi:10.1186/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 [1] . 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 [2, 3] . CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms [4, 5] , 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 [6, 7] . Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis [8, 9] ; 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 [10] . 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) [11] . 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 [11, 12] . 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 [13] [14] [15] [16] [17] . 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. Setting ESCAPED was a multicenter, prospective, interventional study, entitled "Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department (ESCAPED)" [11] , 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 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. 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 [18] . Consecutive adults ( [19] . 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. 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. 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 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. 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 [15, 20, 21] . Several cut-off points for PCT were chosen at the level of 0.10 μg/L [18] , 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. 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 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 . 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. 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. 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) . 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. 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 [21, 22] . 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 [19] . 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 [23] . 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 [21] . 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 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 [15] . The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections [14] . However, this issue is still unresolved [24] , with conflicting positions [14, 15, 25, 26] . 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) [15, 27, 28] . 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 [14, 26, 29] . 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. 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. 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 [30] . 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.

What chest X-ray findings are typically indicative of community-acquired pneumonia?

Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/ SHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b Authors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier Date: 2011-09-29 DOI: 10.1371/journal.pone.0025738 License: cc-by Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001). Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses. Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities. Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] . In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera. The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias. The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC. The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent. Viral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab. Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers. The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate. The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source). Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant. We estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome. Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs). A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five. Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups (P,0.0001). The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001). To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671). We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001). We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1 details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 . Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40, The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] . We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus. Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii) our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus. Lower infection rates were found in adults and the lowest rates were recorded in the elderly. Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance. Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60 yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011. Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level. Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

When did WHO declare a pandemic of pH1N1/2009v influenza?

Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/ SHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b Authors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier Date: 2011-09-29 DOI: 10.1371/journal.pone.0025738 License: cc-by Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001). Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses. Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities. Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] . In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera. The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias. The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC. The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent. Viral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab. Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers. The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate. The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source). Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant. We estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome. Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs). A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five. Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups (P,0.0001). The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001). To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671). We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001). We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1 details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 . Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40, The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] . We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus. Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii) our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus. Lower infection rates were found in adults and the lowest rates were recorded in the elderly. Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance. Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60 yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011. Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level. Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

What is the classical cutoff value for antibody titers?

Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/ SHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b Authors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier Date: 2011-09-29 DOI: 10.1371/journal.pone.0025738 License: cc-by Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001). Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses. Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities. Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] . In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera. The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias. The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC. The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent. Viral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab. Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers. The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate. The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source). Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant. We estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome. Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs). A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five. Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups (P,0.0001). The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001). To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671). We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001). We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1 details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 . Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40, The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] . We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus. Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii) our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus. Lower infection rates were found in adults and the lowest rates were recorded in the elderly. Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance. Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60 yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011. Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level. Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

What is meant by a protective HIA titer?

Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/ SHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b Authors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier Date: 2011-09-29 DOI: 10.1371/journal.pone.0025738 License: cc-by Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001). Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses. Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities. Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] . In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera. The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias. The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC. The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent. Viral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab. Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers. The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate. The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source). Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant. We estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome. Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs). A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five. Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups (P,0.0001). The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001). To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671). We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001). We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1 details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 . Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40, The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] . We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus. Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii) our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus. Lower infection rates were found in adults and the lowest rates were recorded in the elderly. Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance. Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60 yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011. Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level. Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

What are the results of the study?

Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/ SHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b Authors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier Date: 2011-09-29 DOI: 10.1371/journal.pone.0025738 License: cc-by Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001). Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses. Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities. Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] . In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera. The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias. The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC. The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent. Viral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab. Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers. The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate. The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source). Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant. We estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome. Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs). A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five. Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups (P,0.0001). The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001). To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671). We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001). We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1 details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 . Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40, The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] . We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus. Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii) our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus. Lower infection rates were found in adults and the lowest rates were recorded in the elderly. Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance. Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60 yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011. Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level. Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

What was the interpretation for the crossreactive antibodies?

Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/ SHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b Authors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier Date: 2011-09-29 DOI: 10.1371/journal.pone.0025738 License: cc-by Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001). Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses. Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities. Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] . In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera. The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias. The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC. The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent. Viral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab. Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers. The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate. The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source). Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant. We estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome. Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs). A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five. Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups (P,0.0001). The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001). To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671). We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001). We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1 details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 . Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40, The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] . We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus. Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii) our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus. Lower infection rates were found in adults and the lowest rates were recorded in the elderly. Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance. Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60 yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011. Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level. Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

How long did the pH1N1/2009 viral outbreak last?

High Burden of Non-Influenza Viruses in Influenza-Like Illness in the Early Weeks of H1N1v Epidemic in France https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157400/ SHA: f4c1afe385e9e31eb5678e15a3c280ba97326554 Authors: Schnepf, Nathalie; Resche-Rigon, Matthieu; Chaillon, Antoine; Scemla, Anne; Gras, Guillaume; Semoun, Oren; Taboulet, Pierre; Molina, Jean-Michel; Simon, François; Goudeau, Alain; LeGoff, Jérôme Date: 2011-08-17 DOI: 10.1371/journal.pone.0023514 License: cc-by Abstract: BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were positive for H1N1v. In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians. According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] . As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known. The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations. This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application. In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] . In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v. Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] . Total nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample. Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP), Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA). Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] . Data from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests By using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] . All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown). Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ). Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05). Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS. Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379). Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown). In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) . As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%). Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels. In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] . Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose. Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] . Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] . Viral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients. Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] . Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections. In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.

What were the aims of this study?

High Burden of Non-Influenza Viruses in Influenza-Like Illness in the Early Weeks of H1N1v Epidemic in France https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157400/ SHA: f4c1afe385e9e31eb5678e15a3c280ba97326554 Authors: Schnepf, Nathalie; Resche-Rigon, Matthieu; Chaillon, Antoine; Scemla, Anne; Gras, Guillaume; Semoun, Oren; Taboulet, Pierre; Molina, Jean-Michel; Simon, François; Goudeau, Alain; LeGoff, Jérôme Date: 2011-08-17 DOI: 10.1371/journal.pone.0023514 License: cc-by Abstract: BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were positive for H1N1v. In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians. According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] . As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known. The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations. This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application. In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] . In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v. Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] . Total nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample. Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP), Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA). Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] . Data from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests By using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] . All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown). Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ). Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05). Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS. Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379). Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown). In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) . As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%). Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels. In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] . Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose. Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] . Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] . Viral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients. Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] . Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections. In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.

What network of physicians provides real-time clinical data on the spread of influenza in France?

High Burden of Non-Influenza Viruses in Influenza-Like Illness in the Early Weeks of H1N1v Epidemic in France https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157400/ SHA: f4c1afe385e9e31eb5678e15a3c280ba97326554 Authors: Schnepf, Nathalie; Resche-Rigon, Matthieu; Chaillon, Antoine; Scemla, Anne; Gras, Guillaume; Semoun, Oren; Taboulet, Pierre; Molina, Jean-Michel; Simon, François; Goudeau, Alain; LeGoff, Jérôme Date: 2011-08-17 DOI: 10.1371/journal.pone.0023514 License: cc-by Abstract: BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were positive for H1N1v. In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians. According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] . As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known. The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations. This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application. In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] . In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v. Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] . Total nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample. Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP), Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA). Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] . Data from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests By using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] . All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown). Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ). Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05). Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS. Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379). Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown). In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) . As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%). Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels. In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] . Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose. Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] . Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] . Viral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients. Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] . Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections. In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.

What are the criteria used to define an influenza-like illness in France?

High Burden of Non-Influenza Viruses in Influenza-Like Illness in the Early Weeks of H1N1v Epidemic in France https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157400/ SHA: f4c1afe385e9e31eb5678e15a3c280ba97326554 Authors: Schnepf, Nathalie; Resche-Rigon, Matthieu; Chaillon, Antoine; Scemla, Anne; Gras, Guillaume; Semoun, Oren; Taboulet, Pierre; Molina, Jean-Michel; Simon, François; Goudeau, Alain; LeGoff, Jérôme Date: 2011-08-17 DOI: 10.1371/journal.pone.0023514 License: cc-by Abstract: BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were positive for H1N1v. In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians. According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] . As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known. The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations. This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application. In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] . In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v. Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] . Total nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample. Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP), Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA). Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] . Data from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests By using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] . All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown). Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ). Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05). Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS. Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379). Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown). In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) . As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%). Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels. In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] . Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose. Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] . Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] . Viral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients. Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] . Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections. In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.

What virus was the most common among the H1N1v negative patients?

Livestock Drugs and Disease: The Fatal Combination behind Breeding Failure in Endangered Bearded Vultures https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994777/ SHA: f4f804bac7b32c84ad6572776df684df2a2e5fda Authors: Blanco, Guillermo; Lemus, Jesús A. Date: 2010-11-30 DOI: 10.1371/journal.pone.0014163 License: cc-by Abstract: There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health, especially of threatened avian scavengers feeding upon medicated livestock carcasses. We conducted a comprehensive study of failed eggs and dead nestlings in bearded vultures (Gypaetus barbatus) to attempt to elucidate the proximate causes of breeding failure behind the recent decline in productivity in the Spanish Pyrenees. We found high concentrations of multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and nestlings, associated with multiple internal organ damage and livestock pathogens causing disease, especially septicaemia by swine pathogens and infectious bursal disease. The combined impact of drugs and disease as stochastic factors may result in potentially devastating effects exacerbating an already high risk of extinction and should be considered in current conservation programs for bearded vultures and other scavenger species, especially in regards to dangerous veterinary drugs and highly pathogenic poultry viruses. Text: Environmental pollutants are increasingly documented as a driver of wildlife endangerment due to their roles in organ damage, hormonal disruption and alteration of the immune system [1, 2] . Disease may also facilitate endangerment and extinction at global and local scales, especially when pathogens interact with other drivers such as pollutants [3] . There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health [4] [5] [6] , and even causing declines approaching extinction [7] . These threats may be especially detrimental to wildlife as they increasingly concur and interact as a consequence of the elimination of livestock residues containing veterinary pharmaceuticals and resistant pathogens due to growing intensive livestock operations worldwide [6, 8, 9] . In particular, the ingestion of antimicrobials, primarily fluoroquinolones, has been recently related to immunodepression-mediated acquisition of opportunistic pathogens and disease, as well as to organ damage in nestling vultures [6, 10, 11] . Fluoroquinolone residues have also been found in avian scavenger eggs and are associated with severe alterations in the development of embryo cartilage and bones that could preclude embryo movement and subsequently normal development, pre-hatch position and successful hatching [12] . Therefore, antimicrobials and other drugs may negatively affect embryo and nestling health with potentially devastating consequences on breeding success and conservation of vultures and other threatened avian scavengers. The bearded vulture (Gypaetus barbatus) is one of the most endangered birds in Europe, with a main stronghold in the Pyrenees. Increasing declines in productivity (average number of fledglings raised per territorial pair) have recently been reported in the Spanish Pyrenees associated with habitat saturation processes [13, 14] . Given that bearded vultures may raise only one fledgling per breeding attempt, this productivity decline should be linked to increasing breeding failure when the proportion of territorial pairs that are breeding does not greatly vary with time [15] . The proximate mechanisms by which density can affect productivity have been investigated, including habitat heterogeneity, with progressively poorer territories being used, territory shrinkage and interference with breeders and floaters [13] . However, the proximate causes of breeding failure are poorly known despite the long-term interests in the conservation of this species [16] . To evaluate these causes, the examination of failed eggs and dead nestlings is imperative, including the study of the presence and impact of injury, developmental problems, poor nutritional condition, pollutants, organ damage, pathogens causing disease, etc. in order to determine the most likely cause of breeding failure. Here, we conducted a comprehensive study of failed eggs and dead nestling bearded vultures collected during recent years in the Pyrenees. Both the productivity and survival rates of adults and young birds have reached the lowest values since the bovine spongiform encephalopathy (BSE) crisis [13, 14, 17] . This temporal decline could be related to illegal poisoning [17] and recent changes in the abundance, distribution and quality of carrion available to avian scavengers as a consequence of EU regulations derived from the BSE crisis [6, [18] [19] [20] . In particular, the BSE crisis caused the lack or scarcity of unstabled livestock available to scavengers and their subsequent increase in the consumption of carrion from stabled livestock, which is intensively medicated [21] . Therefore, we specifically focused on determining whether breeding failure in bearded vultures is related to the ingestion of veterinary drugs from stabled livestock carrion, as documented in other avian scavenger species [12] . We also assessed the potential effects of veterinary drugs on embryo damage and immunodepression increasing the probability of acquisition and proliferation of pathogens causing fatal disease [6, [10] [11] [12] 21] . Because veterinary drugs should be exclusively acquired from the ingestion of carrion from livestock medicated to combat disease, we predict that their presence should be associated with that of pathogens acquired from the same livestock, especially poultry pathogens more likely transmitted between avian species [22] . Alternatively, if the temporal decline in productivity was primarily associated with breeding failure due to the effects of habitat saturation processes [13, 17] , we should expect egg and nestling mortality to be directly related to developmental and nutritional problems indicating progressively lower quality territories (e.g. embryo emaciation, nestling starvation) and interference by both conspecifics and heterospecifics (e.g. incubation failure, injury due to predation attempts or disturbance). Failed eggs (n = 5) and dead nestlings (n = 4) were collected from bearded vulture nests located in the Spanish Pyrenees between 2005 and 2008. The study of this material did not require of the approval of an ethics committee because it was collected after breeding failure (egg or nestling death) was confirmed in the field. Three of the specimens (two nestlings and one egg) were collected in 2005, 2007 and 2008 from a particular territory. Eggs and nestlings were collected after breeding failure and frozen. Necropsies were performed on all specimens according to standard protocols [12] . The age of embryos and nestlings were estimated according to size and development. Samples of liver, kidney, spleen, large and small intestines, lungs, brain, lymphoid organs (thymus, bursa of Fabricius, Peyer's patches) and knee joints were fixed in 10% buffered formalin, sectioned at 4 mm and stained for histopathological analysis [10, 12] . Liver (dead nestlings and failed embryos) and yolk (failed embryos) were used for the determination of the presence of veterinary drugs, including fluoroquinolones (enrofloxacin and ciprofloxacin), other antimicrobials (amoxicillin and oxytetracycline), non-steroideal anti-inflamatories (NSAIDs) such as diclofenac, flunixin meglumine, ketoprofen, ibuprofen, meloxicam, sodium salicylate, acetaminophen, and antiparasitics (metronidazole, diclazuril, fenbendazole, ivermectin) as described previously [12] . The limits of quantification, percentage recoveries, and interand intra-assay reproducibility were adequate [10, 12] . Other contaminants potentially affecting eggs and embryos were determined in liver, including heavy metals (Cd, Zn, Pb and Hg), following Blanco et al. [23] , dithiocarbamate thiram, disulfuram, polybrominated diphenyl ethers, organochlorines and brominated flame retardants, following Lemus et al. [12] and carbamate and organophosphate pesticides (carbofuran, aldicarb and fenthion) following Elliot et al. [24] . We measured brain cholinesterase activity to assess early exposure to anticholinesterase pesticides [25] . Potential contamination was assessed by comparison with levels from apparently normal wild birds of other species [26] in the absence of basal levels for bearded vultures. Determination of bacterial and fungal pathogens were conducted by sampling oropharynx, lung, liver, kidney, spleen, and intestine with sterile swabs and cultured using standard microbiology protocols [10, 12, 27, 28] . Salmonella serotypes and phage types were determined in the Spanish Reference Laboratory (Laboratorio Central Veterinario, Algete, Madrid). For confirmation of the identification of the alpha hemolytic Streptococcus pneumoniae we used a specific identification test (Accuprobe, Salem, MA) based on the detection of specific ribosomal RNA sequences. Samples of lesions found in internal organs and tissues during necropsies were taken with sterile swabs and cultured using the same standard microbiology protocols. In addition, we determined the presence of selected avian pathogens, including bacterial, viral, fungal, and protozoan pathogens by means of PCR-based methods (see Table S1 for details). The presence of Chlamyophila psittaci and Mycoplasma sp. in blood was determined as described previously [29, 30] . The presence of poxvirus, the paramyxovirus causing Newcastle disease, the serotypes H5, H7 and H9 of avian influenza, falcon adenovirus, circovirus, herpesvirus, polyomavirus, reovirus and West Nile virus were determined following the PCR-based methods available in the literature [31] [32] [33] [34] [35] [36] [37] [38] [39] . We also searched for helminths and protozoans in the gastrointestinal tract by macroscopic and microscopic observations using standard protocols [40] . Specific immunocytochemical procedures were used for detection of mielodepressive virus, including the alphaherpesvirus causing Marek disease [41] in kidney and bursa of Fabricius, the gyrovirus causing infectious chicken anaemia [42] in thymus and bone marrow, the birnavirus causing infectious bursal disease (IBD, [43] ) in bursa of Fabricius, and the coronavirus causing chicken infectious bronchitis in kidney [44] . In addition, we conducted a specific immunocytochemical procedure for West Nile virus antigen detection [45] in brain, spinal medulla, thymus and thyroid. All immunohistochemistry analyses were conducted at the Department of Veterinary Anatomy, Veterinary Faculty, Universidad Complutense de Madrid, Spain and at the Pathology Department of the Veterinary Faculty, University of Utrecht, The Netherlands. The presence of these viruses was also determined by PCR-based methods [43, [46] [47] [48] . All dead nestlings and three of five unhatched embryos showed two to six different veterinary drugs in liver (nestlings) and egg yolk (embryos). In addition, the two embryos with fluoroquinolones in the yolk also had them in the liver (Table 1) . Fluoroquinolones were the most prevalent drugs and showed the highest concentrations (Table 1) . Other drugs such as NSAIDs and antiparasitics were found in most nestlings at variable concentrations, but in no eggs (Table 1) . Other toxic compounds were detected in lower prevalence and concentrations (see Table 1 for those more relevant values; all insecticides were found at concentrations ,0.001 ppb), which was further supported by basal levels of brain cholinesterase (Table 1) . Dead embryos and nestlings showed a moderate to good nutritional state. Major histopathological lesions were primarily located in the kidney, including glomerulonephritis and/or glomerulonephrosis present in all individuals with fluoroquinolones, but not in those without drugs (Table 1 ). All individuals with fluoroquinolones also showed joint lesions, including arthritis and/ or arthrosis of the long bone articulations, as well as massive osseous stroma of the spongeous bones. The fungi Candida albicans was isolated from the oral cavity of five individuals. All individuals showed non-specific mixedbacterial flora. Enterotoxigenic Escherichia coli and Salmonella spp. were isolated in four cases ( Table 1 (cont.) *Samples from the same territory in different years. 1 Veterinary drugs. EN: enrofloxacin (mg/g), CI: ciprofloxacin (mg/g), OX: oxytetracyclin (mg/g), FL: flunixin meglumine (mg/g), AS: sodium salicylate (ng/g), IV: Ivermectin (mg/g). 2 Other toxicants. OR: organochlorines (ng/g), Pb: lead (ng/g). 12. z29 (three cases, see Table 1 ). One individual showed infection by Salmonella enterica enteritidis (see above) and enterotoxigenic Escherichia coli O86 in all examined organs (septicaemia) except brain, which rejected the possibility of post-mortem contamination. Pasteurella multocida was isolated in a single individual that also showed enterotoxigenic Escherichia coli O86 (Table 1) ; all of these individuals contained fluoroquinolones. One of the failed embryos without veterinary drugs showed suppurative myocarditis, multiple microabscesses in head muscles, suppurative leptomeningitis, as well as lower jaw gangrenous inflammation with loss of the osseous stroma due to a mixed infection with Streptococcus suis and Streptococcus pneumoniae in brain, meninges and neck muscles; this embryo also showed infection by chicken infectious bronchitis ( Table 1 ). Both immunocytochemistry for the detection of poultry viruses and PCR pathogen survey were positive to IBDV in six individuals with fluoroquinolones (Table 1) . Immunocytochemical procedures failed to detect West Nile virus antigens in individuals in which PCR for this virus had been positive. Parasitology was negative for all helminths, helminth eggs and protozoans. We found multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and dead nestling bearded vultures from the Pyrenees. They also showed multiple internal organ damage and pathogens potentially acquired from medicated livestock carrion, especially viruses often infecting poultry. Recorded drug concentrations were among the highest reported in avian scavengers [6, [10] [11] [12] 21] . NSAIDs and antiparasitics were found in lower prevalence than fluoroquinolones, but at higher concentrations than those found in other avian scavengers, especially for flunixin meglumine and sodium salicylate [6, 12, 21] . On the contrary, we found no sterile eggs, poor nutritional conditions or injury in any failed embryo or nestling. Other pollutants were found in low prevalence and concentrations posing low risk to embryo and nestling health. Fluoroquinolones may cause generalized direct developmental damage precluding embryo hatching, physiological alterations due to their impact on liver and kidney and immunodepression reducing resistance to opportunistic pathogens [6, [10] [11] [12] 21] . These pathogens may be acquired at the same time that drugs used to treat diseased livestock are ingested, as indicated by their high prevalence in embryos and nestlings. Therefore, despite the relatively small sample size resulting from low abundance, endangerment and logistic difficulties in reaching nests in this species, the results provide evidence of a combined impact of veterinary drugs and livestock disease as the primary cause of breeding failure in the sampled individuals. The presence of West Nile virus is not likely to be associated with nestling disease or mortality because the lack of lesions in target tissues and viral antigen particles in the immunohistochemistry study. Fatal septicaemia caused by Streptococcus suis, one of the most important swine pathogens worldwide [49] , in combination with septicaemia from Streptococcus pneumoniae and infection by chicken infectious bronchitis virus were found in a single embryo. This concentration of livestock pathogens has not been reported before and, to our knowledge, this is the first report of the three pathogens causing disease in a wild bird. Other pathogens recorded in embryos and nestlings, including Salmonella serotypes and phages typical of livestock [50] , and enterotoxigenic Escherichia coli O86 causing septicaemia, were potentially transmitted by consumption of carcasses of infected poultry and other livestock [22, 27, 28] . In addition, we found that the IBD virus infected most individuals alone or together with other pathogens also potentially acquired from livestock carrion. This virus causes a highly contagious immunosuppressive bursal disease in poultry [51] and may be transmitted to wildlife in contact with poultry waste or by ingestion of carcasses [22, 52] . Nestlings are especially susceptible to IBD because of the primary role of bursa of Fabricius in immune function development at this age. In fact, immunosuppression due to IBD was indicated by the inflammation, necrosis and loss of lymphocytes in the bursa of Fabricius together with the presence of viral antigens recorded by means of immunocytochemical procedures. The potential impact of highly pathogenic and contagious poultry viruses has been previously recognized as a threat to wildlife health due to the increasing contact of wildlife with livestock operations in general, and poultry farms and their residues in particular, in natural areas worldwide [52] [53] [54] [55] . However, damage from IBD virus on the bursa of Fabricius represents, to our knowledge, the first evidence of clinical disease compatible with death caused by this poultry virus in wildlife. The presence of IBD has been not previously recorded in embryos of wild birds, probably because vertical transmission has been ruled out in poultry and, as consequence, it has probably not been evaluated in other species until now. This striking and concerning result could be related to the longer egg development and incubation periods of bearded vultures compared with poultry, and/or due to contrasting environmental conditions during incubation between bearded vultures and poultry. Thus, embryo infection with IBD may occurs via the female or during incubation as a consequence of egg contact between the egg and poultry remains in the nests of bearded vultures, which requires more research. Despite their potential effects on population dynamics and conservation through a reduction of productivity and changes in mating behaviour [13, 14] , habitat saturation processes were apparently not directly related to particular proximate causes of egg and nestling failure in this study or in these sampled individuals. As an alternative non-mutually exclusive explanation, we suggest that the recent decline in productivity could also be linked to the increasing ingestion of veterinary drugs and acquisition of pathogens from medicated stabled livestock carcasses due to decreasing availability of unstabled livestock carcasses -the traditional primary food of bearded vultures [16] since the BSE crisis [21] , accompanied by a possible increasing use of antibiotics in stabled livestock operations. In this sense, it is remarkable that bearded vultures primarily feed upon livestock bones, which are one of the major target tissues of fluoroquinolones in medicated animals [56] , therefore, rendering this species especially sensitive to the consequences of an increase in the consumption of stabled intensively medicated livestock. The presence of veterinary drugs in eggs implies their previous presence at least in breeding females [12] , but also probably in breeding males and non-breeders frequently using artificial feeding sites and livestock carcass dumps [17] , where veterinary drugs may be ingested from medicated livestock carcasses [10, 21] . Therefore, further research is required to determine the impact of veterinary drugs and livestock disease on fitness of full-grown individuals, including the potentially subtle, sublethal or indirect effects of these factors on population dynamics. The link between veterinary drugs and livestock disease should be further investigated in scavenger species, because both threats may concur in food and because the immunodepressive effects and other physiological alterations caused by drugs may facilitate the acquisition and proliferation of pathogens [6, 11, 21] . Given that both threats acting together may greatly contribute to breeding failure decreasing productivity, their potential as stochastic factors with potentially devastating effects increasing the risk of extinction should be not overlooked in current conservation programs of bearded vultures and other scavenger species, especially regarding dangerous veterinary drugs and highly pathogenic viruses frequently infecting poultry. In addition, restricted geographic distribution and low genetic variability [57] common to many threatened species may favour pathogen transmission and reduce the ability of a naïve immune system to fight against novel pathogens [3, 28, 58] , making them especially vulnerable to the potential cross-species transmission of highly virulent virus strains able to cause important outbreaks, as reported in poultry [59] [60] [61] . The association of pollution and disease may further increase extinction risk if it interacts with the effects of habitat saturation processes [13, 14, 17] . These processes may facilitate conspecific contact and interactions also likely to increase intra-and interspecific pathogen transmission rates in breeding and feeding areas, especially of highly contagious poultry diseases [22] . This could be further enhanced by the artificially high numbers of bearded vultures and other scavengers attracted to feeding points and carcass refuse dumps, both as a result of management and due to the scarcity of unstabled livestock carcasses since the BSE crisis [17, 21] . Whatever the potential contribution of underlying ultimate mechanisms reducing productivity, our findings highlight the need to determine the proximate causes of breeding failure and mortality in wildlife populations in order to understand the processes regulating demography from an ecological framework perspective. Table S1 Found at: doi:10.1371/journal.pone.0014163.s001 (0.05 MB DOC)

What was the aim of this study?

Livestock Drugs and Disease: The Fatal Combination behind Breeding Failure in Endangered Bearded Vultures https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994777/ SHA: f4f804bac7b32c84ad6572776df684df2a2e5fda Authors: Blanco, Guillermo; Lemus, Jesús A. Date: 2010-11-30 DOI: 10.1371/journal.pone.0014163 License: cc-by Abstract: There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health, especially of threatened avian scavengers feeding upon medicated livestock carcasses. We conducted a comprehensive study of failed eggs and dead nestlings in bearded vultures (Gypaetus barbatus) to attempt to elucidate the proximate causes of breeding failure behind the recent decline in productivity in the Spanish Pyrenees. We found high concentrations of multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and nestlings, associated with multiple internal organ damage and livestock pathogens causing disease, especially septicaemia by swine pathogens and infectious bursal disease. The combined impact of drugs and disease as stochastic factors may result in potentially devastating effects exacerbating an already high risk of extinction and should be considered in current conservation programs for bearded vultures and other scavenger species, especially in regards to dangerous veterinary drugs and highly pathogenic poultry viruses. Text: Environmental pollutants are increasingly documented as a driver of wildlife endangerment due to their roles in organ damage, hormonal disruption and alteration of the immune system [1, 2] . Disease may also facilitate endangerment and extinction at global and local scales, especially when pathogens interact with other drivers such as pollutants [3] . There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health [4] [5] [6] , and even causing declines approaching extinction [7] . These threats may be especially detrimental to wildlife as they increasingly concur and interact as a consequence of the elimination of livestock residues containing veterinary pharmaceuticals and resistant pathogens due to growing intensive livestock operations worldwide [6, 8, 9] . In particular, the ingestion of antimicrobials, primarily fluoroquinolones, has been recently related to immunodepression-mediated acquisition of opportunistic pathogens and disease, as well as to organ damage in nestling vultures [6, 10, 11] . Fluoroquinolone residues have also been found in avian scavenger eggs and are associated with severe alterations in the development of embryo cartilage and bones that could preclude embryo movement and subsequently normal development, pre-hatch position and successful hatching [12] . Therefore, antimicrobials and other drugs may negatively affect embryo and nestling health with potentially devastating consequences on breeding success and conservation of vultures and other threatened avian scavengers. The bearded vulture (Gypaetus barbatus) is one of the most endangered birds in Europe, with a main stronghold in the Pyrenees. Increasing declines in productivity (average number of fledglings raised per territorial pair) have recently been reported in the Spanish Pyrenees associated with habitat saturation processes [13, 14] . Given that bearded vultures may raise only one fledgling per breeding attempt, this productivity decline should be linked to increasing breeding failure when the proportion of territorial pairs that are breeding does not greatly vary with time [15] . The proximate mechanisms by which density can affect productivity have been investigated, including habitat heterogeneity, with progressively poorer territories being used, territory shrinkage and interference with breeders and floaters [13] . However, the proximate causes of breeding failure are poorly known despite the long-term interests in the conservation of this species [16] . To evaluate these causes, the examination of failed eggs and dead nestlings is imperative, including the study of the presence and impact of injury, developmental problems, poor nutritional condition, pollutants, organ damage, pathogens causing disease, etc. in order to determine the most likely cause of breeding failure. Here, we conducted a comprehensive study of failed eggs and dead nestling bearded vultures collected during recent years in the Pyrenees. Both the productivity and survival rates of adults and young birds have reached the lowest values since the bovine spongiform encephalopathy (BSE) crisis [13, 14, 17] . This temporal decline could be related to illegal poisoning [17] and recent changes in the abundance, distribution and quality of carrion available to avian scavengers as a consequence of EU regulations derived from the BSE crisis [6, [18] [19] [20] . In particular, the BSE crisis caused the lack or scarcity of unstabled livestock available to scavengers and their subsequent increase in the consumption of carrion from stabled livestock, which is intensively medicated [21] . Therefore, we specifically focused on determining whether breeding failure in bearded vultures is related to the ingestion of veterinary drugs from stabled livestock carrion, as documented in other avian scavenger species [12] . We also assessed the potential effects of veterinary drugs on embryo damage and immunodepression increasing the probability of acquisition and proliferation of pathogens causing fatal disease [6, [10] [11] [12] 21] . Because veterinary drugs should be exclusively acquired from the ingestion of carrion from livestock medicated to combat disease, we predict that their presence should be associated with that of pathogens acquired from the same livestock, especially poultry pathogens more likely transmitted between avian species [22] . Alternatively, if the temporal decline in productivity was primarily associated with breeding failure due to the effects of habitat saturation processes [13, 17] , we should expect egg and nestling mortality to be directly related to developmental and nutritional problems indicating progressively lower quality territories (e.g. embryo emaciation, nestling starvation) and interference by both conspecifics and heterospecifics (e.g. incubation failure, injury due to predation attempts or disturbance). Failed eggs (n = 5) and dead nestlings (n = 4) were collected from bearded vulture nests located in the Spanish Pyrenees between 2005 and 2008. The study of this material did not require of the approval of an ethics committee because it was collected after breeding failure (egg or nestling death) was confirmed in the field. Three of the specimens (two nestlings and one egg) were collected in 2005, 2007 and 2008 from a particular territory. Eggs and nestlings were collected after breeding failure and frozen. Necropsies were performed on all specimens according to standard protocols [12] . The age of embryos and nestlings were estimated according to size and development. Samples of liver, kidney, spleen, large and small intestines, lungs, brain, lymphoid organs (thymus, bursa of Fabricius, Peyer's patches) and knee joints were fixed in 10% buffered formalin, sectioned at 4 mm and stained for histopathological analysis [10, 12] . Liver (dead nestlings and failed embryos) and yolk (failed embryos) were used for the determination of the presence of veterinary drugs, including fluoroquinolones (enrofloxacin and ciprofloxacin), other antimicrobials (amoxicillin and oxytetracycline), non-steroideal anti-inflamatories (NSAIDs) such as diclofenac, flunixin meglumine, ketoprofen, ibuprofen, meloxicam, sodium salicylate, acetaminophen, and antiparasitics (metronidazole, diclazuril, fenbendazole, ivermectin) as described previously [12] . The limits of quantification, percentage recoveries, and interand intra-assay reproducibility were adequate [10, 12] . Other contaminants potentially affecting eggs and embryos were determined in liver, including heavy metals (Cd, Zn, Pb and Hg), following Blanco et al. [23] , dithiocarbamate thiram, disulfuram, polybrominated diphenyl ethers, organochlorines and brominated flame retardants, following Lemus et al. [12] and carbamate and organophosphate pesticides (carbofuran, aldicarb and fenthion) following Elliot et al. [24] . We measured brain cholinesterase activity to assess early exposure to anticholinesterase pesticides [25] . Potential contamination was assessed by comparison with levels from apparently normal wild birds of other species [26] in the absence of basal levels for bearded vultures. Determination of bacterial and fungal pathogens were conducted by sampling oropharynx, lung, liver, kidney, spleen, and intestine with sterile swabs and cultured using standard microbiology protocols [10, 12, 27, 28] . Salmonella serotypes and phage types were determined in the Spanish Reference Laboratory (Laboratorio Central Veterinario, Algete, Madrid). For confirmation of the identification of the alpha hemolytic Streptococcus pneumoniae we used a specific identification test (Accuprobe, Salem, MA) based on the detection of specific ribosomal RNA sequences. Samples of lesions found in internal organs and tissues during necropsies were taken with sterile swabs and cultured using the same standard microbiology protocols. In addition, we determined the presence of selected avian pathogens, including bacterial, viral, fungal, and protozoan pathogens by means of PCR-based methods (see Table S1 for details). The presence of Chlamyophila psittaci and Mycoplasma sp. in blood was determined as described previously [29, 30] . The presence of poxvirus, the paramyxovirus causing Newcastle disease, the serotypes H5, H7 and H9 of avian influenza, falcon adenovirus, circovirus, herpesvirus, polyomavirus, reovirus and West Nile virus were determined following the PCR-based methods available in the literature [31] [32] [33] [34] [35] [36] [37] [38] [39] . We also searched for helminths and protozoans in the gastrointestinal tract by macroscopic and microscopic observations using standard protocols [40] . Specific immunocytochemical procedures were used for detection of mielodepressive virus, including the alphaherpesvirus causing Marek disease [41] in kidney and bursa of Fabricius, the gyrovirus causing infectious chicken anaemia [42] in thymus and bone marrow, the birnavirus causing infectious bursal disease (IBD, [43] ) in bursa of Fabricius, and the coronavirus causing chicken infectious bronchitis in kidney [44] . In addition, we conducted a specific immunocytochemical procedure for West Nile virus antigen detection [45] in brain, spinal medulla, thymus and thyroid. All immunohistochemistry analyses were conducted at the Department of Veterinary Anatomy, Veterinary Faculty, Universidad Complutense de Madrid, Spain and at the Pathology Department of the Veterinary Faculty, University of Utrecht, The Netherlands. The presence of these viruses was also determined by PCR-based methods [43, [46] [47] [48] . All dead nestlings and three of five unhatched embryos showed two to six different veterinary drugs in liver (nestlings) and egg yolk (embryos). In addition, the two embryos with fluoroquinolones in the yolk also had them in the liver (Table 1) . Fluoroquinolones were the most prevalent drugs and showed the highest concentrations (Table 1) . Other drugs such as NSAIDs and antiparasitics were found in most nestlings at variable concentrations, but in no eggs (Table 1) . Other toxic compounds were detected in lower prevalence and concentrations (see Table 1 for those more relevant values; all insecticides were found at concentrations ,0.001 ppb), which was further supported by basal levels of brain cholinesterase (Table 1) . Dead embryos and nestlings showed a moderate to good nutritional state. Major histopathological lesions were primarily located in the kidney, including glomerulonephritis and/or glomerulonephrosis present in all individuals with fluoroquinolones, but not in those without drugs (Table 1 ). All individuals with fluoroquinolones also showed joint lesions, including arthritis and/ or arthrosis of the long bone articulations, as well as massive osseous stroma of the spongeous bones. The fungi Candida albicans was isolated from the oral cavity of five individuals. All individuals showed non-specific mixedbacterial flora. Enterotoxigenic Escherichia coli and Salmonella spp. were isolated in four cases ( Table 1 (cont.) *Samples from the same territory in different years. 1 Veterinary drugs. EN: enrofloxacin (mg/g), CI: ciprofloxacin (mg/g), OX: oxytetracyclin (mg/g), FL: flunixin meglumine (mg/g), AS: sodium salicylate (ng/g), IV: Ivermectin (mg/g). 2 Other toxicants. OR: organochlorines (ng/g), Pb: lead (ng/g). 12. z29 (three cases, see Table 1 ). One individual showed infection by Salmonella enterica enteritidis (see above) and enterotoxigenic Escherichia coli O86 in all examined organs (septicaemia) except brain, which rejected the possibility of post-mortem contamination. Pasteurella multocida was isolated in a single individual that also showed enterotoxigenic Escherichia coli O86 (Table 1) ; all of these individuals contained fluoroquinolones. One of the failed embryos without veterinary drugs showed suppurative myocarditis, multiple microabscesses in head muscles, suppurative leptomeningitis, as well as lower jaw gangrenous inflammation with loss of the osseous stroma due to a mixed infection with Streptococcus suis and Streptococcus pneumoniae in brain, meninges and neck muscles; this embryo also showed infection by chicken infectious bronchitis ( Table 1 ). Both immunocytochemistry for the detection of poultry viruses and PCR pathogen survey were positive to IBDV in six individuals with fluoroquinolones (Table 1) . Immunocytochemical procedures failed to detect West Nile virus antigens in individuals in which PCR for this virus had been positive. Parasitology was negative for all helminths, helminth eggs and protozoans. We found multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and dead nestling bearded vultures from the Pyrenees. They also showed multiple internal organ damage and pathogens potentially acquired from medicated livestock carrion, especially viruses often infecting poultry. Recorded drug concentrations were among the highest reported in avian scavengers [6, [10] [11] [12] 21] . NSAIDs and antiparasitics were found in lower prevalence than fluoroquinolones, but at higher concentrations than those found in other avian scavengers, especially for flunixin meglumine and sodium salicylate [6, 12, 21] . On the contrary, we found no sterile eggs, poor nutritional conditions or injury in any failed embryo or nestling. Other pollutants were found in low prevalence and concentrations posing low risk to embryo and nestling health. Fluoroquinolones may cause generalized direct developmental damage precluding embryo hatching, physiological alterations due to their impact on liver and kidney and immunodepression reducing resistance to opportunistic pathogens [6, [10] [11] [12] 21] . These pathogens may be acquired at the same time that drugs used to treat diseased livestock are ingested, as indicated by their high prevalence in embryos and nestlings. Therefore, despite the relatively small sample size resulting from low abundance, endangerment and logistic difficulties in reaching nests in this species, the results provide evidence of a combined impact of veterinary drugs and livestock disease as the primary cause of breeding failure in the sampled individuals. The presence of West Nile virus is not likely to be associated with nestling disease or mortality because the lack of lesions in target tissues and viral antigen particles in the immunohistochemistry study. Fatal septicaemia caused by Streptococcus suis, one of the most important swine pathogens worldwide [49] , in combination with septicaemia from Streptococcus pneumoniae and infection by chicken infectious bronchitis virus were found in a single embryo. This concentration of livestock pathogens has not been reported before and, to our knowledge, this is the first report of the three pathogens causing disease in a wild bird. Other pathogens recorded in embryos and nestlings, including Salmonella serotypes and phages typical of livestock [50] , and enterotoxigenic Escherichia coli O86 causing septicaemia, were potentially transmitted by consumption of carcasses of infected poultry and other livestock [22, 27, 28] . In addition, we found that the IBD virus infected most individuals alone or together with other pathogens also potentially acquired from livestock carrion. This virus causes a highly contagious immunosuppressive bursal disease in poultry [51] and may be transmitted to wildlife in contact with poultry waste or by ingestion of carcasses [22, 52] . Nestlings are especially susceptible to IBD because of the primary role of bursa of Fabricius in immune function development at this age. In fact, immunosuppression due to IBD was indicated by the inflammation, necrosis and loss of lymphocytes in the bursa of Fabricius together with the presence of viral antigens recorded by means of immunocytochemical procedures. The potential impact of highly pathogenic and contagious poultry viruses has been previously recognized as a threat to wildlife health due to the increasing contact of wildlife with livestock operations in general, and poultry farms and their residues in particular, in natural areas worldwide [52] [53] [54] [55] . However, damage from IBD virus on the bursa of Fabricius represents, to our knowledge, the first evidence of clinical disease compatible with death caused by this poultry virus in wildlife. The presence of IBD has been not previously recorded in embryos of wild birds, probably because vertical transmission has been ruled out in poultry and, as consequence, it has probably not been evaluated in other species until now. This striking and concerning result could be related to the longer egg development and incubation periods of bearded vultures compared with poultry, and/or due to contrasting environmental conditions during incubation between bearded vultures and poultry. Thus, embryo infection with IBD may occurs via the female or during incubation as a consequence of egg contact between the egg and poultry remains in the nests of bearded vultures, which requires more research. Despite their potential effects on population dynamics and conservation through a reduction of productivity and changes in mating behaviour [13, 14] , habitat saturation processes were apparently not directly related to particular proximate causes of egg and nestling failure in this study or in these sampled individuals. As an alternative non-mutually exclusive explanation, we suggest that the recent decline in productivity could also be linked to the increasing ingestion of veterinary drugs and acquisition of pathogens from medicated stabled livestock carcasses due to decreasing availability of unstabled livestock carcasses -the traditional primary food of bearded vultures [16] since the BSE crisis [21] , accompanied by a possible increasing use of antibiotics in stabled livestock operations. In this sense, it is remarkable that bearded vultures primarily feed upon livestock bones, which are one of the major target tissues of fluoroquinolones in medicated animals [56] , therefore, rendering this species especially sensitive to the consequences of an increase in the consumption of stabled intensively medicated livestock. The presence of veterinary drugs in eggs implies their previous presence at least in breeding females [12] , but also probably in breeding males and non-breeders frequently using artificial feeding sites and livestock carcass dumps [17] , where veterinary drugs may be ingested from medicated livestock carcasses [10, 21] . Therefore, further research is required to determine the impact of veterinary drugs and livestock disease on fitness of full-grown individuals, including the potentially subtle, sublethal or indirect effects of these factors on population dynamics. The link between veterinary drugs and livestock disease should be further investigated in scavenger species, because both threats may concur in food and because the immunodepressive effects and other physiological alterations caused by drugs may facilitate the acquisition and proliferation of pathogens [6, 11, 21] . Given that both threats acting together may greatly contribute to breeding failure decreasing productivity, their potential as stochastic factors with potentially devastating effects increasing the risk of extinction should be not overlooked in current conservation programs of bearded vultures and other scavenger species, especially regarding dangerous veterinary drugs and highly pathogenic viruses frequently infecting poultry. In addition, restricted geographic distribution and low genetic variability [57] common to many threatened species may favour pathogen transmission and reduce the ability of a naïve immune system to fight against novel pathogens [3, 28, 58] , making them especially vulnerable to the potential cross-species transmission of highly virulent virus strains able to cause important outbreaks, as reported in poultry [59] [60] [61] . The association of pollution and disease may further increase extinction risk if it interacts with the effects of habitat saturation processes [13, 14, 17] . These processes may facilitate conspecific contact and interactions also likely to increase intra-and interspecific pathogen transmission rates in breeding and feeding areas, especially of highly contagious poultry diseases [22] . This could be further enhanced by the artificially high numbers of bearded vultures and other scavengers attracted to feeding points and carcass refuse dumps, both as a result of management and due to the scarcity of unstabled livestock carcasses since the BSE crisis [17, 21] . Whatever the potential contribution of underlying ultimate mechanisms reducing productivity, our findings highlight the need to determine the proximate causes of breeding failure and mortality in wildlife populations in order to understand the processes regulating demography from an ecological framework perspective. Table S1 Found at: doi:10.1371/journal.pone.0014163.s001 (0.05 MB DOC)

Where is the bearded vulture (Gypaetus barbatus) commonly found?

A Schiff Base-Derived Copper (II) Complex Is a Potent Inducer of Apoptosis in Colon Cancer Cells by Activating the Intrinsic Pathway https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967396/ SHA: f1f24521928f5d8565a15a17bd7f79239a3d4116 Authors: Hajrezaie, Maryam; Paydar, Mohammadjavad; Zorofchian Moghadamtousi, Soheil; Hassandarvish, Pouya; Gwaram, Nura Suleiman; Zahedifard, Maryam; Rouhollahi, Elham; Karimian, Hamed; Looi, Chung Yeng; Ali, Hapipah Mohd; Abdul Majid, Nazia; Abdulla, Mahmood Ameen Date: 2014-03-05 DOI: 10.1155/2014/540463 License: cc-by Abstract: Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper (II) complex on HT-29 colon cancer cells. The Cu(BrHAP)(2 ) Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC(50 )value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu (II) complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G(1 ) cell population. At a concentration of 6.25 μg/ml, the Cu(BrHAP)(2 ) compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu (II) complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu(BrHAP)(2 ) compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern [1] . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively [2] . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success [3] . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated [4] . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis [5] . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells [6, 7] . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects [8] . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications [9, 10] . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds [11] . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application [12, 13] . Schiff base Cu(II) complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity [14] [15] [16] [17] [18] . This study evaluated the anticancer potential of a copper (II) complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu(BrHAP) 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville, MD) containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks (Corning, USA) according to each experimental scale. Cell viability was measured by a conventional MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] reduction assay. After 48 h exposure to six concentrations of Cu(BrHAP) 2 , cells were treated with MTT solution (2 mg/mL) for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader (Hidex, Turku, Finland). The IC 50 value was determined as the concentration of Cu(BrHAP) 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase (LDH) release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 and Triton X-100 (positive control) for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu(BrHAP) 2 was expressed as a percentage of the positive control. A propidium iodide (PI) and acridine orange (AO) double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope (Leica attached with Q-Floro software) according to a standard procedure. HT-29 cells (5 × 10 4 cells/mL in a 25 mL culture flask) were plated, treated with Cu(BrHAP) 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope (Olympus BX51) within 30 min. In brief, HT-29 cells (1 × 10 4 cells/well in 96-well plate) were supplemented with Cu(BrHAP) 2 (2 g/mL) or DMSO (negative control) for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader (Thermo Scientific). The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells (5 × 10 4 cells/mL) were treated with Cu(BrHAP) 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol (90%) and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A (10 mg/mL) and 50 L of propidium iodide (PI) (1 mg/mL) were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer (BD FACSCanto II). The oxygen radical antioxidant capacity (ORAC) assay was carried out based on the protocols described in detail previously [19] . In brief, Cu(BrHAP) 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank (solvent/PBS), standard (trolox), or positive control (quercetin). The plate was then supplemented with the working fluorescein solution (150 L), followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve (AUC) of samples and blank. The values were Trolox equivalents (TE). In brief, HT-29 cells (1 × 10 4 cells/mL) were seeded in 96-well plates and treated with different concentrations of Cu(BrHAP) 2 and DMSO (negative control) for 24 h. After 30 min treatment with dihydroethidium (DHE) dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously [20] . Plates with stained cells were analyzed using the ArrayScan HCS system (Cellomics, PA, USA). Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit (Promega, Madison, WI). HT-29 cells (1.0 × 10 4 cells/well) were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu(BrHAP) 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent (100 L) and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. In brief, HT-29 cells (1.0 × 10 4 cells/well in a 96-well plate) were treated with different concentrations of Cu(BrHAP) 2 for 3 h, followed by stimulation with TNF-(1 ng/mL) for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B (NF-B) activation kit (Thermo Scientific) according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation (SD) of the number of experiments shown in the legends. An analysis of variance (ANOVA) was carried out using the prism statistical package (GraphPad Software, USA). < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu(BrHAP) 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu(BrHAP) 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase (LDH) release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu(BrHAP) 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu(BrHAP) 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells ( Figure 2 ). Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu(BrHAP) 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu(BrHAP) 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment ( Figure 3) . The results showed that the Cu(BrHAP) 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment ( Figure 5 ). Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve (AUC) technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu(BrHAP) 2 exhibited low to moderate antioxidant activity compared to quercetin ( Table 2) . Formation. HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu(BrHAP) 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu(BrHAP) 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate (ATP) and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu(BrHAP) 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu(BrHAP) 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu(BrHAP) 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor-(TNF-). The Cu(BrHAP) 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus (Figure 9 ). Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression [21] . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation [22, 23]. The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence [24] , and agents that can induce apoptosis are known to have potential anticancer effects [25, 26] . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways [27] . In the present study, the Cu(BrHAP) 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies [28] [29] [30] . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line (CCD 841) showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu(II) compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu(BrHAP) 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway [31] . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation [32, 33] . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu(II) complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry [34] [35] [36] . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis [37, 38] . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis [39] . As the main source of cellular ROS and adenosine triphosphate (ATP), mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu(BrHAP) 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu(II) compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS [40] . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential (MMP) fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu(BrHAP) 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu(II) Schiff base complexes may be associated with the mitochondrial pathway [26, 41, 42] . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP [30] . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets [43] . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 [44] . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 [45, 46] . In our study, the Cu(BrHAP) 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu(BrHAP) 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.

What was the focus of this study?

A Schiff Base-Derived Copper (II) Complex Is a Potent Inducer of Apoptosis in Colon Cancer Cells by Activating the Intrinsic Pathway https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967396/ SHA: f1f24521928f5d8565a15a17bd7f79239a3d4116 Authors: Hajrezaie, Maryam; Paydar, Mohammadjavad; Zorofchian Moghadamtousi, Soheil; Hassandarvish, Pouya; Gwaram, Nura Suleiman; Zahedifard, Maryam; Rouhollahi, Elham; Karimian, Hamed; Looi, Chung Yeng; Ali, Hapipah Mohd; Abdul Majid, Nazia; Abdulla, Mahmood Ameen Date: 2014-03-05 DOI: 10.1155/2014/540463 License: cc-by Abstract: Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper (II) complex on HT-29 colon cancer cells. The Cu(BrHAP)(2 ) Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC(50 )value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu (II) complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G(1 ) cell population. At a concentration of 6.25 μg/ml, the Cu(BrHAP)(2 ) compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu (II) complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu(BrHAP)(2 ) compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern [1] . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively [2] . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success [3] . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated [4] . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis [5] . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells [6, 7] . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects [8] . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications [9, 10] . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds [11] . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application [12, 13] . Schiff base Cu(II) complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity [14] [15] [16] [17] [18] . This study evaluated the anticancer potential of a copper (II) complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu(BrHAP) 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville, MD) containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks (Corning, USA) according to each experimental scale. Cell viability was measured by a conventional MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] reduction assay. After 48 h exposure to six concentrations of Cu(BrHAP) 2 , cells were treated with MTT solution (2 mg/mL) for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader (Hidex, Turku, Finland). The IC 50 value was determined as the concentration of Cu(BrHAP) 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase (LDH) release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 and Triton X-100 (positive control) for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu(BrHAP) 2 was expressed as a percentage of the positive control. A propidium iodide (PI) and acridine orange (AO) double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope (Leica attached with Q-Floro software) according to a standard procedure. HT-29 cells (5 × 10 4 cells/mL in a 25 mL culture flask) were plated, treated with Cu(BrHAP) 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope (Olympus BX51) within 30 min. In brief, HT-29 cells (1 × 10 4 cells/well in 96-well plate) were supplemented with Cu(BrHAP) 2 (2 g/mL) or DMSO (negative control) for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader (Thermo Scientific). The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells (5 × 10 4 cells/mL) were treated with Cu(BrHAP) 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol (90%) and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A (10 mg/mL) and 50 L of propidium iodide (PI) (1 mg/mL) were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer (BD FACSCanto II). The oxygen radical antioxidant capacity (ORAC) assay was carried out based on the protocols described in detail previously [19] . In brief, Cu(BrHAP) 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank (solvent/PBS), standard (trolox), or positive control (quercetin). The plate was then supplemented with the working fluorescein solution (150 L), followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve (AUC) of samples and blank. The values were Trolox equivalents (TE). In brief, HT-29 cells (1 × 10 4 cells/mL) were seeded in 96-well plates and treated with different concentrations of Cu(BrHAP) 2 and DMSO (negative control) for 24 h. After 30 min treatment with dihydroethidium (DHE) dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously [20] . Plates with stained cells were analyzed using the ArrayScan HCS system (Cellomics, PA, USA). Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit (Promega, Madison, WI). HT-29 cells (1.0 × 10 4 cells/well) were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu(BrHAP) 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent (100 L) and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. In brief, HT-29 cells (1.0 × 10 4 cells/well in a 96-well plate) were treated with different concentrations of Cu(BrHAP) 2 for 3 h, followed by stimulation with TNF-(1 ng/mL) for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B (NF-B) activation kit (Thermo Scientific) according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation (SD) of the number of experiments shown in the legends. An analysis of variance (ANOVA) was carried out using the prism statistical package (GraphPad Software, USA). < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu(BrHAP) 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu(BrHAP) 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase (LDH) release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu(BrHAP) 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu(BrHAP) 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells ( Figure 2 ). Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu(BrHAP) 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu(BrHAP) 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment ( Figure 3) . The results showed that the Cu(BrHAP) 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment ( Figure 5 ). Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve (AUC) technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu(BrHAP) 2 exhibited low to moderate antioxidant activity compared to quercetin ( Table 2) . Formation. HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu(BrHAP) 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu(BrHAP) 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate (ATP) and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu(BrHAP) 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu(BrHAP) 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu(BrHAP) 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor-(TNF-). The Cu(BrHAP) 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus (Figure 9 ). Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression [21] . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation [22, 23]. The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence [24] , and agents that can induce apoptosis are known to have potential anticancer effects [25, 26] . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways [27] . In the present study, the Cu(BrHAP) 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies [28] [29] [30] . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line (CCD 841) showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu(II) compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu(BrHAP) 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway [31] . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation [32, 33] . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu(II) complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry [34] [35] [36] . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis [37, 38] . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis [39] . As the main source of cellular ROS and adenosine triphosphate (ATP), mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu(BrHAP) 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu(II) compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS [40] . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential (MMP) fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu(BrHAP) 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu(II) Schiff base complexes may be associated with the mitochondrial pathway [26, 41, 42] . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP [30] . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets [43] . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 [44] . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 [45, 46] . In our study, the Cu(BrHAP) 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu(BrHAP) 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.

What is the third most prevalent cancer in females in the United States?

A Schiff Base-Derived Copper (II) Complex Is a Potent Inducer of Apoptosis in Colon Cancer Cells by Activating the Intrinsic Pathway https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967396/ SHA: f1f24521928f5d8565a15a17bd7f79239a3d4116 Authors: Hajrezaie, Maryam; Paydar, Mohammadjavad; Zorofchian Moghadamtousi, Soheil; Hassandarvish, Pouya; Gwaram, Nura Suleiman; Zahedifard, Maryam; Rouhollahi, Elham; Karimian, Hamed; Looi, Chung Yeng; Ali, Hapipah Mohd; Abdul Majid, Nazia; Abdulla, Mahmood Ameen Date: 2014-03-05 DOI: 10.1155/2014/540463 License: cc-by Abstract: Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper (II) complex on HT-29 colon cancer cells. The Cu(BrHAP)(2 ) Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC(50 )value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu (II) complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G(1 ) cell population. At a concentration of 6.25 μg/ml, the Cu(BrHAP)(2 ) compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu (II) complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu(BrHAP)(2 ) compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern [1] . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively [2] . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success [3] . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated [4] . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis [5] . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells [6, 7] . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects [8] . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications [9, 10] . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds [11] . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application [12, 13] . Schiff base Cu(II) complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity [14] [15] [16] [17] [18] . This study evaluated the anticancer potential of a copper (II) complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu(BrHAP) 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville, MD) containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks (Corning, USA) according to each experimental scale. Cell viability was measured by a conventional MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] reduction assay. After 48 h exposure to six concentrations of Cu(BrHAP) 2 , cells were treated with MTT solution (2 mg/mL) for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader (Hidex, Turku, Finland). The IC 50 value was determined as the concentration of Cu(BrHAP) 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase (LDH) release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 and Triton X-100 (positive control) for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu(BrHAP) 2 was expressed as a percentage of the positive control. A propidium iodide (PI) and acridine orange (AO) double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope (Leica attached with Q-Floro software) according to a standard procedure. HT-29 cells (5 × 10 4 cells/mL in a 25 mL culture flask) were plated, treated with Cu(BrHAP) 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope (Olympus BX51) within 30 min. In brief, HT-29 cells (1 × 10 4 cells/well in 96-well plate) were supplemented with Cu(BrHAP) 2 (2 g/mL) or DMSO (negative control) for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader (Thermo Scientific). The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells (5 × 10 4 cells/mL) were treated with Cu(BrHAP) 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol (90%) and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A (10 mg/mL) and 50 L of propidium iodide (PI) (1 mg/mL) were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer (BD FACSCanto II). The oxygen radical antioxidant capacity (ORAC) assay was carried out based on the protocols described in detail previously [19] . In brief, Cu(BrHAP) 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank (solvent/PBS), standard (trolox), or positive control (quercetin). The plate was then supplemented with the working fluorescein solution (150 L), followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve (AUC) of samples and blank. The values were Trolox equivalents (TE). In brief, HT-29 cells (1 × 10 4 cells/mL) were seeded in 96-well plates and treated with different concentrations of Cu(BrHAP) 2 and DMSO (negative control) for 24 h. After 30 min treatment with dihydroethidium (DHE) dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously [20] . Plates with stained cells were analyzed using the ArrayScan HCS system (Cellomics, PA, USA). Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit (Promega, Madison, WI). HT-29 cells (1.0 × 10 4 cells/well) were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu(BrHAP) 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent (100 L) and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. In brief, HT-29 cells (1.0 × 10 4 cells/well in a 96-well plate) were treated with different concentrations of Cu(BrHAP) 2 for 3 h, followed by stimulation with TNF-(1 ng/mL) for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B (NF-B) activation kit (Thermo Scientific) according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation (SD) of the number of experiments shown in the legends. An analysis of variance (ANOVA) was carried out using the prism statistical package (GraphPad Software, USA). < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu(BrHAP) 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu(BrHAP) 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase (LDH) release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu(BrHAP) 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu(BrHAP) 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells ( Figure 2 ). Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu(BrHAP) 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu(BrHAP) 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment ( Figure 3) . The results showed that the Cu(BrHAP) 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment ( Figure 5 ). Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve (AUC) technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu(BrHAP) 2 exhibited low to moderate antioxidant activity compared to quercetin ( Table 2) . Formation. HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu(BrHAP) 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu(BrHAP) 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate (ATP) and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu(BrHAP) 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu(BrHAP) 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu(BrHAP) 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor-(TNF-). The Cu(BrHAP) 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus (Figure 9 ). Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression [21] . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation [22, 23]. The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence [24] , and agents that can induce apoptosis are known to have potential anticancer effects [25, 26] . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways [27] . In the present study, the Cu(BrHAP) 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies [28] [29] [30] . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line (CCD 841) showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu(II) compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu(BrHAP) 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway [31] . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation [32, 33] . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu(II) complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry [34] [35] [36] . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis [37, 38] . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis [39] . As the main source of cellular ROS and adenosine triphosphate (ATP), mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu(BrHAP) 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu(II) compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS [40] . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential (MMP) fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu(BrHAP) 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu(II) Schiff base complexes may be associated with the mitochondrial pathway [26, 41, 42] . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP [30] . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets [43] . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 [44] . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 [45, 46] . In our study, the Cu(BrHAP) 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu(BrHAP) 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.

What is the 1-year survival rate for colorectal cancer patients?

A Schiff Base-Derived Copper (II) Complex Is a Potent Inducer of Apoptosis in Colon Cancer Cells by Activating the Intrinsic Pathway https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967396/ SHA: f1f24521928f5d8565a15a17bd7f79239a3d4116 Authors: Hajrezaie, Maryam; Paydar, Mohammadjavad; Zorofchian Moghadamtousi, Soheil; Hassandarvish, Pouya; Gwaram, Nura Suleiman; Zahedifard, Maryam; Rouhollahi, Elham; Karimian, Hamed; Looi, Chung Yeng; Ali, Hapipah Mohd; Abdul Majid, Nazia; Abdulla, Mahmood Ameen Date: 2014-03-05 DOI: 10.1155/2014/540463 License: cc-by Abstract: Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper (II) complex on HT-29 colon cancer cells. The Cu(BrHAP)(2 ) Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC(50 )value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu (II) complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G(1 ) cell population. At a concentration of 6.25 μg/ml, the Cu(BrHAP)(2 ) compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu (II) complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu(BrHAP)(2 ) compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern [1] . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively [2] . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success [3] . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated [4] . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis [5] . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells [6, 7] . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects [8] . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications [9, 10] . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds [11] . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application [12, 13] . Schiff base Cu(II) complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity [14] [15] [16] [17] [18] . This study evaluated the anticancer potential of a copper (II) complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu(BrHAP) 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville, MD) containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks (Corning, USA) according to each experimental scale. Cell viability was measured by a conventional MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] reduction assay. After 48 h exposure to six concentrations of Cu(BrHAP) 2 , cells were treated with MTT solution (2 mg/mL) for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader (Hidex, Turku, Finland). The IC 50 value was determined as the concentration of Cu(BrHAP) 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase (LDH) release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 and Triton X-100 (positive control) for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu(BrHAP) 2 was expressed as a percentage of the positive control. A propidium iodide (PI) and acridine orange (AO) double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope (Leica attached with Q-Floro software) according to a standard procedure. HT-29 cells (5 × 10 4 cells/mL in a 25 mL culture flask) were plated, treated with Cu(BrHAP) 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope (Olympus BX51) within 30 min. In brief, HT-29 cells (1 × 10 4 cells/well in 96-well plate) were supplemented with Cu(BrHAP) 2 (2 g/mL) or DMSO (negative control) for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader (Thermo Scientific). The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells (5 × 10 4 cells/mL) were treated with Cu(BrHAP) 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol (90%) and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A (10 mg/mL) and 50 L of propidium iodide (PI) (1 mg/mL) were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer (BD FACSCanto II). The oxygen radical antioxidant capacity (ORAC) assay was carried out based on the protocols described in detail previously [19] . In brief, Cu(BrHAP) 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank (solvent/PBS), standard (trolox), or positive control (quercetin). The plate was then supplemented with the working fluorescein solution (150 L), followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve (AUC) of samples and blank. The values were Trolox equivalents (TE). In brief, HT-29 cells (1 × 10 4 cells/mL) were seeded in 96-well plates and treated with different concentrations of Cu(BrHAP) 2 and DMSO (negative control) for 24 h. After 30 min treatment with dihydroethidium (DHE) dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously [20] . Plates with stained cells were analyzed using the ArrayScan HCS system (Cellomics, PA, USA). Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit (Promega, Madison, WI). HT-29 cells (1.0 × 10 4 cells/well) were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu(BrHAP) 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent (100 L) and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. In brief, HT-29 cells (1.0 × 10 4 cells/well in a 96-well plate) were treated with different concentrations of Cu(BrHAP) 2 for 3 h, followed by stimulation with TNF-(1 ng/mL) for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B (NF-B) activation kit (Thermo Scientific) according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation (SD) of the number of experiments shown in the legends. An analysis of variance (ANOVA) was carried out using the prism statistical package (GraphPad Software, USA). < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu(BrHAP) 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu(BrHAP) 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase (LDH) release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu(BrHAP) 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu(BrHAP) 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells ( Figure 2 ). Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu(BrHAP) 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu(BrHAP) 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment ( Figure 3) . The results showed that the Cu(BrHAP) 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment ( Figure 5 ). Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve (AUC) technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu(BrHAP) 2 exhibited low to moderate antioxidant activity compared to quercetin ( Table 2) . Formation. HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu(BrHAP) 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu(BrHAP) 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate (ATP) and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu(BrHAP) 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu(BrHAP) 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu(BrHAP) 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor-(TNF-). The Cu(BrHAP) 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus (Figure 9 ). Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression [21] . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation [22, 23]. The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence [24] , and agents that can induce apoptosis are known to have potential anticancer effects [25, 26] . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways [27] . In the present study, the Cu(BrHAP) 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies [28] [29] [30] . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line (CCD 841) showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu(II) compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu(BrHAP) 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway [31] . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation [32, 33] . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu(II) complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry [34] [35] [36] . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis [37, 38] . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis [39] . As the main source of cellular ROS and adenosine triphosphate (ATP), mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu(BrHAP) 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu(II) compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS [40] . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential (MMP) fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu(BrHAP) 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu(II) Schiff base complexes may be associated with the mitochondrial pathway [26, 41, 42] . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP [30] . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets [43] . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 [44] . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 [45, 46] . In our study, the Cu(BrHAP) 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu(BrHAP) 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.

What is the 5-year survival rate for colorectal cancer patients?

A Schiff Base-Derived Copper (II) Complex Is a Potent Inducer of Apoptosis in Colon Cancer Cells by Activating the Intrinsic Pathway https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967396/ SHA: f1f24521928f5d8565a15a17bd7f79239a3d4116 Authors: Hajrezaie, Maryam; Paydar, Mohammadjavad; Zorofchian Moghadamtousi, Soheil; Hassandarvish, Pouya; Gwaram, Nura Suleiman; Zahedifard, Maryam; Rouhollahi, Elham; Karimian, Hamed; Looi, Chung Yeng; Ali, Hapipah Mohd; Abdul Majid, Nazia; Abdulla, Mahmood Ameen Date: 2014-03-05 DOI: 10.1155/2014/540463 License: cc-by Abstract: Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper (II) complex on HT-29 colon cancer cells. The Cu(BrHAP)(2 ) Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC(50 )value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu (II) complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G(1 ) cell population. At a concentration of 6.25 μg/ml, the Cu(BrHAP)(2 ) compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu (II) complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu(BrHAP)(2 ) compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern [1] . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively [2] . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success [3] . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated [4] . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis [5] . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells [6, 7] . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects [8] . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications [9, 10] . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds [11] . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application [12, 13] . Schiff base Cu(II) complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity [14] [15] [16] [17] [18] . This study evaluated the anticancer potential of a copper (II) complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu(BrHAP) 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville, MD) containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks (Corning, USA) according to each experimental scale. Cell viability was measured by a conventional MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] reduction assay. After 48 h exposure to six concentrations of Cu(BrHAP) 2 , cells were treated with MTT solution (2 mg/mL) for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader (Hidex, Turku, Finland). The IC 50 value was determined as the concentration of Cu(BrHAP) 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase (LDH) release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 and Triton X-100 (positive control) for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu(BrHAP) 2 was expressed as a percentage of the positive control. A propidium iodide (PI) and acridine orange (AO) double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope (Leica attached with Q-Floro software) according to a standard procedure. HT-29 cells (5 × 10 4 cells/mL in a 25 mL culture flask) were plated, treated with Cu(BrHAP) 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope (Olympus BX51) within 30 min. In brief, HT-29 cells (1 × 10 4 cells/well in 96-well plate) were supplemented with Cu(BrHAP) 2 (2 g/mL) or DMSO (negative control) for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader (Thermo Scientific). The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells (5 × 10 4 cells/mL) were treated with Cu(BrHAP) 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol (90%) and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A (10 mg/mL) and 50 L of propidium iodide (PI) (1 mg/mL) were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer (BD FACSCanto II). The oxygen radical antioxidant capacity (ORAC) assay was carried out based on the protocols described in detail previously [19] . In brief, Cu(BrHAP) 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank (solvent/PBS), standard (trolox), or positive control (quercetin). The plate was then supplemented with the working fluorescein solution (150 L), followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve (AUC) of samples and blank. The values were Trolox equivalents (TE). In brief, HT-29 cells (1 × 10 4 cells/mL) were seeded in 96-well plates and treated with different concentrations of Cu(BrHAP) 2 and DMSO (negative control) for 24 h. After 30 min treatment with dihydroethidium (DHE) dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously [20] . Plates with stained cells were analyzed using the ArrayScan HCS system (Cellomics, PA, USA). Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit (Promega, Madison, WI). HT-29 cells (1.0 × 10 4 cells/well) were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu(BrHAP) 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent (100 L) and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. In brief, HT-29 cells (1.0 × 10 4 cells/well in a 96-well plate) were treated with different concentrations of Cu(BrHAP) 2 for 3 h, followed by stimulation with TNF-(1 ng/mL) for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B (NF-B) activation kit (Thermo Scientific) according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation (SD) of the number of experiments shown in the legends. An analysis of variance (ANOVA) was carried out using the prism statistical package (GraphPad Software, USA). < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu(BrHAP) 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu(BrHAP) 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase (LDH) release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu(BrHAP) 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu(BrHAP) 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells ( Figure 2 ). Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu(BrHAP) 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu(BrHAP) 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment ( Figure 3) . The results showed that the Cu(BrHAP) 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment ( Figure 5 ). Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve (AUC) technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu(BrHAP) 2 exhibited low to moderate antioxidant activity compared to quercetin ( Table 2) . Formation. HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu(BrHAP) 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu(BrHAP) 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate (ATP) and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu(BrHAP) 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu(BrHAP) 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu(BrHAP) 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor-(TNF-). The Cu(BrHAP) 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus (Figure 9 ). Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression [21] . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation [22, 23]. The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence [24] , and agents that can induce apoptosis are known to have potential anticancer effects [25, 26] . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways [27] . In the present study, the Cu(BrHAP) 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies [28] [29] [30] . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line (CCD 841) showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu(II) compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu(BrHAP) 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway [31] . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation [32, 33] . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu(II) complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry [34] [35] [36] . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis [37, 38] . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis [39] . As the main source of cellular ROS and adenosine triphosphate (ATP), mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu(BrHAP) 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu(II) compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS [40] . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential (MMP) fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu(BrHAP) 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu(II) Schiff base complexes may be associated with the mitochondrial pathway [26, 41, 42] . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP [30] . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets [43] . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 [44] . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 [45, 46] . In our study, the Cu(BrHAP) 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu(BrHAP) 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.

How were nuclear morphological changes in HT-29 cells measured?

A Schiff Base-Derived Copper (II) Complex Is a Potent Inducer of Apoptosis in Colon Cancer Cells by Activating the Intrinsic Pathway https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967396/ SHA: f1f24521928f5d8565a15a17bd7f79239a3d4116 Authors: Hajrezaie, Maryam; Paydar, Mohammadjavad; Zorofchian Moghadamtousi, Soheil; Hassandarvish, Pouya; Gwaram, Nura Suleiman; Zahedifard, Maryam; Rouhollahi, Elham; Karimian, Hamed; Looi, Chung Yeng; Ali, Hapipah Mohd; Abdul Majid, Nazia; Abdulla, Mahmood Ameen Date: 2014-03-05 DOI: 10.1155/2014/540463 License: cc-by Abstract: Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper (II) complex on HT-29 colon cancer cells. The Cu(BrHAP)(2 ) Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC(50 )value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu (II) complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G(1 ) cell population. At a concentration of 6.25 μg/ml, the Cu(BrHAP)(2 ) compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu (II) complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu(BrHAP)(2 ) compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern [1] . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively [2] . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success [3] . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated [4] . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis [5] . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells [6, 7] . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects [8] . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications [9, 10] . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds [11] . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application [12, 13] . Schiff base Cu(II) complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity [14] [15] [16] [17] [18] . This study evaluated the anticancer potential of a copper (II) complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu(BrHAP) 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville, MD) containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks (Corning, USA) according to each experimental scale. Cell viability was measured by a conventional MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] reduction assay. After 48 h exposure to six concentrations of Cu(BrHAP) 2 , cells were treated with MTT solution (2 mg/mL) for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader (Hidex, Turku, Finland). The IC 50 value was determined as the concentration of Cu(BrHAP) 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase (LDH) release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 and Triton X-100 (positive control) for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu(BrHAP) 2 was expressed as a percentage of the positive control. A propidium iodide (PI) and acridine orange (AO) double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope (Leica attached with Q-Floro software) according to a standard procedure. HT-29 cells (5 × 10 4 cells/mL in a 25 mL culture flask) were plated, treated with Cu(BrHAP) 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope (Olympus BX51) within 30 min. In brief, HT-29 cells (1 × 10 4 cells/well in 96-well plate) were supplemented with Cu(BrHAP) 2 (2 g/mL) or DMSO (negative control) for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader (Thermo Scientific). The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells (5 × 10 4 cells/mL) were treated with Cu(BrHAP) 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol (90%) and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A (10 mg/mL) and 50 L of propidium iodide (PI) (1 mg/mL) were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer (BD FACSCanto II). The oxygen radical antioxidant capacity (ORAC) assay was carried out based on the protocols described in detail previously [19] . In brief, Cu(BrHAP) 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank (solvent/PBS), standard (trolox), or positive control (quercetin). The plate was then supplemented with the working fluorescein solution (150 L), followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve (AUC) of samples and blank. The values were Trolox equivalents (TE). In brief, HT-29 cells (1 × 10 4 cells/mL) were seeded in 96-well plates and treated with different concentrations of Cu(BrHAP) 2 and DMSO (negative control) for 24 h. After 30 min treatment with dihydroethidium (DHE) dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously [20] . Plates with stained cells were analyzed using the ArrayScan HCS system (Cellomics, PA, USA). Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit (Promega, Madison, WI). HT-29 cells (1.0 × 10 4 cells/well) were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu(BrHAP) 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent (100 L) and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. In brief, HT-29 cells (1.0 × 10 4 cells/well in a 96-well plate) were treated with different concentrations of Cu(BrHAP) 2 for 3 h, followed by stimulation with TNF-(1 ng/mL) for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B (NF-B) activation kit (Thermo Scientific) according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation (SD) of the number of experiments shown in the legends. An analysis of variance (ANOVA) was carried out using the prism statistical package (GraphPad Software, USA). < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu(BrHAP) 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu(BrHAP) 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase (LDH) release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu(BrHAP) 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu(BrHAP) 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells ( Figure 2 ). Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu(BrHAP) 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu(BrHAP) 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment ( Figure 3) . The results showed that the Cu(BrHAP) 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment ( Figure 5 ). Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve (AUC) technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu(BrHAP) 2 exhibited low to moderate antioxidant activity compared to quercetin ( Table 2) . Formation. HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu(BrHAP) 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu(BrHAP) 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate (ATP) and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu(BrHAP) 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu(BrHAP) 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu(BrHAP) 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor-(TNF-). The Cu(BrHAP) 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus (Figure 9 ). Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression [21] . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation [22, 23]. The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence [24] , and agents that can induce apoptosis are known to have potential anticancer effects [25, 26] . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways [27] . In the present study, the Cu(BrHAP) 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies [28] [29] [30] . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line (CCD 841) showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu(II) compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu(BrHAP) 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway [31] . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation [32, 33] . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu(II) complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry [34] [35] [36] . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis [37, 38] . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis [39] . As the main source of cellular ROS and adenosine triphosphate (ATP), mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu(BrHAP) 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu(II) compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS [40] . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential (MMP) fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu(BrHAP) 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu(II) Schiff base complexes may be associated with the mitochondrial pathway [26, 41, 42] . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP [30] . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets [43] . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 [44] . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 [45, 46] . In our study, the Cu(BrHAP) 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu(BrHAP) 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.

What is directly related to nuclear condensation?

A Schiff Base-Derived Copper (II) Complex Is a Potent Inducer of Apoptosis in Colon Cancer Cells by Activating the Intrinsic Pathway https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967396/ SHA: f1f24521928f5d8565a15a17bd7f79239a3d4116 Authors: Hajrezaie, Maryam; Paydar, Mohammadjavad; Zorofchian Moghadamtousi, Soheil; Hassandarvish, Pouya; Gwaram, Nura Suleiman; Zahedifard, Maryam; Rouhollahi, Elham; Karimian, Hamed; Looi, Chung Yeng; Ali, Hapipah Mohd; Abdul Majid, Nazia; Abdulla, Mahmood Ameen Date: 2014-03-05 DOI: 10.1155/2014/540463 License: cc-by Abstract: Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper (II) complex on HT-29 colon cancer cells. The Cu(BrHAP)(2 ) Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC(50 )value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu (II) complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G(1 ) cell population. At a concentration of 6.25 μg/ml, the Cu(BrHAP)(2 ) compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu (II) complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu(BrHAP)(2 ) compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern [1] . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively [2] . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success [3] . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated [4] . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis [5] . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells [6, 7] . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects [8] . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications [9, 10] . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds [11] . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application [12, 13] . Schiff base Cu(II) complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity [14] [15] [16] [17] [18] . This study evaluated the anticancer potential of a copper (II) complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu(BrHAP) 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville, MD) containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks (Corning, USA) according to each experimental scale. Cell viability was measured by a conventional MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] reduction assay. After 48 h exposure to six concentrations of Cu(BrHAP) 2 , cells were treated with MTT solution (2 mg/mL) for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader (Hidex, Turku, Finland). The IC 50 value was determined as the concentration of Cu(BrHAP) 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase (LDH) release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 and Triton X-100 (positive control) for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu(BrHAP) 2 was expressed as a percentage of the positive control. A propidium iodide (PI) and acridine orange (AO) double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope (Leica attached with Q-Floro software) according to a standard procedure. HT-29 cells (5 × 10 4 cells/mL in a 25 mL culture flask) were plated, treated with Cu(BrHAP) 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope (Olympus BX51) within 30 min. In brief, HT-29 cells (1 × 10 4 cells/well in 96-well plate) were supplemented with Cu(BrHAP) 2 (2 g/mL) or DMSO (negative control) for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader (Thermo Scientific). The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells (5 × 10 4 cells/mL) were treated with Cu(BrHAP) 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol (90%) and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A (10 mg/mL) and 50 L of propidium iodide (PI) (1 mg/mL) were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer (BD FACSCanto II). The oxygen radical antioxidant capacity (ORAC) assay was carried out based on the protocols described in detail previously [19] . In brief, Cu(BrHAP) 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank (solvent/PBS), standard (trolox), or positive control (quercetin). The plate was then supplemented with the working fluorescein solution (150 L), followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve (AUC) of samples and blank. The values were Trolox equivalents (TE). In brief, HT-29 cells (1 × 10 4 cells/mL) were seeded in 96-well plates and treated with different concentrations of Cu(BrHAP) 2 and DMSO (negative control) for 24 h. After 30 min treatment with dihydroethidium (DHE) dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously [20] . Plates with stained cells were analyzed using the ArrayScan HCS system (Cellomics, PA, USA). Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit (Promega, Madison, WI). HT-29 cells (1.0 × 10 4 cells/well) were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu(BrHAP) 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent (100 L) and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. In brief, HT-29 cells (1.0 × 10 4 cells/well in a 96-well plate) were treated with different concentrations of Cu(BrHAP) 2 for 3 h, followed by stimulation with TNF-(1 ng/mL) for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B (NF-B) activation kit (Thermo Scientific) according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation (SD) of the number of experiments shown in the legends. An analysis of variance (ANOVA) was carried out using the prism statistical package (GraphPad Software, USA). < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu(BrHAP) 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu(BrHAP) 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase (LDH) release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu(BrHAP) 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu(BrHAP) 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells ( Figure 2 ). Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu(BrHAP) 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu(BrHAP) 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment ( Figure 3) . The results showed that the Cu(BrHAP) 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment ( Figure 5 ). Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve (AUC) technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu(BrHAP) 2 exhibited low to moderate antioxidant activity compared to quercetin ( Table 2) . Formation. HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu(BrHAP) 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu(BrHAP) 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate (ATP) and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu(BrHAP) 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu(BrHAP) 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu(BrHAP) 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor-(TNF-). The Cu(BrHAP) 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus (Figure 9 ). Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression [21] . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation [22, 23]. The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence [24] , and agents that can induce apoptosis are known to have potential anticancer effects [25, 26] . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways [27] . In the present study, the Cu(BrHAP) 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies [28] [29] [30] . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line (CCD 841) showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu(II) compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu(BrHAP) 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway [31] . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation [32, 33] . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu(II) complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry [34] [35] [36] . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis [37, 38] . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis [39] . As the main source of cellular ROS and adenosine triphosphate (ATP), mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu(BrHAP) 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu(II) compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS [40] . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential (MMP) fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu(BrHAP) 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu(II) Schiff base complexes may be associated with the mitochondrial pathway [26, 41, 42] . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP [30] . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets [43] . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 [44] . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 [45, 46] . In our study, the Cu(BrHAP) 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu(BrHAP) 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.

What morphological cell changes are most associated with apoptosis?

A Schiff Base-Derived Copper (II) Complex Is a Potent Inducer of Apoptosis in Colon Cancer Cells by Activating the Intrinsic Pathway https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967396/ SHA: f1f24521928f5d8565a15a17bd7f79239a3d4116 Authors: Hajrezaie, Maryam; Paydar, Mohammadjavad; Zorofchian Moghadamtousi, Soheil; Hassandarvish, Pouya; Gwaram, Nura Suleiman; Zahedifard, Maryam; Rouhollahi, Elham; Karimian, Hamed; Looi, Chung Yeng; Ali, Hapipah Mohd; Abdul Majid, Nazia; Abdulla, Mahmood Ameen Date: 2014-03-05 DOI: 10.1155/2014/540463 License: cc-by Abstract: Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper (II) complex on HT-29 colon cancer cells. The Cu(BrHAP)(2 ) Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC(50 )value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu (II) complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G(1 ) cell population. At a concentration of 6.25 μg/ml, the Cu(BrHAP)(2 ) compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu (II) complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu(BrHAP)(2 ) compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern [1] . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively [2] . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success [3] . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated [4] . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis [5] . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells [6, 7] . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects [8] . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications [9, 10] . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds [11] . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application [12, 13] . Schiff base Cu(II) complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity [14] [15] [16] [17] [18] . This study evaluated the anticancer potential of a copper (II) complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu(BrHAP) 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville, MD) containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks (Corning, USA) according to each experimental scale. Cell viability was measured by a conventional MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] reduction assay. After 48 h exposure to six concentrations of Cu(BrHAP) 2 , cells were treated with MTT solution (2 mg/mL) for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader (Hidex, Turku, Finland). The IC 50 value was determined as the concentration of Cu(BrHAP) 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase (LDH) release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 and Triton X-100 (positive control) for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu(BrHAP) 2 was expressed as a percentage of the positive control. A propidium iodide (PI) and acridine orange (AO) double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope (Leica attached with Q-Floro software) according to a standard procedure. HT-29 cells (5 × 10 4 cells/mL in a 25 mL culture flask) were plated, treated with Cu(BrHAP) 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope (Olympus BX51) within 30 min. In brief, HT-29 cells (1 × 10 4 cells/well in 96-well plate) were supplemented with Cu(BrHAP) 2 (2 g/mL) or DMSO (negative control) for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader (Thermo Scientific). The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells (5 × 10 4 cells/mL) were treated with Cu(BrHAP) 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol (90%) and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A (10 mg/mL) and 50 L of propidium iodide (PI) (1 mg/mL) were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer (BD FACSCanto II). The oxygen radical antioxidant capacity (ORAC) assay was carried out based on the protocols described in detail previously [19] . In brief, Cu(BrHAP) 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank (solvent/PBS), standard (trolox), or positive control (quercetin). The plate was then supplemented with the working fluorescein solution (150 L), followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve (AUC) of samples and blank. The values were Trolox equivalents (TE). In brief, HT-29 cells (1 × 10 4 cells/mL) were seeded in 96-well plates and treated with different concentrations of Cu(BrHAP) 2 and DMSO (negative control) for 24 h. After 30 min treatment with dihydroethidium (DHE) dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously [20] . Plates with stained cells were analyzed using the ArrayScan HCS system (Cellomics, PA, USA). Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit (Promega, Madison, WI). HT-29 cells (1.0 × 10 4 cells/well) were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu(BrHAP) 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent (100 L) and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. In brief, HT-29 cells (1.0 × 10 4 cells/well in a 96-well plate) were treated with different concentrations of Cu(BrHAP) 2 for 3 h, followed by stimulation with TNF-(1 ng/mL) for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B (NF-B) activation kit (Thermo Scientific) according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation (SD) of the number of experiments shown in the legends. An analysis of variance (ANOVA) was carried out using the prism statistical package (GraphPad Software, USA). < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu(BrHAP) 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu(BrHAP) 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase (LDH) release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu(BrHAP) 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu(BrHAP) 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells ( Figure 2 ). Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu(BrHAP) 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu(BrHAP) 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment ( Figure 3) . The results showed that the Cu(BrHAP) 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment ( Figure 5 ). Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve (AUC) technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu(BrHAP) 2 exhibited low to moderate antioxidant activity compared to quercetin ( Table 2) . Formation. HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu(BrHAP) 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu(BrHAP) 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate (ATP) and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu(BrHAP) 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu(BrHAP) 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu(BrHAP) 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor-(TNF-). The Cu(BrHAP) 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus (Figure 9 ). Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression [21] . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation [22, 23]. The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence [24] , and agents that can induce apoptosis are known to have potential anticancer effects [25, 26] . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways [27] . In the present study, the Cu(BrHAP) 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies [28] [29] [30] . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line (CCD 841) showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu(II) compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu(BrHAP) 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway [31] . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation [32, 33] . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu(II) complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry [34] [35] [36] . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis [37, 38] . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis [39] . As the main source of cellular ROS and adenosine triphosphate (ATP), mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu(BrHAP) 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu(II) compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS [40] . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential (MMP) fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu(BrHAP) 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu(II) Schiff base complexes may be associated with the mitochondrial pathway [26, 41, 42] . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP [30] . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets [43] . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 [44] . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 [45, 46] . In our study, the Cu(BrHAP) 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu(BrHAP) 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.

What types of cells are suitable for colon cancer studies?

Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2657001/ SHA: ef872b80cf38917f64c42bfa52a57beb4399897a Authors: Rao, Srinivas; Kong, Wing-Pui; Wei, Chih-Jen; Yang, Zhi-Yong; Nason, Martha; Styles, Darrel; DeTolla, Louis J.; Sorrell, Erin M.; Song, Haichen; Wan, Hongquan; Ramirez-Nieto, Gloria C.; Perez, Daniel; Nabel, Gary J. Date: 2008-06-18 DOI: 10.1371/journal.pone.0002432 License: cc0 Abstract: BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates. Text: The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. The greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers. DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . In human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, SARS, SIV and HIV. In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses [1] [2] [3] 6, 12, 13] . DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle (against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis) [14, 15] , pigs (against classical swine fever virus and mycoplasmosis) [16] , and horses (against West Nile virus and rabies) [17] . In addition, DNA vaccines have been tested against avian plasmodium infection in penguins [18] and against influenza and infectious bursal disease in chickens [7, 8, 19] , duck hepatitis B virus in ducks [6] , and avian metapneumovirus and Chlamydia psittaci in turkeys [20, 21] (reviewed in ref. [22] ). While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain [23] , there is only one previous report of a monovalent DNA vaccine effective against H5N1 (and that only against a matched H5N1 isolate) [24] ; no protection with multivalent DNA vaccines against heterologous strains has been reported. Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks. The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species [25] [26] [27] . While there is marked diversity in the host range of type A influenza viruses, many experts have speculated that a pandemic strain of type A influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment [28, 29] . Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses [7, 30, 31] . In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals [32] so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies. In this study, we used an automated high capacity needle-free injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO) to explore the feasibility of DNA vaccination of poultry. After optimization of injection conditions, alternative multivalent DNA vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of HPAI H5N1 infection. The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses. Immunogenicity and neutralizing antibody specificity of alternative HA DNA vaccines in mice To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice. Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses [33] were generated by synthesis of cDNAs (see Materials and Methods) in plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . Animals were immunized with each expression vector intramuscularly (IM) at three week intervals, and the antisera were evaluated on day 14 after the third immunization for their ability to neutralize HPAI H5N1 pseudotyped lentiviral vectors as previously described [35, 36] . We have previously shown that lentiviral assay inhibition (LAI) yields similar results to microneutralization and HAI analyses with higher sensitivity in mice [35, 36] To determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration (1.5 mg per immunogen) into groups of 10 mice (see Materials and Methods). Remarkably, despite a log lower DNA concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 H5 HA pseudoviruses at dilutions of up to 1:400 ( Fig. 2A) . To evaluate whether similar breadth of immunity could be generated with fewer immunogens, two different combinations of 5 immunogens were selected, based on the phylogenetic diversity of HA among the avian influenza viruses [33] and the crossreactivity of the neutralizing antibody responses of select individual immunogens (Fig. 1 ). As expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines (Fig. 2 , B vs. C). In one set, while neutralization of homologous strains was comparable to the monovalent and the 10 immunogen multivalent immune response, fewer cross-reactive antibodies were detected, directed most prominently against A/Iraq Protection of DNA-vaccinated mice against challenge with heterologous H5N1 A/Vietnam/1203/2004 influenza virus Mice immunized as described above were challenged with a heterologous H5N1 virus 68 weeks after the final DNA vaccination. Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. The control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection. Complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent DNA vaccines (Fig. 3) . Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%. In contrast, animals injected with HA plasmid DNA derived from A/ Anhui/1/2005, which has diverged more from A/Vietnam/ 1203/2004, showed a lower percent survival (70%) after lethal viral challenge. Survival differences between groups were assessed using a log-rank test and the Gehan-Wilcoxon test on the survival curves for pairs of groups. A test was deemed significant if the pvalue was ,0.01. Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA (Set 1), or 5 HA (Set 2) showed a significant difference compared to control (all p values,0.001). Among the HA-immunized groups, there was no significant difference between any two groups (p.0.08 for all comparisons). For example, no significant difference was observed between the A/Anhui/1/05 group, which had the least survival among the HA immunized groups (7 out of 10), and other HA groups: A/Indonesia/5/05 (p = 0.377), 10 HA (p = 0.082), 5 HA (Set 1) (p = 0.101), or 5 HA (Set 2) (p = .411). Therefore, we cannot exclude the possibility that the 3 deaths in the A/Anhui/1/05 group may have been due to random chance. Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens. The ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies (A/Vietnam/1203/2004, A/Anhui/ 1/2005 and A/Indonesia/05/2005), administered individually or in combination, by different injection methods. In addition to needle injection, a needle-free repetitive injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO), was analyzed. This device disperses the 0.1 to 5 ml injection doses into the dermal, subcutaneous, or intramuscular tissue depending upon the pressure adjustments, powered by a CO 2 gas pressure plunger [39] . The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues (Fig. S1 ). Immunization of chickens with the control plasmid (CMV/R) without an HA gene insert elicited minimal neutralizing antibody titers compared to HA-immunized animals 1 week after 3 DNA immunizations. Nearly all chickens immunized with either monovalent or multivalent HA DNA vaccines generated significant neutralization titers ( Fig. 4 and Table S1 ). In general, there was a progressive increase in the amount of neutralization after each successive DNA vaccination (data not shown) with maximal response at 1 week after the 3 rd DNA immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the Agro-JetH device. Neutralization of the Indonesia HA strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200. Both the monovalent and multivalent vaccines elicited robust homologous ( vaccine (Fig. 4 ). Even though one chicken (238) in the multivalent vaccine group produced almost the same degree of neutralization at each time point and was protected, it did not produce a high neutralizing antibody titer for reasons that were uncertain but possibly related to a non-specific inhibitor in the sera. To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice (n = 10) were immunized with 15 mg of plasmid (3 mg each) three times at 3 week intervals. Serum pools from the immunized animals were collected 14 days after the third immunization. The antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions. Error bars at each point indicate the standard deviation; each sample was evaluated in triplicate. In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. doi:10.1371/journal.pone.0002432.g002 inoculated with 20 LD 50 of highly pathogenic A/Vietnam/1203/ 2004 heterologous virus intranasally using standard methods [25, 40] and monitored for morbidity, mortality, viral shedding and serum antibodies. While all the control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens (Fig. 5A ). The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. There was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation (data not shown: egg infectious dose 50 (EID 50 ) limit of detection ,100 virus particles). To compare the relative efficacy of DNA vaccines delivered IM by needle and syringe versus the needle-free Agro-JetH device injection, a dose-response study was performed with amounts of DNA vaccine ranging from 500 to 0.5 mg with two inoculations. In these experiments, the HA derived from A/chicken/Nigeria/641/ 2006 was substituted for A/Vietnam/1203/2004 since it represented a more contemporary isolate. The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet (8/8) than by IM injection (6/8) (Fig. 5, B vs. C). Both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses (Fig. 5B, C) . Survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups. A test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg (p = .047). In the same group there was a significant difference between control and 5, 50 and 500 mg (p,.001 for all comparisons) and the difference between control and 0.5 mg was marginally significant (p = .016). Chickens that were injected using Agro-JetH showed a significant difference between 0.5 and 5 mg (p = .004) and between control and 5, 50, and 500 mg (p,.001 for all comparisons). There were no differences between control and 0.5 mg or between 5, 50, and 500 mg. Lastly, the survival differences between Agro-JetH and IM for each dose group were not significant. The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle (Table S2) . We assessed viable viral shedding after inoculation by chick embryo inoculation three days after virus challenge (Week 8). While we noted some embryonic lethality at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups (data not shown). Since the HPAI H5N1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in Asia, Africa, and Europe [28, [41] [42] [43] [44] . In addition to its effects on human health by crossspecies transmission [28, 45, 46] and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species. The pandemic potential of this virus, especially as it relates to the poultry industry and for reservoir avian hosts, underscores the need for a vaccine that offers broad spectrum immunity and protection against lethal viral challenge. While the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission [28, 47] , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain. One approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species. We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice [36] . DNA vaccines offer several advantages, including the ability to express diverse antigens, tolerability in various hosts, ease of delivery, and stability for storage and distribution without the necessity of maintaining a cold chain; they have been shown to be safe and efficacious in a variety of animal models [2, 4, 12, 22, 48] . Because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals. There is evidence that DNA vaccination elicits cell-mediated immunity against influenza HA in addition to inducing an antibody response [36] , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization. Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains. A multivalent H5 vaccine containing diverse serotypes could expand the antigenic breadth sufficiently to provide protection against heterologous challenge and may preclude the emergence of vaccine-resistant strains that may arise due to evolutionary vaccine pressure on the virus. Due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak. DNA vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. The mutations promote a focused and enhanced immune response [3, 49, 50] that may be particularly important in the event of an outbreak where specificity is the key to epidemic control. The use of modified codons ensures maximal expression in the host and eliminates the possibility of recombination with influenza viruses that might potentially generate new strains. A more broadly protective murine vaccine was developed here by including more HAs from varying strains in the multivalent vaccine (Figs. 2 and 3) . However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine. Therefore, we simplified the vaccine regimen based on cross-neutralization studies and phylogenetic relationships. A trivalent vaccine was subsequently identified for further studies. Due [51] . While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice [36] , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device. In addition, based on the chick embryo inoculation data, we believe that there is effective neutralization of the virus and lack of infectious viral shedding in chicken vaccinated with as little as 5 mg of DNA. The device's capacity for rapid repetitive injection and the lower quantity and stability of DNA enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals. A/Vietnam/1203/2004 (H5N1) (A/VN/1203/04) was obtained from the repository at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia. The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use. The virus was titrated by the Reed and Muench method to determine EID 50 [52] . GenBank ABD28180) were synthesized using human-preferred codons (GeneArt, Regensburg, Germany) [36] . HA cDNAs from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006. The immunogens used in DNA vaccination contained a cleavage site mutation (PQRERRRKKRG to PQRETRG) as previously described [35, 36] . This mutation was generated by site-directed mutagenesis using a QuickChange kit (Stratagene, La Jolla, CA). DNA immunization of mice [6] [7] [8] week old female BALB/c mice were purchased from The Jackson Laboratory and maintained in the AAALAC-accredited Vaccine Research Center Animal Care Facility (Bethesda, MD) under specific pathogen-free conditions. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee. The mice were immunized as previously described [5] . Briefly, mice (10 animals for all test groups, 20 animals for the The study was carried out in the AAALAC-accredited animal facility at the University of Maryland School of Medicine. Six groups of 8 one-day-old male and female SPAFAS White Leghorn Chickens, Gallus domesticus, were obtained from Charles River Laboratories (Connecticut). The animals were housed in brooder and grower cages (McMurray Hatcheries, Iowa). Feed (Teklad Japanese Quail Diet -3050, Harlan-Teklad, WI) and water were provided to the animals ad libitum. The study was performed in strict accordance with the ''Guide'' after approvals from the Animal Care and Use Committees of the Vaccine Research Center, NIH and the University of Maryland. DNA immunizations were performed as described at 0, 3 and 6 weeks. A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA Agro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry. The device is semiautomatic and requires a small CO 2 tank or compressed air for low pressure delivery. Upon trigger activation, CO 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use. We used an effective volume of 0.1 ml in our injectate [39] . In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi. Sixty-eight weeks after the last immunization, female BALB/c mice were lightly anesthetized with Ketamine/Xylazine and inoculated intranasally with 10 LD 50 of A/Vietnam/1203/2004 virus diluted in phosphate-buffered saline in a 50 ul volume. Mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection. Any mouse that had lost more than 25% of its body weight was euthanized. All experiments involving the HPAI virus were conducted in an AAALAC accredited facility (BioQual Inc., Gaithersburg, MD) under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program. White Leghorn chickens were challenged one week after the last immunization with 20 lethal dose 50 (LD 50 ) of A/Vietnam/1203/04 (H5N1) influenza A virus, equivalent to 2610 4 EID 50 based on previous challenges [53] . Chickens were infected with 200 ml virus intranasally. Tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing BHI medium (BBL TM Brain Heart Infusion, Becton Dickinson) at 280uC. Blood was collected 14 days post-challenge and serum was titered by microneutralization assay. Chickens were observed and scored daily for clinical signs of infection, morbidity and mortality. Chickens that survived the study were bled and humanely euthanized at day 14 post-challenge. Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. Experiments were carried out under BSL3+ conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal Care and Use Committee-approved protocols and under Animal Welfare Act regulations at the University of Maryland, College Park, Maryland. Representative tracheal and cloacal swabs were chosen to run an EID 50 assay for comparison and virus titers were determine by the method of Reed and Meunch [52] . Briefly, swabs were used to infect 10 day-old embryonated chicken eggs in 10-fold dilutions. Three eggs were inoculated per dilution and incubated for 48 hours before titration. Neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge. The microneutralization assay was performed using a 96-well plate format. Serum was treated with receptor-destroying enzyme (Denka Seiken Co.) and treated at 37uC per the manufacturer's instructions. After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well. The plates were then incubated at 37uC, 5% CO 2 for 1-2 hours. Following incubation, supernatants were used to infect a second 96-well plate of MDCK cells. Microplates were incubated at 4uC for 15 minutes and then 37uC, 5% CO 2 for 45 minutes. Supernatants of serum and virus were then discarded and 200 ml of OptiMEM (containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin) was added and incubated at 37uC, 5% CO 2 for 3 days. After 3 days, 50 ml of the supernatant from each well was transferred into a new 96-well microplate, and an HA assay was performed to calculate the antibody titers. Virus and cell controls were included in the assay. Two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay as previously described [54] for the presence of antibodies that neutralized the infectivity of 100 TCID 50 (50% tissue culture infectious dose) of the A/Vietnam/ 1203/2004 H5N1 virus on MDCK cell monolayers by using two wells per dilution on a 96-well plate. The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described [35, 36] . For the neutralization assay, antisera from immunized animals were heat-inactivated at 55uC for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions. The sera/virus mixture was then added to 293A cells in 96-well B&W TC Isoplates (Wallac, Turku, Finland; 12,000 cells/well). Two hours later, the plates were washed and fresh medium was added. Cells were lysed in mammalian cell lysis buffer (Promega, Madison, WI) 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI). The following strains were used for the production of pseudotyped viruses: for HA we used A/Thailand/1(KAN- The HA/HI titers were determined as previously described [54] . Briefly, HA titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in PBS added to 50 ml of twofold dilutions of virus in PBS. This mix was incubated at room temperature for 30 minutes. The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of A/Vietnam/1203/2004 virus (four hemagglutinating doses) was added to each well. Wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added. HI titers were calculated after 30 minutes as the reciprocal of the serum dilution that inhibited hemagglutination. Table S1 Hemagglutination inhibition (HI), microneutralization titer (NT), and LAI of sera from individual chickens immunized with different vaccines. Sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by HI, microneutralization (NT) and LAI (shown as IC 50 ). Individual animal serum of each group is shown and was analyzed as described in the Materials and Methods section. Figure S1 Characterization of needle-free (Agro-JetH) DNA immunization in chickens. To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg. Three sites (thigh, wing and breast) were used, and biopsies were taken for routine hematoxylin and eosin staining. Representative sections of thigh injections are shown from 7 week old chickens and were similar at 4 weeks (data not shown). While the 48 mm Hg pressure deposited the injectate into the dermis/subcutaneous region (left), the higher pressure injections, 52 and 58 mm Hg, deposited the injectate into the subcutaneous and muscle layers (middle, right). 48 mm Hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all AgroJetH immunizations. Found at: doi:10.1371/journal.pone.0002432.s003 (10.74 MB DOC)

What was the goal of this study?

Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2657001/ SHA: ef872b80cf38917f64c42bfa52a57beb4399897a Authors: Rao, Srinivas; Kong, Wing-Pui; Wei, Chih-Jen; Yang, Zhi-Yong; Nason, Martha; Styles, Darrel; DeTolla, Louis J.; Sorrell, Erin M.; Song, Haichen; Wan, Hongquan; Ramirez-Nieto, Gloria C.; Perez, Daniel; Nabel, Gary J. Date: 2008-06-18 DOI: 10.1371/journal.pone.0002432 License: cc0 Abstract: BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates. Text: The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. The greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers. DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . In human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, SARS, SIV and HIV. In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses [1] [2] [3] 6, 12, 13] . DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle (against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis) [14, 15] , pigs (against classical swine fever virus and mycoplasmosis) [16] , and horses (against West Nile virus and rabies) [17] . In addition, DNA vaccines have been tested against avian plasmodium infection in penguins [18] and against influenza and infectious bursal disease in chickens [7, 8, 19] , duck hepatitis B virus in ducks [6] , and avian metapneumovirus and Chlamydia psittaci in turkeys [20, 21] (reviewed in ref. [22] ). While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain [23] , there is only one previous report of a monovalent DNA vaccine effective against H5N1 (and that only against a matched H5N1 isolate) [24] ; no protection with multivalent DNA vaccines against heterologous strains has been reported. Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks. The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species [25] [26] [27] . While there is marked diversity in the host range of type A influenza viruses, many experts have speculated that a pandemic strain of type A influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment [28, 29] . Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses [7, 30, 31] . In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals [32] so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies. In this study, we used an automated high capacity needle-free injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO) to explore the feasibility of DNA vaccination of poultry. After optimization of injection conditions, alternative multivalent DNA vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of HPAI H5N1 infection. The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses. Immunogenicity and neutralizing antibody specificity of alternative HA DNA vaccines in mice To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice. Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses [33] were generated by synthesis of cDNAs (see Materials and Methods) in plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . Animals were immunized with each expression vector intramuscularly (IM) at three week intervals, and the antisera were evaluated on day 14 after the third immunization for their ability to neutralize HPAI H5N1 pseudotyped lentiviral vectors as previously described [35, 36] . We have previously shown that lentiviral assay inhibition (LAI) yields similar results to microneutralization and HAI analyses with higher sensitivity in mice [35, 36] To determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration (1.5 mg per immunogen) into groups of 10 mice (see Materials and Methods). Remarkably, despite a log lower DNA concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 H5 HA pseudoviruses at dilutions of up to 1:400 ( Fig. 2A) . To evaluate whether similar breadth of immunity could be generated with fewer immunogens, two different combinations of 5 immunogens were selected, based on the phylogenetic diversity of HA among the avian influenza viruses [33] and the crossreactivity of the neutralizing antibody responses of select individual immunogens (Fig. 1 ). As expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines (Fig. 2 , B vs. C). In one set, while neutralization of homologous strains was comparable to the monovalent and the 10 immunogen multivalent immune response, fewer cross-reactive antibodies were detected, directed most prominently against A/Iraq Protection of DNA-vaccinated mice against challenge with heterologous H5N1 A/Vietnam/1203/2004 influenza virus Mice immunized as described above were challenged with a heterologous H5N1 virus 68 weeks after the final DNA vaccination. Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. The control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection. Complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent DNA vaccines (Fig. 3) . Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%. In contrast, animals injected with HA plasmid DNA derived from A/ Anhui/1/2005, which has diverged more from A/Vietnam/ 1203/2004, showed a lower percent survival (70%) after lethal viral challenge. Survival differences between groups were assessed using a log-rank test and the Gehan-Wilcoxon test on the survival curves for pairs of groups. A test was deemed significant if the pvalue was ,0.01. Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA (Set 1), or 5 HA (Set 2) showed a significant difference compared to control (all p values,0.001). Among the HA-immunized groups, there was no significant difference between any two groups (p.0.08 for all comparisons). For example, no significant difference was observed between the A/Anhui/1/05 group, which had the least survival among the HA immunized groups (7 out of 10), and other HA groups: A/Indonesia/5/05 (p = 0.377), 10 HA (p = 0.082), 5 HA (Set 1) (p = 0.101), or 5 HA (Set 2) (p = .411). Therefore, we cannot exclude the possibility that the 3 deaths in the A/Anhui/1/05 group may have been due to random chance. Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens. The ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies (A/Vietnam/1203/2004, A/Anhui/ 1/2005 and A/Indonesia/05/2005), administered individually or in combination, by different injection methods. In addition to needle injection, a needle-free repetitive injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO), was analyzed. This device disperses the 0.1 to 5 ml injection doses into the dermal, subcutaneous, or intramuscular tissue depending upon the pressure adjustments, powered by a CO 2 gas pressure plunger [39] . The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues (Fig. S1 ). Immunization of chickens with the control plasmid (CMV/R) without an HA gene insert elicited minimal neutralizing antibody titers compared to HA-immunized animals 1 week after 3 DNA immunizations. Nearly all chickens immunized with either monovalent or multivalent HA DNA vaccines generated significant neutralization titers ( Fig. 4 and Table S1 ). In general, there was a progressive increase in the amount of neutralization after each successive DNA vaccination (data not shown) with maximal response at 1 week after the 3 rd DNA immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the Agro-JetH device. Neutralization of the Indonesia HA strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200. Both the monovalent and multivalent vaccines elicited robust homologous ( vaccine (Fig. 4 ). Even though one chicken (238) in the multivalent vaccine group produced almost the same degree of neutralization at each time point and was protected, it did not produce a high neutralizing antibody titer for reasons that were uncertain but possibly related to a non-specific inhibitor in the sera. To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice (n = 10) were immunized with 15 mg of plasmid (3 mg each) three times at 3 week intervals. Serum pools from the immunized animals were collected 14 days after the third immunization. The antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions. Error bars at each point indicate the standard deviation; each sample was evaluated in triplicate. In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. doi:10.1371/journal.pone.0002432.g002 inoculated with 20 LD 50 of highly pathogenic A/Vietnam/1203/ 2004 heterologous virus intranasally using standard methods [25, 40] and monitored for morbidity, mortality, viral shedding and serum antibodies. While all the control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens (Fig. 5A ). The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. There was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation (data not shown: egg infectious dose 50 (EID 50 ) limit of detection ,100 virus particles). To compare the relative efficacy of DNA vaccines delivered IM by needle and syringe versus the needle-free Agro-JetH device injection, a dose-response study was performed with amounts of DNA vaccine ranging from 500 to 0.5 mg with two inoculations. In these experiments, the HA derived from A/chicken/Nigeria/641/ 2006 was substituted for A/Vietnam/1203/2004 since it represented a more contemporary isolate. The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet (8/8) than by IM injection (6/8) (Fig. 5, B vs. C). Both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses (Fig. 5B, C) . Survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups. A test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg (p = .047). In the same group there was a significant difference between control and 5, 50 and 500 mg (p,.001 for all comparisons) and the difference between control and 0.5 mg was marginally significant (p = .016). Chickens that were injected using Agro-JetH showed a significant difference between 0.5 and 5 mg (p = .004) and between control and 5, 50, and 500 mg (p,.001 for all comparisons). There were no differences between control and 0.5 mg or between 5, 50, and 500 mg. Lastly, the survival differences between Agro-JetH and IM for each dose group were not significant. The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle (Table S2) . We assessed viable viral shedding after inoculation by chick embryo inoculation three days after virus challenge (Week 8). While we noted some embryonic lethality at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups (data not shown). Since the HPAI H5N1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in Asia, Africa, and Europe [28, [41] [42] [43] [44] . In addition to its effects on human health by crossspecies transmission [28, 45, 46] and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species. The pandemic potential of this virus, especially as it relates to the poultry industry and for reservoir avian hosts, underscores the need for a vaccine that offers broad spectrum immunity and protection against lethal viral challenge. While the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission [28, 47] , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain. One approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species. We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice [36] . DNA vaccines offer several advantages, including the ability to express diverse antigens, tolerability in various hosts, ease of delivery, and stability for storage and distribution without the necessity of maintaining a cold chain; they have been shown to be safe and efficacious in a variety of animal models [2, 4, 12, 22, 48] . Because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals. There is evidence that DNA vaccination elicits cell-mediated immunity against influenza HA in addition to inducing an antibody response [36] , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization. Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains. A multivalent H5 vaccine containing diverse serotypes could expand the antigenic breadth sufficiently to provide protection against heterologous challenge and may preclude the emergence of vaccine-resistant strains that may arise due to evolutionary vaccine pressure on the virus. Due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak. DNA vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. The mutations promote a focused and enhanced immune response [3, 49, 50] that may be particularly important in the event of an outbreak where specificity is the key to epidemic control. The use of modified codons ensures maximal expression in the host and eliminates the possibility of recombination with influenza viruses that might potentially generate new strains. A more broadly protective murine vaccine was developed here by including more HAs from varying strains in the multivalent vaccine (Figs. 2 and 3) . However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine. Therefore, we simplified the vaccine regimen based on cross-neutralization studies and phylogenetic relationships. A trivalent vaccine was subsequently identified for further studies. Due [51] . While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice [36] , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device. In addition, based on the chick embryo inoculation data, we believe that there is effective neutralization of the virus and lack of infectious viral shedding in chicken vaccinated with as little as 5 mg of DNA. The device's capacity for rapid repetitive injection and the lower quantity and stability of DNA enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals. A/Vietnam/1203/2004 (H5N1) (A/VN/1203/04) was obtained from the repository at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia. The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use. The virus was titrated by the Reed and Muench method to determine EID 50 [52] . GenBank ABD28180) were synthesized using human-preferred codons (GeneArt, Regensburg, Germany) [36] . HA cDNAs from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006. The immunogens used in DNA vaccination contained a cleavage site mutation (PQRERRRKKRG to PQRETRG) as previously described [35, 36] . This mutation was generated by site-directed mutagenesis using a QuickChange kit (Stratagene, La Jolla, CA). DNA immunization of mice [6] [7] [8] week old female BALB/c mice were purchased from The Jackson Laboratory and maintained in the AAALAC-accredited Vaccine Research Center Animal Care Facility (Bethesda, MD) under specific pathogen-free conditions. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee. The mice were immunized as previously described [5] . Briefly, mice (10 animals for all test groups, 20 animals for the The study was carried out in the AAALAC-accredited animal facility at the University of Maryland School of Medicine. Six groups of 8 one-day-old male and female SPAFAS White Leghorn Chickens, Gallus domesticus, were obtained from Charles River Laboratories (Connecticut). The animals were housed in brooder and grower cages (McMurray Hatcheries, Iowa). Feed (Teklad Japanese Quail Diet -3050, Harlan-Teklad, WI) and water were provided to the animals ad libitum. The study was performed in strict accordance with the ''Guide'' after approvals from the Animal Care and Use Committees of the Vaccine Research Center, NIH and the University of Maryland. DNA immunizations were performed as described at 0, 3 and 6 weeks. A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA Agro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry. The device is semiautomatic and requires a small CO 2 tank or compressed air for low pressure delivery. Upon trigger activation, CO 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use. We used an effective volume of 0.1 ml in our injectate [39] . In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi. Sixty-eight weeks after the last immunization, female BALB/c mice were lightly anesthetized with Ketamine/Xylazine and inoculated intranasally with 10 LD 50 of A/Vietnam/1203/2004 virus diluted in phosphate-buffered saline in a 50 ul volume. Mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection. Any mouse that had lost more than 25% of its body weight was euthanized. All experiments involving the HPAI virus were conducted in an AAALAC accredited facility (BioQual Inc., Gaithersburg, MD) under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program. White Leghorn chickens were challenged one week after the last immunization with 20 lethal dose 50 (LD 50 ) of A/Vietnam/1203/04 (H5N1) influenza A virus, equivalent to 2610 4 EID 50 based on previous challenges [53] . Chickens were infected with 200 ml virus intranasally. Tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing BHI medium (BBL TM Brain Heart Infusion, Becton Dickinson) at 280uC. Blood was collected 14 days post-challenge and serum was titered by microneutralization assay. Chickens were observed and scored daily for clinical signs of infection, morbidity and mortality. Chickens that survived the study were bled and humanely euthanized at day 14 post-challenge. Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. Experiments were carried out under BSL3+ conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal Care and Use Committee-approved protocols and under Animal Welfare Act regulations at the University of Maryland, College Park, Maryland. Representative tracheal and cloacal swabs were chosen to run an EID 50 assay for comparison and virus titers were determine by the method of Reed and Meunch [52] . Briefly, swabs were used to infect 10 day-old embryonated chicken eggs in 10-fold dilutions. Three eggs were inoculated per dilution and incubated for 48 hours before titration. Neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge. The microneutralization assay was performed using a 96-well plate format. Serum was treated with receptor-destroying enzyme (Denka Seiken Co.) and treated at 37uC per the manufacturer's instructions. After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well. The plates were then incubated at 37uC, 5% CO 2 for 1-2 hours. Following incubation, supernatants were used to infect a second 96-well plate of MDCK cells. Microplates were incubated at 4uC for 15 minutes and then 37uC, 5% CO 2 for 45 minutes. Supernatants of serum and virus were then discarded and 200 ml of OptiMEM (containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin) was added and incubated at 37uC, 5% CO 2 for 3 days. After 3 days, 50 ml of the supernatant from each well was transferred into a new 96-well microplate, and an HA assay was performed to calculate the antibody titers. Virus and cell controls were included in the assay. Two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay as previously described [54] for the presence of antibodies that neutralized the infectivity of 100 TCID 50 (50% tissue culture infectious dose) of the A/Vietnam/ 1203/2004 H5N1 virus on MDCK cell monolayers by using two wells per dilution on a 96-well plate. The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described [35, 36] . For the neutralization assay, antisera from immunized animals were heat-inactivated at 55uC for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions. The sera/virus mixture was then added to 293A cells in 96-well B&W TC Isoplates (Wallac, Turku, Finland; 12,000 cells/well). Two hours later, the plates were washed and fresh medium was added. Cells were lysed in mammalian cell lysis buffer (Promega, Madison, WI) 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI). The following strains were used for the production of pseudotyped viruses: for HA we used A/Thailand/1(KAN- The HA/HI titers were determined as previously described [54] . Briefly, HA titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in PBS added to 50 ml of twofold dilutions of virus in PBS. This mix was incubated at room temperature for 30 minutes. The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of A/Vietnam/1203/2004 virus (four hemagglutinating doses) was added to each well. Wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added. HI titers were calculated after 30 minutes as the reciprocal of the serum dilution that inhibited hemagglutination. Table S1 Hemagglutination inhibition (HI), microneutralization titer (NT), and LAI of sera from individual chickens immunized with different vaccines. Sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by HI, microneutralization (NT) and LAI (shown as IC 50 ). Individual animal serum of each group is shown and was analyzed as described in the Materials and Methods section. Figure S1 Characterization of needle-free (Agro-JetH) DNA immunization in chickens. To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg. Three sites (thigh, wing and breast) were used, and biopsies were taken for routine hematoxylin and eosin staining. Representative sections of thigh injections are shown from 7 week old chickens and were similar at 4 weeks (data not shown). While the 48 mm Hg pressure deposited the injectate into the dermis/subcutaneous region (left), the higher pressure injections, 52 and 58 mm Hg, deposited the injectate into the subcutaneous and muscle layers (middle, right). 48 mm Hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all AgroJetH immunizations. Found at: doi:10.1371/journal.pone.0002432.s003 (10.74 MB DOC)

What is the conclusion of this study?

Metabolic engineering of Escherichia coli into a versatile glycosylation platform: production of bio-active quercetin glycosides https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573293/ SHA: f4cd52975e6aa33e8c947082eda9b261952b0f8f Authors: De Bruyn, Frederik; Van Brempt, Maarten; Maertens, Jo; Van Bellegem, Wouter; Duchi, Dries; De Mey, Marjan Date: 2015-09-16 DOI: 10.1186/s12934-015-0326-1 License: cc-by Abstract: BACKGROUND: Flavonoids are bio-active specialized plant metabolites which mainly occur as different glycosides. Due to the increasing market demand, various biotechnological approaches have been developed which use Escherichia coli as a microbial catalyst for the stereospecific glycosylation of flavonoids. Despite these efforts, most processes still display low production rates and titers, which render them unsuitable for large-scale applications. RESULTS: In this contribution, we expanded a previously developed in vivo glucosylation platform in E. coli W, into an efficient system for selective galactosylation and rhamnosylation. The rational of the novel metabolic engineering strategy constitutes of the introduction of an alternative sucrose metabolism in the form of a sucrose phosphorylase, which cleaves sucrose into fructose and glucose 1-phosphate as precursor for UDP-glucose. To preserve these intermediates for glycosylation purposes, metabolization reactions were knocked-out. Due to the pivotal role of UDP-glucose, overexpression of the interconverting enzymes galE and MUM4 ensured the formation of both UDP-galactose and UDP-rhamnose, respectively. By additionally supplying exogenously fed quercetin and overexpressing a flavonol galactosyltransferase (F3GT) or a rhamnosyltransferase (RhaGT), 0.94 g/L hyperoside (quercetin 3-O-galactoside) and 1.12 g/L quercitrin (quercetin 3-O-rhamnoside) could be produced, respectively. In addition, both strains showed activity towards other promising dietary flavonols like kaempferol, fisetin, morin and myricetin. CONCLUSIONS: Two E. coli W mutants were engineered that could effectively produce the bio-active flavonol glycosides hyperoside and quercitrin starting from the cheap substrates sucrose and quercetin. This novel fermentation-based glycosylation strategy will allow the economically viable production of various glycosides. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0326-1) contains supplementary material, which is available to authorized users. Text: Flavonoids are a class of plant secondary metabolites, which are chemically characterized by a 15-carbon backbone that consists of two phenyl rings and a heterocyclic ring. To date, over 10,000 flavonoids have been characterized from various plants, which are classified according to their chemical structure, i.e., the number and presence of hydroxyl groups and further functional group modifications into various subgroups, such as anthoxanthins, flavanones, and flavanonols [1, 2] . In recent years flavonoids have garnered much attention from various application domains because of the various beneficial effects on human health that have been attributed to them, such as anticancer [3] and antioxidant [4] to anti-inflammatory [5] , antimicrobial [6] and antiviral [6, 7] effects. As final step in their biosynthesis, flavonoids are often glycosylated which has a profound effect on their solubility, stability and bio-activity [8, 9] . For example, the best studied flavonol quercetin, which makes up to 75 % of our daily flavonoid intake, predominantly occurs as different glycosides. Over 350 different quercetin glycoforms have been reported to date with varying pharmacological properties [10, 11] . In this context, hyperoside (quercetin 3-O-galactoside) and quercitrin (quercetin 3-O-rhamnoside) ( Fig. 1) have gained a lot of attention as valuable products for the pharmaceutical industry e.g., as powerful antioxidants with cytoprotective effects [12] [13] [14] [15] and as promising antiviral agents that block replication of the influenza virus [16] or inhibit the viruses hepatitis B [17] and SARS [18] . Furthermore, they have been attributed with anti-inflammatory [19, 20] , antidepressant [21, 22] , apoptotic [23] and antifungal [24] activities, rendering them interesting therapeutics resulting in a steadily increasing market demand. To date, the majority of quercetin and its glycosides are extracted from plant material, which is generally a laborious and low-yielding process requiring many purification steps [25] . In vitro plant cell cultures or engineered plants can be used to overcome the low yields and improve production [26] [27] [28] , however since metabolic engineering of plants is both very controversial and still in its infancy [29] , this approach is often restricted to small-scale production. Although chemical synthesis of quercetin (glycosides) has proven to be feasible [30] [31] [32] , stereoselective formation of glycosidic linkages is often hampered by the presence of various reactive groups [33] , which requires many protecting and deprotecting steps [34] . In addition, the generation of toxic waste and a low atomefficiency [35] render these production processes neither sustainable nor economically viable. As a result, in the last two decades enormous efforts have been invested in the development of alternative production methods for these specialized (secondary) plant metabolites [36] . Advances in the fields of protein engineering, systems and synthetic biology have accelerated these efforts to transform model organisms like Escherichia coli and Saccharomyces cerevisiae in real microbial cell factories for the sustainable production of flavonoids [37] [38] [39] . Subsequently, strategies for the in vivo glycosylation of flavonoids have also been developed. These are typically based on both the overexpression of specific glycosyltransferases, which transfer a sugar residue from an activated nucleotide sugar to an aglycon in a stereoand regioselective way, and the engineering or introduction of the targeted nucleotide sugar pathway. In this way, Fig. 1 Transformation of E. coli W into a sucrose-based galactosylation and rhamnosylation platform. The metabolic engineering strategy applied makes use of several gene deletions (indicated in red) and overexpressions of genes (indicated in green). The rational of a split metabolism is applied, whereby sucrose is divided by sucrose phosphorylase (BaSP) in fructose to be used for growth and a glucose 1-phosphate as activated precursor for UDP-glucose. The latter is a universal pivot molecule for the formation of UDP-galactose and UDP-rhamnose, interconversions catalyzed by the enzymes GalE and MUM4, respectively. To ensure growth-coupled production, various genes, involved in the metabolization of these UDPsugars and their precursors, were knocked out (shown in red). The production of the bioactive quercetin glycosides hyperoside and quercitrin was chosen to evaluate the versatility of the engineered production platform. Finally, the introduction of either the glycosyltransferase F3GT or RhaGT ensures efficient galactosylation or rhamnosylation, respectively various quercetin glycosides have already been produced in E. coli such as the naturally occurring 3-O-glucoside [40] , 3-O-xyloside [41] and 3,7-O-bisrhamnoside [42] , or the new-to-nature quercetin 3-O-(6-deoxytalose) [43] . However, despite these engineering efforts, the reported product rates and titers are still in the milligram range, rendering these microbial production hosts unsuitable for industrial applications. The developed production processes are typically biphasic bioconversion processes using resting cells, which makes it difficult to improve production rates [44] . Furthermore, such systems often entail expensive growth media or the addition of enzyme inducers, making the overall process very costly. To tackle these problems, we previously developed an efficient platform for the glucosylation of small molecules in E. coli W [45] . Through metabolic engineering, a mutant was created which couples the production of glucosides to growth, using sucrose as a cheap and sustainable carbon source. By introducing the sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) sucrose can be split into fructose to be used for growth purposes and glucose 1-phosphate (glc1P) to be used as precursor for UDP-glucose (UDP-glc) formation ( Fig. 1) . To impede the conversion of glc1P into biomass precursors, several endogenous genes involved in its metabolization such as phosphoglucomutase (pgm) and glucose-1-phosphatase (agp) were knocked out. Subsequently, glc1P can efficiently be channeled towards UDP-glc by overexpressing the uridylyltransferase from Bifidobacterium bifidum (ugpA). Metabolization of UDP-glc is prevented by knocking out the UDP-sugar hydrolase (ushA) and the galactose operon (galETKM). However, in view of the pivotal role of UDP-glc in the production of a large variety of UDP-sugars, this glucosylation system can easily be extended towards other UDP-sugars, such as UDP-galactose (UDP-gal), UDPrhamnose (UDP-rha) and UDP-glucuronate. In the present contribution, this previously developed E. coli W-based glucosylation platform is transformed into a platform for galactosylation and rhamnosylation ( Fig. 1) , whose potential is demonstrated using the galactosylation and rhamnosylation of exogenously fed quercetin yielding hyperoside and quercitrin, respectively, as case study. Escherichia coli W is a fast-growing non-pathogenic strain which tolerates osmotic stress, acidic conditions, and can be cultured to high cell densities, making it an attractive host for industrial fermentations [46] . Moreover, E. coli W is able to grow on sucrose as sole carbon source [46] , which is an emerging feedstock for the production of bio-products. Hence, E. coli W was selected as host for sucrose-based in vivo glycosylation. Prior to the production of the glycosides hyperoside and quercitrin in E. coli W, the toxicity of their aglycon quercetin was investigated. To this end, the wild type (WT) strain was grown on minimal sucrose medium containing different concentrations of quercetin (0, 0.15 and 1.5 g/L). The specific growth rates (h −1 ) (0.96 ± 0.06, 0.92 ± 0.05 and 0.87 ± 0.06, respectively) were not significantly different (p ANOVA = 0.12) nor from the one previously determined for the WT [45] (p = 0.69, p = 0.98 and p = 0.68, respectively). On the other hand, the optical density at 600 nm after 24 h incubation (6.36 ± 0.12, 5.18 ± 0.16 and 4.77 ± 0.20, respectively) was lower (about 20 %) when quercetin was added (p = 0.0002 and p = 0.0001). No significant difference in optical density could be observed between 0.15 and 1.5 g/L quercetin (p = 0.14). In view of the above, it was opted to add 1.5 g/L quercetin to evaluate the potential of the developed glycosylation platform. To evaluate the in vivo glycosylation potential, strains sGAL1 and sRHA1, which constitutively express the flavonol 3-O-galactosyltransferase from Petunia hybrida and the flavonol 3-O-rhamnosyltransferase from A. thaliana, respectively, were cultured in minimal medium with 1.5 g/L of quercetin for 16 h. TLC analysis of the supernatants of both cultures yielded two new yellow product spots. The TLC spot obtained from the sGAL1 culture, which had the same retention time as the hyperoside standard (R f = 0.5), was subsequently purified and analyzed. Both NMR and MS analysis confirmed the production of quercetin 3-O-galactoside. However, the product spot obtained from the sRHA1 culture had a different retention factor (R f = 0.55) than the quercitrin standard (R f = 0.74), and was identified as isoquercitrin (quercetin 3-O-glucoside). As opposed to other reports on wild type E. coli strains expressing RhaGT, which simultaneously produced quercitrin (quercetin 3-O-rhamnoside) and isoquercitrin [47, 48] , no rhamnoside could be detected. Examination of the E. coli W genome revealed that the gene cluster responsible for the endogenous production of dTDP-rhamnose, which functions as an alternative rhamnosyldonor for RhaGT in E. coli B and K12 derivatives [47] , was not present [46, 49] . In a follow-up experiment, sGAL1 and sRHA1 were grown on minimal medium with two different concentrations (0.15 and 1.5 g/L) of quercetin. Growth and glycoside formation were monitored during 30 h. The final titers (C p ) and specific productivities (q p ) are shown in Fig. 2 . Remarkably, an increase in quercetin concentration resulted in a two to threefold increase in productivity and titer, indicating that quercetin supply is rate-limiting and crucial for efficient in vivo glycosylation. However, while sGAL1 continuously produced hyperoside during the exponential phase, which is also reflected in the relatively high specific productivity, sRHA1 only started to accumulate significant amounts of isoquercitrin at the end of the exponential phase. This production start coincides with a reduction in specific growth rate, which dropped from 0.35 ± 0.04 to 0.06 ± 0.01 h −1 . As described in detail in the Background section, we previously metabolically engineered E. coli W to create a platform for in vivo glucosylation of small molecules [45] . In the original base glucosylation strain, sucrose phosphorylase encoded by BaSP was located on a mediumcopy plasmid and transcribed from a medium-strong constitutive promoter (P22) [50] . For reasons of comparison and flexibility, it was opted to integrate BaSP in the genome of E. coli W. In addition, chromosomal integration is advantageous because of a significant increase in gene stability. Since the level of gene expression can considerably be impacted by the genome integration site [51] due to structural differences such as supercoiling DNA regions, two different DNA sites were assessed for BaSP integration, i.e., melA and glgC, which encode an α-galactosidase and a glucose-1-phosphate adenylyltransferase, respectively. To this end, an adapted knockin-knockout procedure for large DNA fragments was applied, which is schematically shown in Additional file 1: Figure S2 . BaSP under control of promoter P22 was knocked in at the two different loci in E. coli W ΔcscAR, which resulted in the E. coli W strains ΔcscAR ΔmelA::L4-P22-BaSP-L5 and ΔcscAR ΔglgC::L4-P22-BaSP-L5. Their maximal specific growth rate (µ max ) on minimal sucrose medium, which is shown in Fig. 3 , was compared to the original strain ΔcscAR + pBaSP. The influence of the knockin locus on the maximal specific growth rate is clear. Interestingly, integration at the melA locus resulted in a strain with a µ max which was not significantly different from the reference strain ΔcscAR + pBaSP. In view of the latter and considering the aimed growth-coupled production, it was opted to integrate BaSP at the melA locus leading to the final production base strain E. coli W ΔcscAR Δpgm Δagp ΔushA ΔlacZYA::P22-lacY ΔgalETKM ΔmelA::L4-P22-BaSP-L5 (sGLYC) as shown in Table 1 . In nature, UDP-glc serves as a pivot molecule in the formation of a variety of UDP-sugars [44] . For example, using the interconverting enzymes UDP-glucose 4-epimerase (GalE) and UDP-rhamnose synthase (MUM4) UDP-glc can be converted to UDP-gal and UDP-rha, respectively. Though GalE is natively present in E. coli W an alternative homologous epimerase (GalE2) from B. bifidum was also selected and cloned due to the Fig. 2 Comparison of the specific glycoside productivities (q p ) and glycoside titers (C p ) for strains sGAL1, which produces the 3-O-galactoside, and sRHA1, which produces the 3-O-glucoside, when grown for 30 h on minimal medium containing 0.15 or 1.5 g/L of quercetin. Error bars represent standard deviations tight and complex regulation of GalE expression in E. coli W. On the other hand, UDP-rhamnose synthesis is restricted to plants. Due to lack of the rfb cluster [46] E. coli W is even unable to form endogenous dTDP-rhamnose as alternative rhamnosyl donor. Hence, the MUM4 gene from A. thaliana was expressed from plasmid pMUM4 to achieve UDP-rhamnose formation in E. coli (Fig. 1) . The constructed galactosylation (sGAL) and rhamnosylation (sRHA) strains were grown on minimal medium with two levels (0.15 and 1.5 g/L) of quercetin. Growth and production were monitored to determine the specific productivities, as shown in Fig. 4 . Again, higher extracellular quercetin concentrations resulted in a fivefold increase in q p . However, no significant difference in productivity was observed between sGAL2 and sGAL3 at 1.5 g/L quercetin, indicating that UDP-galactose formation is as efficient with both GalE homologs and not likely the rate limiting step. With sGAL3, the highest hyperoside productivity (68.7 mg/g CDW/h) and titer (0.94 g/L) were obtained, the latter being 3.5-fold higher compared to sGAL1. In contrast to sRHA1, TLC analysis of the supernatant of the cultures of sRHA2 and sRHA3 resulted in a product spot with a retention factor that corresponds to quercitrin, which was confirmed by MS analysis, thus showing in vivo activity of MUM4. A quercitrin titer of 1.18 g/L and specific productivity of 47.8 mg/g CDW/h were obtained after 30 h incubation of sRHA3 when 1.5 g/L quercetin was added to the medium, which corresponded to a 53 % conversion. Also 51 mg/L of isoquercitrin was produced extracellularly which corresponds with a quercitrin:isoquercitrin production ratio of 24:1. This suggests the preference of RhaGT for UDP-rhamnose when different UDP-sugar donors are present. Possible explanations for the significantly lower specific productivity (fivefold decrease) of sRHA2 as compared to sRHA3 are either a higher metabolic burden [52] caused by the two plasmid system or a too limited activity of the native GalU, which could be insufficient for adequate UDP-glc formation [45] . To demonstrate the scalability of the developed bioprocess, strain sGAL3 was cultured in a 1-L bioreactor, which also ensures a constant pH set at 6.80 and avoid oxygen limitation. A detailed overview of the consumption of sucrose, growth and hyperoside production is given in Fig. 5 . After a lag-phase, the strain displayed a growth rate of 0.32 ± 0.02 h −1 while simultaneously producing hyperoside. The observed specific productivity (65.9 ± 2.6 mg/g CDW/h) was comparable to the one obtained on shake flask scale. When nearly all quercetin was converted, hyperoside formation slowed down, which can be explained either by the observed correlation between quercetin concentration and q p , or by the reported reversibility of F3GT [53] . It is likely that further improvements in titer and productivity can be realized by optimizing the supply of quercetin using a fed-batch system. To the best of our knowledge, the results obtained in this study with the engineered sGAL and sRHA strains for the production of hyperoside and quercitrin are the highest reported to date both in terms of titer and production rate. The maximal production rate obtained in this contribution was 6 to 50-fold higher compared to the maximal production rates (r p,max ) of processes reported in the literature [47, 54] as is illustrated in Fig. 6 . The increased performance, in terms of titer and productivity, obtained with the developed platform can be attributed to the use of a split metabolism in combination with optimally rerouting the flux from glucose 1-phosphate towards UDP-galactose and UDP-rhamnose. The undesired conversion of the activated sugars into biomass Fig. 3 Effect of the chromosomal integration locus of the knockin of BaSP on the growth rate. Strains were grown in shake flasks and the resulting maximal growth rates (µ max ) were compared with E. coli W ΔcscAR with plasmid-based BaSP expression (+pBaSP). Error bars represent standard deviations is impeded by gene deletions, which guarantees a high product yield. In addition, since biomass formation, which is fueled by the fructose moiety of sucrose, and glycoside synthesis go hand in hand and subsequently are performed at the same time at a high rate, a high productivity is equally guaranteed (one-step fermentation process). Besides quercetin also other flavonols such as kaempferol, fisetin, morin and myricetin significantly contribute to our daily flavonoid intake, which also have extremely diverse beneficial effects [55, 56] . As the sugar moiety is a major determinant of the intestinal absorption of dietary flavonoids and their subsequent bioactivity [57, 58] , the To this end, strains sGAL3 and sRHA3 were grown in tubes with 5 mL minimal medium, each containing 1.5 g/L of either kaempferol, myricetin, morin or fisetin. Growth and production were monitored over 48 h and various spots were observed on TLC with similar retention factors as hyperoside and quercitrin. Mass spectrometry was used to identify the compounds produced, which confirmed the in vivo galactosylation of myricetin, kaempferol, morin and fisetin ( Table 2 ). All compounds occurred with an m/z of [M + 114], due to complexation with trifluoroacetic acid from the mobile phase. The galactoside of morin was produced at a slow rate, which is in accordance to the very low in vitro activity of F3GT towards this flavonol [53] . A possible explanation for this limited activity may be the presence of an unusual hydroxyl group at the 2′ position, which may sterically hinder deprotonation and consequent galactosylation of morin at hydroxyl group 3 [59] . Incubation of sRHA3 with the different flavonols investigated showed two distinct glycoside spots on TLC, which corresponded to the 3-O-rhamnoside and 3-O-glucoside. Kaempferol proved to be the best substrate for RhaGT and was predominantly rhamnosylated (8:1 ratio), with a titer exceeding 400 mg/L, which is twofold higher than previously reported [47] . Fisetin on the other hand was efficiently glucosylated, yet the formation of its rhamnoside was not as efficient, with a titer below 5 mg/L. A similar preference towards glucoside formation was also observed with myricetin and morin, which indicates that the positioning of the hydroxyl groups is the determining factor for glycosylation with RhaGT. The production of the desired rhamnosides, galactosides or glucosides may be improved considerably by using UGTs that are more specific towards certain flavonols and UDP-sugars. Transformation of the corresponding UGTs in the developed in vivo glycosylation strains presents a promising alternative for the large-scale production of various flavonol glycoforms, which are to date mainly extracted from plant material. On the other hand, due to the pivotal role of UDP-glc, various other UDP-sugars can be formed in vivo (e.g. UDP-glucuronate, UDP-xylose, UDP-arabinose). In combination with the modularity of the developed glycosylation platform, which permits rapid introduction of any UGT or UDP-sugar pathway, virtually any glycoside can be produced. Hence, this demonstrates that the proposed microbial platform is a robust, versatile and efficient microbial cell factory for the glycosylation (e.g. glucosylation, rhamnosylation, galactosylation) of small molecules. Although obtained productivities are the highest reported today and compete with the current production processes, further improvement can be limited due to solubility issues of the aglycon or of the glycoside. To this end follow-up research can focus on further metabolic engineering (e.g. introduction of the aglycon pathway allowing in vivo gradually production of the aglycon) or on process optimization [e.g. 2-phase (bilayer) fermentation which enables in situ recovery] to improve these issues. In this contribution, a biotechnological platform was developed for the galactosylation and rhamnosylation of small molecules, such as secondary metabolite natural products, starting from a previously created glucosylation host. To this end, the routes to convert UDP-glucose into UDP-galactose and UDP-rhamnose were introduced by expressing a UDP-glucose epimerase (galE) and a UDP-rhamnose synthase (MUM4), respectively. As a proof of concept, the bio-active flavonol quercetin was selected for galactosylation and rhamnosylation, yielding hyperoside (quercetin 3-O-galactoside) and quercitrin (quercetin 3-O-rhamnoside), respectively. Next, the flavonol 3-O-galactosyltransferase (F3GT) from Petunia hybrida and the flavonol 3-O-rhamnosyltransferase from Arabidopsis thaliana (RhaGT) were overexpressed in the metabolically engineered E. coli W mutants. The strains created were able to produce 940 mg/L of hyperoside and 1176 mg/L of quercitrin at specific production rates of 68.7 mg/g CDW/h and 47.8 mg/g CDW/h, respectively, which are the highest reported to date. Interestingly, both GTs showed in vivo activity towards other dietary flavonols, whereby for example over 400 mg/L of kaempferol 3-O-rhamnoside could be formed extracellularly. All plasmids used were constructed using Gibson assembly [60] or CLIVA [61] . All PCR fragments were amplified using Q5 polymerase from New England Biolabs (Ipswich, Massachusetts). Oligonucleotides were purchased from IDT (Leuven, Belgium). The plasmids and bacterial strains used in this study are listed in Table 1 . A list of primers for the creation of gene knockouts/knockins and for the cloning of the expression plasmids is given in Additional file 2: Table S1 . E. coli DH5α was used for plasmid cloning and propagation, while E. coli W was used for expression of the production plasmids and the creation of gene knockouts and knockins. Hyperoside, quercitrin, isoquercitrin, kaempferol and myricetin were purchased from Carbosynth (Berkshire, UK). All other chemicals used were purchased from Sigma Aldrich (Germany) unless otherwise indicated. The expression plasmids for the prod uction of hyperoside and quercitrin were constructed as depicted in Additional file 3: Figure S1A Figure S1D ). The galE [Genbank: JW0742] and galE2 [Genbank: KJ543703] sequences were amplified from the genomic DNA of E. coli and Bifidobacterium bifidum, respectively. CLIVA assembly resulted in the intermediary plasmid pBaSP/F3GT/UgpA ( Figure S1A ), which was subsequently used for the amplification of the F3GT/ UgpA backbone. Gibson assembly of the GalE or GalE2 inserts with this backbone resulted in the final galactosylation plasmids pGalE/F3GT/UgpA and pGalE2/F3GT/ UgpA, respectively ( Figure S1B ). Similarly, MUM4 and RhaGT were introduced using a 3-pieces Gibson assembly ( Figure S1C ), resulting in the final rhamnosylation plasmid pMUM4/RhaGT/UgpA. The overall E. coli W knockout mutants were created using the one step deletion system of Datsenko and Wanner [62] . The strategy for chromosomal integration of BaSP under control of the constitutive promoter P22 flanked by L4 and L5 at the melA and glgC loci is depicted and explained in Additional file 1: Figure S2 . Transformants were plated on minimal sucrose medium agar plates and grown overnight for screening. The in-house strain E. coli W ΔcscAR Δpgm Δagp ΔushA ΔlacZYA::P22-lacY ΔgalETKM [45] was used for the chromosomal integration of L4-P22-BaSP-L5 at the melA site, yielding the base strain sGLYC (Table 1) . This strain and the E. coli W wild type were transformed with the production plasmids described above, resulting in the galactosylation (sGAL) and rhamnosylation (sRHA) strains given in Table 1 . Composition of LB and minimal sucrose medium was described previously [45] . Minimal medium agar plates with sucrose (50 g/L) had the same composition as minimal sucrose medium, but contained additionally 15 g/L of agarose. The agarose and salts were autoclaved separately at 121 °C for 21 min. Sucrose was filter sterilized through a 0.22 µm corning filter (Fisher, Belgium) and heated for 1 min in a microwave oven at 800 W prior to mixing it with the warm agarose and salt solutions. 1 mL/L of mineral solution [45] was sterilely added prior to pouring the plates. Escherichia coli W mutant precultures were grown in 5 mL LB medium with the antibiotics (50 μg/mL kanamycin or carbenicillin) required for maintenance and selection of the plasmids. The cultures were grown for 16 h at 37 °C and 200 rpm and used for the 2 % inoculation of 100 mL minimal sucrose medium in 500 mL shake flasks. For the production of hyperoside and quercitrin, quercetin was added to the minimal medium at a concentration of 0.15 or 1.5 g/L. Growth conditions were the same as previously described [45] . Samples were taken at regular intervals from the broth and, after centrifugation, the supernatant was used for the analysis and quantification of sugars. For the analysis of quercetin and its glycosides, 200 µL of the culture was collected and extracted with 800 µL ethyl acetate. The organic layer was collected, evaporated in a SpeedVac ™ vacuum concentrator (Thermo Fisher, USA) and dissolved in 200 µL of DMSO for HPLC quantification. The bioreactor set-up and fermentation conditions used are the same as previously described [45] . Production experiments were performed on minimal sucrose medium without MOPS buffer and with the addition of quercetin as acceptor. Culture samples were primarily analyzed by TLC on Silica gel 60 F 254 precoated plates (Merck, Germany). All plates were run in a closed TLC chamber and developed using standard visualization techniques and agents: UV fluorescence (254 nm) or by staining with 10 % (v/v) H 2 SO 4 and subsequent charring. The mobile phase for detecting the various flavonols and corresponding glycosides consisted of an ethyl acetate:acetic acid:formic acid:water (100:11:11:27 v/v) mixture [63] . Product spot intensities of other flavonol glycosides were processed and quantified using ImageJ [64] . HPLC quantification of sucrose, fructose and glucose was performed using an X-bridge Amide column (35 μm, Waters, USA) as described previously [45] . Quercetin, hyperoside, quercitrin and isoquercitrin were detected with the method described by Pandey et al. [41] using a Varian HPLC system (Agilent technologies, California). Mass spectrometry for determination of the various flavonol glycosides was performed with a Micromass Quattro LC (McKinley Scientific, USA). Detection was performed in negative mode ESI-224 MS with a capillary voltage of 2.53 kV, a cone voltage of 20 V, cone and desolvation gas flows of 93 and 420 L/h, and source and cone temperatures of 150 and 350 °C, respectively. Quercetin glycosides were extracted from the broth with an equal volume of ethyl acetate after which the organic layer was evaporated to dryness. The remaining product was dissolved in the solvent system described above and run on a preparative TLC plate. The band containing hyperoside (R f 0.53) or quercitrin (R f 0.75) was scraped off, extracted with ethyl acetate and evaporated to yield a bright yellow powder. Products were confirmed by NMR. Spectra were reported elsewhere [47, 65] .

What was the conclusion of this study?

Metabolic engineering of Escherichia coli into a versatile glycosylation platform: production of bio-active quercetin glycosides https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573293/ SHA: f4cd52975e6aa33e8c947082eda9b261952b0f8f Authors: De Bruyn, Frederik; Van Brempt, Maarten; Maertens, Jo; Van Bellegem, Wouter; Duchi, Dries; De Mey, Marjan Date: 2015-09-16 DOI: 10.1186/s12934-015-0326-1 License: cc-by Abstract: BACKGROUND: Flavonoids are bio-active specialized plant metabolites which mainly occur as different glycosides. Due to the increasing market demand, various biotechnological approaches have been developed which use Escherichia coli as a microbial catalyst for the stereospecific glycosylation of flavonoids. Despite these efforts, most processes still display low production rates and titers, which render them unsuitable for large-scale applications. RESULTS: In this contribution, we expanded a previously developed in vivo glucosylation platform in E. coli W, into an efficient system for selective galactosylation and rhamnosylation. The rational of the novel metabolic engineering strategy constitutes of the introduction of an alternative sucrose metabolism in the form of a sucrose phosphorylase, which cleaves sucrose into fructose and glucose 1-phosphate as precursor for UDP-glucose. To preserve these intermediates for glycosylation purposes, metabolization reactions were knocked-out. Due to the pivotal role of UDP-glucose, overexpression of the interconverting enzymes galE and MUM4 ensured the formation of both UDP-galactose and UDP-rhamnose, respectively. By additionally supplying exogenously fed quercetin and overexpressing a flavonol galactosyltransferase (F3GT) or a rhamnosyltransferase (RhaGT), 0.94 g/L hyperoside (quercetin 3-O-galactoside) and 1.12 g/L quercitrin (quercetin 3-O-rhamnoside) could be produced, respectively. In addition, both strains showed activity towards other promising dietary flavonols like kaempferol, fisetin, morin and myricetin. CONCLUSIONS: Two E. coli W mutants were engineered that could effectively produce the bio-active flavonol glycosides hyperoside and quercitrin starting from the cheap substrates sucrose and quercetin. This novel fermentation-based glycosylation strategy will allow the economically viable production of various glycosides. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0326-1) contains supplementary material, which is available to authorized users. Text: Flavonoids are a class of plant secondary metabolites, which are chemically characterized by a 15-carbon backbone that consists of two phenyl rings and a heterocyclic ring. To date, over 10,000 flavonoids have been characterized from various plants, which are classified according to their chemical structure, i.e., the number and presence of hydroxyl groups and further functional group modifications into various subgroups, such as anthoxanthins, flavanones, and flavanonols [1, 2] . In recent years flavonoids have garnered much attention from various application domains because of the various beneficial effects on human health that have been attributed to them, such as anticancer [3] and antioxidant [4] to anti-inflammatory [5] , antimicrobial [6] and antiviral [6, 7] effects. As final step in their biosynthesis, flavonoids are often glycosylated which has a profound effect on their solubility, stability and bio-activity [8, 9] . For example, the best studied flavonol quercetin, which makes up to 75 % of our daily flavonoid intake, predominantly occurs as different glycosides. Over 350 different quercetin glycoforms have been reported to date with varying pharmacological properties [10, 11] . In this context, hyperoside (quercetin 3-O-galactoside) and quercitrin (quercetin 3-O-rhamnoside) ( Fig. 1) have gained a lot of attention as valuable products for the pharmaceutical industry e.g., as powerful antioxidants with cytoprotective effects [12] [13] [14] [15] and as promising antiviral agents that block replication of the influenza virus [16] or inhibit the viruses hepatitis B [17] and SARS [18] . Furthermore, they have been attributed with anti-inflammatory [19, 20] , antidepressant [21, 22] , apoptotic [23] and antifungal [24] activities, rendering them interesting therapeutics resulting in a steadily increasing market demand. To date, the majority of quercetin and its glycosides are extracted from plant material, which is generally a laborious and low-yielding process requiring many purification steps [25] . In vitro plant cell cultures or engineered plants can be used to overcome the low yields and improve production [26] [27] [28] , however since metabolic engineering of plants is both very controversial and still in its infancy [29] , this approach is often restricted to small-scale production. Although chemical synthesis of quercetin (glycosides) has proven to be feasible [30] [31] [32] , stereoselective formation of glycosidic linkages is often hampered by the presence of various reactive groups [33] , which requires many protecting and deprotecting steps [34] . In addition, the generation of toxic waste and a low atomefficiency [35] render these production processes neither sustainable nor economically viable. As a result, in the last two decades enormous efforts have been invested in the development of alternative production methods for these specialized (secondary) plant metabolites [36] . Advances in the fields of protein engineering, systems and synthetic biology have accelerated these efforts to transform model organisms like Escherichia coli and Saccharomyces cerevisiae in real microbial cell factories for the sustainable production of flavonoids [37] [38] [39] . Subsequently, strategies for the in vivo glycosylation of flavonoids have also been developed. These are typically based on both the overexpression of specific glycosyltransferases, which transfer a sugar residue from an activated nucleotide sugar to an aglycon in a stereoand regioselective way, and the engineering or introduction of the targeted nucleotide sugar pathway. In this way, Fig. 1 Transformation of E. coli W into a sucrose-based galactosylation and rhamnosylation platform. The metabolic engineering strategy applied makes use of several gene deletions (indicated in red) and overexpressions of genes (indicated in green). The rational of a split metabolism is applied, whereby sucrose is divided by sucrose phosphorylase (BaSP) in fructose to be used for growth and a glucose 1-phosphate as activated precursor for UDP-glucose. The latter is a universal pivot molecule for the formation of UDP-galactose and UDP-rhamnose, interconversions catalyzed by the enzymes GalE and MUM4, respectively. To ensure growth-coupled production, various genes, involved in the metabolization of these UDPsugars and their precursors, were knocked out (shown in red). The production of the bioactive quercetin glycosides hyperoside and quercitrin was chosen to evaluate the versatility of the engineered production platform. Finally, the introduction of either the glycosyltransferase F3GT or RhaGT ensures efficient galactosylation or rhamnosylation, respectively various quercetin glycosides have already been produced in E. coli such as the naturally occurring 3-O-glucoside [40] , 3-O-xyloside [41] and 3,7-O-bisrhamnoside [42] , or the new-to-nature quercetin 3-O-(6-deoxytalose) [43] . However, despite these engineering efforts, the reported product rates and titers are still in the milligram range, rendering these microbial production hosts unsuitable for industrial applications. The developed production processes are typically biphasic bioconversion processes using resting cells, which makes it difficult to improve production rates [44] . Furthermore, such systems often entail expensive growth media or the addition of enzyme inducers, making the overall process very costly. To tackle these problems, we previously developed an efficient platform for the glucosylation of small molecules in E. coli W [45] . Through metabolic engineering, a mutant was created which couples the production of glucosides to growth, using sucrose as a cheap and sustainable carbon source. By introducing the sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) sucrose can be split into fructose to be used for growth purposes and glucose 1-phosphate (glc1P) to be used as precursor for UDP-glucose (UDP-glc) formation ( Fig. 1) . To impede the conversion of glc1P into biomass precursors, several endogenous genes involved in its metabolization such as phosphoglucomutase (pgm) and glucose-1-phosphatase (agp) were knocked out. Subsequently, glc1P can efficiently be channeled towards UDP-glc by overexpressing the uridylyltransferase from Bifidobacterium bifidum (ugpA). Metabolization of UDP-glc is prevented by knocking out the UDP-sugar hydrolase (ushA) and the galactose operon (galETKM). However, in view of the pivotal role of UDP-glc in the production of a large variety of UDP-sugars, this glucosylation system can easily be extended towards other UDP-sugars, such as UDP-galactose (UDP-gal), UDPrhamnose (UDP-rha) and UDP-glucuronate. In the present contribution, this previously developed E. coli W-based glucosylation platform is transformed into a platform for galactosylation and rhamnosylation ( Fig. 1) , whose potential is demonstrated using the galactosylation and rhamnosylation of exogenously fed quercetin yielding hyperoside and quercitrin, respectively, as case study. Escherichia coli W is a fast-growing non-pathogenic strain which tolerates osmotic stress, acidic conditions, and can be cultured to high cell densities, making it an attractive host for industrial fermentations [46] . Moreover, E. coli W is able to grow on sucrose as sole carbon source [46] , which is an emerging feedstock for the production of bio-products. Hence, E. coli W was selected as host for sucrose-based in vivo glycosylation. Prior to the production of the glycosides hyperoside and quercitrin in E. coli W, the toxicity of their aglycon quercetin was investigated. To this end, the wild type (WT) strain was grown on minimal sucrose medium containing different concentrations of quercetin (0, 0.15 and 1.5 g/L). The specific growth rates (h −1 ) (0.96 ± 0.06, 0.92 ± 0.05 and 0.87 ± 0.06, respectively) were not significantly different (p ANOVA = 0.12) nor from the one previously determined for the WT [45] (p = 0.69, p = 0.98 and p = 0.68, respectively). On the other hand, the optical density at 600 nm after 24 h incubation (6.36 ± 0.12, 5.18 ± 0.16 and 4.77 ± 0.20, respectively) was lower (about 20 %) when quercetin was added (p = 0.0002 and p = 0.0001). No significant difference in optical density could be observed between 0.15 and 1.5 g/L quercetin (p = 0.14). In view of the above, it was opted to add 1.5 g/L quercetin to evaluate the potential of the developed glycosylation platform. To evaluate the in vivo glycosylation potential, strains sGAL1 and sRHA1, which constitutively express the flavonol 3-O-galactosyltransferase from Petunia hybrida and the flavonol 3-O-rhamnosyltransferase from A. thaliana, respectively, were cultured in minimal medium with 1.5 g/L of quercetin for 16 h. TLC analysis of the supernatants of both cultures yielded two new yellow product spots. The TLC spot obtained from the sGAL1 culture, which had the same retention time as the hyperoside standard (R f = 0.5), was subsequently purified and analyzed. Both NMR and MS analysis confirmed the production of quercetin 3-O-galactoside. However, the product spot obtained from the sRHA1 culture had a different retention factor (R f = 0.55) than the quercitrin standard (R f = 0.74), and was identified as isoquercitrin (quercetin 3-O-glucoside). As opposed to other reports on wild type E. coli strains expressing RhaGT, which simultaneously produced quercitrin (quercetin 3-O-rhamnoside) and isoquercitrin [47, 48] , no rhamnoside could be detected. Examination of the E. coli W genome revealed that the gene cluster responsible for the endogenous production of dTDP-rhamnose, which functions as an alternative rhamnosyldonor for RhaGT in E. coli B and K12 derivatives [47] , was not present [46, 49] . In a follow-up experiment, sGAL1 and sRHA1 were grown on minimal medium with two different concentrations (0.15 and 1.5 g/L) of quercetin. Growth and glycoside formation were monitored during 30 h. The final titers (C p ) and specific productivities (q p ) are shown in Fig. 2 . Remarkably, an increase in quercetin concentration resulted in a two to threefold increase in productivity and titer, indicating that quercetin supply is rate-limiting and crucial for efficient in vivo glycosylation. However, while sGAL1 continuously produced hyperoside during the exponential phase, which is also reflected in the relatively high specific productivity, sRHA1 only started to accumulate significant amounts of isoquercitrin at the end of the exponential phase. This production start coincides with a reduction in specific growth rate, which dropped from 0.35 ± 0.04 to 0.06 ± 0.01 h −1 . As described in detail in the Background section, we previously metabolically engineered E. coli W to create a platform for in vivo glucosylation of small molecules [45] . In the original base glucosylation strain, sucrose phosphorylase encoded by BaSP was located on a mediumcopy plasmid and transcribed from a medium-strong constitutive promoter (P22) [50] . For reasons of comparison and flexibility, it was opted to integrate BaSP in the genome of E. coli W. In addition, chromosomal integration is advantageous because of a significant increase in gene stability. Since the level of gene expression can considerably be impacted by the genome integration site [51] due to structural differences such as supercoiling DNA regions, two different DNA sites were assessed for BaSP integration, i.e., melA and glgC, which encode an α-galactosidase and a glucose-1-phosphate adenylyltransferase, respectively. To this end, an adapted knockin-knockout procedure for large DNA fragments was applied, which is schematically shown in Additional file 1: Figure S2 . BaSP under control of promoter P22 was knocked in at the two different loci in E. coli W ΔcscAR, which resulted in the E. coli W strains ΔcscAR ΔmelA::L4-P22-BaSP-L5 and ΔcscAR ΔglgC::L4-P22-BaSP-L5. Their maximal specific growth rate (µ max ) on minimal sucrose medium, which is shown in Fig. 3 , was compared to the original strain ΔcscAR + pBaSP. The influence of the knockin locus on the maximal specific growth rate is clear. Interestingly, integration at the melA locus resulted in a strain with a µ max which was not significantly different from the reference strain ΔcscAR + pBaSP. In view of the latter and considering the aimed growth-coupled production, it was opted to integrate BaSP at the melA locus leading to the final production base strain E. coli W ΔcscAR Δpgm Δagp ΔushA ΔlacZYA::P22-lacY ΔgalETKM ΔmelA::L4-P22-BaSP-L5 (sGLYC) as shown in Table 1 . In nature, UDP-glc serves as a pivot molecule in the formation of a variety of UDP-sugars [44] . For example, using the interconverting enzymes UDP-glucose 4-epimerase (GalE) and UDP-rhamnose synthase (MUM4) UDP-glc can be converted to UDP-gal and UDP-rha, respectively. Though GalE is natively present in E. coli W an alternative homologous epimerase (GalE2) from B. bifidum was also selected and cloned due to the Fig. 2 Comparison of the specific glycoside productivities (q p ) and glycoside titers (C p ) for strains sGAL1, which produces the 3-O-galactoside, and sRHA1, which produces the 3-O-glucoside, when grown for 30 h on minimal medium containing 0.15 or 1.5 g/L of quercetin. Error bars represent standard deviations tight and complex regulation of GalE expression in E. coli W. On the other hand, UDP-rhamnose synthesis is restricted to plants. Due to lack of the rfb cluster [46] E. coli W is even unable to form endogenous dTDP-rhamnose as alternative rhamnosyl donor. Hence, the MUM4 gene from A. thaliana was expressed from plasmid pMUM4 to achieve UDP-rhamnose formation in E. coli (Fig. 1) . The constructed galactosylation (sGAL) and rhamnosylation (sRHA) strains were grown on minimal medium with two levels (0.15 and 1.5 g/L) of quercetin. Growth and production were monitored to determine the specific productivities, as shown in Fig. 4 . Again, higher extracellular quercetin concentrations resulted in a fivefold increase in q p . However, no significant difference in productivity was observed between sGAL2 and sGAL3 at 1.5 g/L quercetin, indicating that UDP-galactose formation is as efficient with both GalE homologs and not likely the rate limiting step. With sGAL3, the highest hyperoside productivity (68.7 mg/g CDW/h) and titer (0.94 g/L) were obtained, the latter being 3.5-fold higher compared to sGAL1. In contrast to sRHA1, TLC analysis of the supernatant of the cultures of sRHA2 and sRHA3 resulted in a product spot with a retention factor that corresponds to quercitrin, which was confirmed by MS analysis, thus showing in vivo activity of MUM4. A quercitrin titer of 1.18 g/L and specific productivity of 47.8 mg/g CDW/h were obtained after 30 h incubation of sRHA3 when 1.5 g/L quercetin was added to the medium, which corresponded to a 53 % conversion. Also 51 mg/L of isoquercitrin was produced extracellularly which corresponds with a quercitrin:isoquercitrin production ratio of 24:1. This suggests the preference of RhaGT for UDP-rhamnose when different UDP-sugar donors are present. Possible explanations for the significantly lower specific productivity (fivefold decrease) of sRHA2 as compared to sRHA3 are either a higher metabolic burden [52] caused by the two plasmid system or a too limited activity of the native GalU, which could be insufficient for adequate UDP-glc formation [45] . To demonstrate the scalability of the developed bioprocess, strain sGAL3 was cultured in a 1-L bioreactor, which also ensures a constant pH set at 6.80 and avoid oxygen limitation. A detailed overview of the consumption of sucrose, growth and hyperoside production is given in Fig. 5 . After a lag-phase, the strain displayed a growth rate of 0.32 ± 0.02 h −1 while simultaneously producing hyperoside. The observed specific productivity (65.9 ± 2.6 mg/g CDW/h) was comparable to the one obtained on shake flask scale. When nearly all quercetin was converted, hyperoside formation slowed down, which can be explained either by the observed correlation between quercetin concentration and q p , or by the reported reversibility of F3GT [53] . It is likely that further improvements in titer and productivity can be realized by optimizing the supply of quercetin using a fed-batch system. To the best of our knowledge, the results obtained in this study with the engineered sGAL and sRHA strains for the production of hyperoside and quercitrin are the highest reported to date both in terms of titer and production rate. The maximal production rate obtained in this contribution was 6 to 50-fold higher compared to the maximal production rates (r p,max ) of processes reported in the literature [47, 54] as is illustrated in Fig. 6 . The increased performance, in terms of titer and productivity, obtained with the developed platform can be attributed to the use of a split metabolism in combination with optimally rerouting the flux from glucose 1-phosphate towards UDP-galactose and UDP-rhamnose. The undesired conversion of the activated sugars into biomass Fig. 3 Effect of the chromosomal integration locus of the knockin of BaSP on the growth rate. Strains were grown in shake flasks and the resulting maximal growth rates (µ max ) were compared with E. coli W ΔcscAR with plasmid-based BaSP expression (+pBaSP). Error bars represent standard deviations is impeded by gene deletions, which guarantees a high product yield. In addition, since biomass formation, which is fueled by the fructose moiety of sucrose, and glycoside synthesis go hand in hand and subsequently are performed at the same time at a high rate, a high productivity is equally guaranteed (one-step fermentation process). Besides quercetin also other flavonols such as kaempferol, fisetin, morin and myricetin significantly contribute to our daily flavonoid intake, which also have extremely diverse beneficial effects [55, 56] . As the sugar moiety is a major determinant of the intestinal absorption of dietary flavonoids and their subsequent bioactivity [57, 58] , the To this end, strains sGAL3 and sRHA3 were grown in tubes with 5 mL minimal medium, each containing 1.5 g/L of either kaempferol, myricetin, morin or fisetin. Growth and production were monitored over 48 h and various spots were observed on TLC with similar retention factors as hyperoside and quercitrin. Mass spectrometry was used to identify the compounds produced, which confirmed the in vivo galactosylation of myricetin, kaempferol, morin and fisetin ( Table 2 ). All compounds occurred with an m/z of [M + 114], due to complexation with trifluoroacetic acid from the mobile phase. The galactoside of morin was produced at a slow rate, which is in accordance to the very low in vitro activity of F3GT towards this flavonol [53] . A possible explanation for this limited activity may be the presence of an unusual hydroxyl group at the 2′ position, which may sterically hinder deprotonation and consequent galactosylation of morin at hydroxyl group 3 [59] . Incubation of sRHA3 with the different flavonols investigated showed two distinct glycoside spots on TLC, which corresponded to the 3-O-rhamnoside and 3-O-glucoside. Kaempferol proved to be the best substrate for RhaGT and was predominantly rhamnosylated (8:1 ratio), with a titer exceeding 400 mg/L, which is twofold higher than previously reported [47] . Fisetin on the other hand was efficiently glucosylated, yet the formation of its rhamnoside was not as efficient, with a titer below 5 mg/L. A similar preference towards glucoside formation was also observed with myricetin and morin, which indicates that the positioning of the hydroxyl groups is the determining factor for glycosylation with RhaGT. The production of the desired rhamnosides, galactosides or glucosides may be improved considerably by using UGTs that are more specific towards certain flavonols and UDP-sugars. Transformation of the corresponding UGTs in the developed in vivo glycosylation strains presents a promising alternative for the large-scale production of various flavonol glycoforms, which are to date mainly extracted from plant material. On the other hand, due to the pivotal role of UDP-glc, various other UDP-sugars can be formed in vivo (e.g. UDP-glucuronate, UDP-xylose, UDP-arabinose). In combination with the modularity of the developed glycosylation platform, which permits rapid introduction of any UGT or UDP-sugar pathway, virtually any glycoside can be produced. Hence, this demonstrates that the proposed microbial platform is a robust, versatile and efficient microbial cell factory for the glycosylation (e.g. glucosylation, rhamnosylation, galactosylation) of small molecules. Although obtained productivities are the highest reported today and compete with the current production processes, further improvement can be limited due to solubility issues of the aglycon or of the glycoside. To this end follow-up research can focus on further metabolic engineering (e.g. introduction of the aglycon pathway allowing in vivo gradually production of the aglycon) or on process optimization [e.g. 2-phase (bilayer) fermentation which enables in situ recovery] to improve these issues. In this contribution, a biotechnological platform was developed for the galactosylation and rhamnosylation of small molecules, such as secondary metabolite natural products, starting from a previously created glucosylation host. To this end, the routes to convert UDP-glucose into UDP-galactose and UDP-rhamnose were introduced by expressing a UDP-glucose epimerase (galE) and a UDP-rhamnose synthase (MUM4), respectively. As a proof of concept, the bio-active flavonol quercetin was selected for galactosylation and rhamnosylation, yielding hyperoside (quercetin 3-O-galactoside) and quercitrin (quercetin 3-O-rhamnoside), respectively. Next, the flavonol 3-O-galactosyltransferase (F3GT) from Petunia hybrida and the flavonol 3-O-rhamnosyltransferase from Arabidopsis thaliana (RhaGT) were overexpressed in the metabolically engineered E. coli W mutants. The strains created were able to produce 940 mg/L of hyperoside and 1176 mg/L of quercitrin at specific production rates of 68.7 mg/g CDW/h and 47.8 mg/g CDW/h, respectively, which are the highest reported to date. Interestingly, both GTs showed in vivo activity towards other dietary flavonols, whereby for example over 400 mg/L of kaempferol 3-O-rhamnoside could be formed extracellularly. All plasmids used were constructed using Gibson assembly [60] or CLIVA [61] . All PCR fragments were amplified using Q5 polymerase from New England Biolabs (Ipswich, Massachusetts). Oligonucleotides were purchased from IDT (Leuven, Belgium). The plasmids and bacterial strains used in this study are listed in Table 1 . A list of primers for the creation of gene knockouts/knockins and for the cloning of the expression plasmids is given in Additional file 2: Table S1 . E. coli DH5α was used for plasmid cloning and propagation, while E. coli W was used for expression of the production plasmids and the creation of gene knockouts and knockins. Hyperoside, quercitrin, isoquercitrin, kaempferol and myricetin were purchased from Carbosynth (Berkshire, UK). All other chemicals used were purchased from Sigma Aldrich (Germany) unless otherwise indicated. The expression plasmids for the prod uction of hyperoside and quercitrin were constructed as depicted in Additional file 3: Figure S1A Figure S1D ). The galE [Genbank: JW0742] and galE2 [Genbank: KJ543703] sequences were amplified from the genomic DNA of E. coli and Bifidobacterium bifidum, respectively. CLIVA assembly resulted in the intermediary plasmid pBaSP/F3GT/UgpA ( Figure S1A ), which was subsequently used for the amplification of the F3GT/ UgpA backbone. Gibson assembly of the GalE or GalE2 inserts with this backbone resulted in the final galactosylation plasmids pGalE/F3GT/UgpA and pGalE2/F3GT/ UgpA, respectively ( Figure S1B ). Similarly, MUM4 and RhaGT were introduced using a 3-pieces Gibson assembly ( Figure S1C ), resulting in the final rhamnosylation plasmid pMUM4/RhaGT/UgpA. The overall E. coli W knockout mutants were created using the one step deletion system of Datsenko and Wanner [62] . The strategy for chromosomal integration of BaSP under control of the constitutive promoter P22 flanked by L4 and L5 at the melA and glgC loci is depicted and explained in Additional file 1: Figure S2 . Transformants were plated on minimal sucrose medium agar plates and grown overnight for screening. The in-house strain E. coli W ΔcscAR Δpgm Δagp ΔushA ΔlacZYA::P22-lacY ΔgalETKM [45] was used for the chromosomal integration of L4-P22-BaSP-L5 at the melA site, yielding the base strain sGLYC (Table 1) . This strain and the E. coli W wild type were transformed with the production plasmids described above, resulting in the galactosylation (sGAL) and rhamnosylation (sRHA) strains given in Table 1 . Composition of LB and minimal sucrose medium was described previously [45] . Minimal medium agar plates with sucrose (50 g/L) had the same composition as minimal sucrose medium, but contained additionally 15 g/L of agarose. The agarose and salts were autoclaved separately at 121 °C for 21 min. Sucrose was filter sterilized through a 0.22 µm corning filter (Fisher, Belgium) and heated for 1 min in a microwave oven at 800 W prior to mixing it with the warm agarose and salt solutions. 1 mL/L of mineral solution [45] was sterilely added prior to pouring the plates. Escherichia coli W mutant precultures were grown in 5 mL LB medium with the antibiotics (50 μg/mL kanamycin or carbenicillin) required for maintenance and selection of the plasmids. The cultures were grown for 16 h at 37 °C and 200 rpm and used for the 2 % inoculation of 100 mL minimal sucrose medium in 500 mL shake flasks. For the production of hyperoside and quercitrin, quercetin was added to the minimal medium at a concentration of 0.15 or 1.5 g/L. Growth conditions were the same as previously described [45] . Samples were taken at regular intervals from the broth and, after centrifugation, the supernatant was used for the analysis and quantification of sugars. For the analysis of quercetin and its glycosides, 200 µL of the culture was collected and extracted with 800 µL ethyl acetate. The organic layer was collected, evaporated in a SpeedVac ™ vacuum concentrator (Thermo Fisher, USA) and dissolved in 200 µL of DMSO for HPLC quantification. The bioreactor set-up and fermentation conditions used are the same as previously described [45] . Production experiments were performed on minimal sucrose medium without MOPS buffer and with the addition of quercetin as acceptor. Culture samples were primarily analyzed by TLC on Silica gel 60 F 254 precoated plates (Merck, Germany). All plates were run in a closed TLC chamber and developed using standard visualization techniques and agents: UV fluorescence (254 nm) or by staining with 10 % (v/v) H 2 SO 4 and subsequent charring. The mobile phase for detecting the various flavonols and corresponding glycosides consisted of an ethyl acetate:acetic acid:formic acid:water (100:11:11:27 v/v) mixture [63] . Product spot intensities of other flavonol glycosides were processed and quantified using ImageJ [64] . HPLC quantification of sucrose, fructose and glucose was performed using an X-bridge Amide column (35 μm, Waters, USA) as described previously [45] . Quercetin, hyperoside, quercitrin and isoquercitrin were detected with the method described by Pandey et al. [41] using a Varian HPLC system (Agilent technologies, California). Mass spectrometry for determination of the various flavonol glycosides was performed with a Micromass Quattro LC (McKinley Scientific, USA). Detection was performed in negative mode ESI-224 MS with a capillary voltage of 2.53 kV, a cone voltage of 20 V, cone and desolvation gas flows of 93 and 420 L/h, and source and cone temperatures of 150 and 350 °C, respectively. Quercetin glycosides were extracted from the broth with an equal volume of ethyl acetate after which the organic layer was evaporated to dryness. The remaining product was dissolved in the solvent system described above and run on a preparative TLC plate. The band containing hyperoside (R f 0.53) or quercitrin (R f 0.75) was scraped off, extracted with ethyl acetate and evaporated to yield a bright yellow powder. Products were confirmed by NMR. Spectra were reported elsewhere [47, 65] .

What are the implications of the novel fermentation-based glycosylation strategy described in this study?

Metabolic engineering of Escherichia coli into a versatile glycosylation platform: production of bio-active quercetin glycosides https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573293/ SHA: f4cd52975e6aa33e8c947082eda9b261952b0f8f Authors: De Bruyn, Frederik; Van Brempt, Maarten; Maertens, Jo; Van Bellegem, Wouter; Duchi, Dries; De Mey, Marjan Date: 2015-09-16 DOI: 10.1186/s12934-015-0326-1 License: cc-by Abstract: BACKGROUND: Flavonoids are bio-active specialized plant metabolites which mainly occur as different glycosides. Due to the increasing market demand, various biotechnological approaches have been developed which use Escherichia coli as a microbial catalyst for the stereospecific glycosylation of flavonoids. Despite these efforts, most processes still display low production rates and titers, which render them unsuitable for large-scale applications. RESULTS: In this contribution, we expanded a previously developed in vivo glucosylation platform in E. coli W, into an efficient system for selective galactosylation and rhamnosylation. The rational of the novel metabolic engineering strategy constitutes of the introduction of an alternative sucrose metabolism in the form of a sucrose phosphorylase, which cleaves sucrose into fructose and glucose 1-phosphate as precursor for UDP-glucose. To preserve these intermediates for glycosylation purposes, metabolization reactions were knocked-out. Due to the pivotal role of UDP-glucose, overexpression of the interconverting enzymes galE and MUM4 ensured the formation of both UDP-galactose and UDP-rhamnose, respectively. By additionally supplying exogenously fed quercetin and overexpressing a flavonol galactosyltransferase (F3GT) or a rhamnosyltransferase (RhaGT), 0.94 g/L hyperoside (quercetin 3-O-galactoside) and 1.12 g/L quercitrin (quercetin 3-O-rhamnoside) could be produced, respectively. In addition, both strains showed activity towards other promising dietary flavonols like kaempferol, fisetin, morin and myricetin. CONCLUSIONS: Two E. coli W mutants were engineered that could effectively produce the bio-active flavonol glycosides hyperoside and quercitrin starting from the cheap substrates sucrose and quercetin. This novel fermentation-based glycosylation strategy will allow the economically viable production of various glycosides. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0326-1) contains supplementary material, which is available to authorized users. Text: Flavonoids are a class of plant secondary metabolites, which are chemically characterized by a 15-carbon backbone that consists of two phenyl rings and a heterocyclic ring. To date, over 10,000 flavonoids have been characterized from various plants, which are classified according to their chemical structure, i.e., the number and presence of hydroxyl groups and further functional group modifications into various subgroups, such as anthoxanthins, flavanones, and flavanonols [1, 2] . In recent years flavonoids have garnered much attention from various application domains because of the various beneficial effects on human health that have been attributed to them, such as anticancer [3] and antioxidant [4] to anti-inflammatory [5] , antimicrobial [6] and antiviral [6, 7] effects. As final step in their biosynthesis, flavonoids are often glycosylated which has a profound effect on their solubility, stability and bio-activity [8, 9] . For example, the best studied flavonol quercetin, which makes up to 75 % of our daily flavonoid intake, predominantly occurs as different glycosides. Over 350 different quercetin glycoforms have been reported to date with varying pharmacological properties [10, 11] . In this context, hyperoside (quercetin 3-O-galactoside) and quercitrin (quercetin 3-O-rhamnoside) ( Fig. 1) have gained a lot of attention as valuable products for the pharmaceutical industry e.g., as powerful antioxidants with cytoprotective effects [12] [13] [14] [15] and as promising antiviral agents that block replication of the influenza virus [16] or inhibit the viruses hepatitis B [17] and SARS [18] . Furthermore, they have been attributed with anti-inflammatory [19, 20] , antidepressant [21, 22] , apoptotic [23] and antifungal [24] activities, rendering them interesting therapeutics resulting in a steadily increasing market demand. To date, the majority of quercetin and its glycosides are extracted from plant material, which is generally a laborious and low-yielding process requiring many purification steps [25] . In vitro plant cell cultures or engineered plants can be used to overcome the low yields and improve production [26] [27] [28] , however since metabolic engineering of plants is both very controversial and still in its infancy [29] , this approach is often restricted to small-scale production. Although chemical synthesis of quercetin (glycosides) has proven to be feasible [30] [31] [32] , stereoselective formation of glycosidic linkages is often hampered by the presence of various reactive groups [33] , which requires many protecting and deprotecting steps [34] . In addition, the generation of toxic waste and a low atomefficiency [35] render these production processes neither sustainable nor economically viable. As a result, in the last two decades enormous efforts have been invested in the development of alternative production methods for these specialized (secondary) plant metabolites [36] . Advances in the fields of protein engineering, systems and synthetic biology have accelerated these efforts to transform model organisms like Escherichia coli and Saccharomyces cerevisiae in real microbial cell factories for the sustainable production of flavonoids [37] [38] [39] . Subsequently, strategies for the in vivo glycosylation of flavonoids have also been developed. These are typically based on both the overexpression of specific glycosyltransferases, which transfer a sugar residue from an activated nucleotide sugar to an aglycon in a stereoand regioselective way, and the engineering or introduction of the targeted nucleotide sugar pathway. In this way, Fig. 1 Transformation of E. coli W into a sucrose-based galactosylation and rhamnosylation platform. The metabolic engineering strategy applied makes use of several gene deletions (indicated in red) and overexpressions of genes (indicated in green). The rational of a split metabolism is applied, whereby sucrose is divided by sucrose phosphorylase (BaSP) in fructose to be used for growth and a glucose 1-phosphate as activated precursor for UDP-glucose. The latter is a universal pivot molecule for the formation of UDP-galactose and UDP-rhamnose, interconversions catalyzed by the enzymes GalE and MUM4, respectively. To ensure growth-coupled production, various genes, involved in the metabolization of these UDPsugars and their precursors, were knocked out (shown in red). The production of the bioactive quercetin glycosides hyperoside and quercitrin was chosen to evaluate the versatility of the engineered production platform. Finally, the introduction of either the glycosyltransferase F3GT or RhaGT ensures efficient galactosylation or rhamnosylation, respectively various quercetin glycosides have already been produced in E. coli such as the naturally occurring 3-O-glucoside [40] , 3-O-xyloside [41] and 3,7-O-bisrhamnoside [42] , or the new-to-nature quercetin 3-O-(6-deoxytalose) [43] . However, despite these engineering efforts, the reported product rates and titers are still in the milligram range, rendering these microbial production hosts unsuitable for industrial applications. The developed production processes are typically biphasic bioconversion processes using resting cells, which makes it difficult to improve production rates [44] . Furthermore, such systems often entail expensive growth media or the addition of enzyme inducers, making the overall process very costly. To tackle these problems, we previously developed an efficient platform for the glucosylation of small molecules in E. coli W [45] . Through metabolic engineering, a mutant was created which couples the production of glucosides to growth, using sucrose as a cheap and sustainable carbon source. By introducing the sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) sucrose can be split into fructose to be used for growth purposes and glucose 1-phosphate (glc1P) to be used as precursor for UDP-glucose (UDP-glc) formation ( Fig. 1) . To impede the conversion of glc1P into biomass precursors, several endogenous genes involved in its metabolization such as phosphoglucomutase (pgm) and glucose-1-phosphatase (agp) were knocked out. Subsequently, glc1P can efficiently be channeled towards UDP-glc by overexpressing the uridylyltransferase from Bifidobacterium bifidum (ugpA). Metabolization of UDP-glc is prevented by knocking out the UDP-sugar hydrolase (ushA) and the galactose operon (galETKM). However, in view of the pivotal role of UDP-glc in the production of a large variety of UDP-sugars, this glucosylation system can easily be extended towards other UDP-sugars, such as UDP-galactose (UDP-gal), UDPrhamnose (UDP-rha) and UDP-glucuronate. In the present contribution, this previously developed E. coli W-based glucosylation platform is transformed into a platform for galactosylation and rhamnosylation ( Fig. 1) , whose potential is demonstrated using the galactosylation and rhamnosylation of exogenously fed quercetin yielding hyperoside and quercitrin, respectively, as case study. Escherichia coli W is a fast-growing non-pathogenic strain which tolerates osmotic stress, acidic conditions, and can be cultured to high cell densities, making it an attractive host for industrial fermentations [46] . Moreover, E. coli W is able to grow on sucrose as sole carbon source [46] , which is an emerging feedstock for the production of bio-products. Hence, E. coli W was selected as host for sucrose-based in vivo glycosylation. Prior to the production of the glycosides hyperoside and quercitrin in E. coli W, the toxicity of their aglycon quercetin was investigated. To this end, the wild type (WT) strain was grown on minimal sucrose medium containing different concentrations of quercetin (0, 0.15 and 1.5 g/L). The specific growth rates (h −1 ) (0.96 ± 0.06, 0.92 ± 0.05 and 0.87 ± 0.06, respectively) were not significantly different (p ANOVA = 0.12) nor from the one previously determined for the WT [45] (p = 0.69, p = 0.98 and p = 0.68, respectively). On the other hand, the optical density at 600 nm after 24 h incubation (6.36 ± 0.12, 5.18 ± 0.16 and 4.77 ± 0.20, respectively) was lower (about 20 %) when quercetin was added (p = 0.0002 and p = 0.0001). No significant difference in optical density could be observed between 0.15 and 1.5 g/L quercetin (p = 0.14). In view of the above, it was opted to add 1.5 g/L quercetin to evaluate the potential of the developed glycosylation platform. To evaluate the in vivo glycosylation potential, strains sGAL1 and sRHA1, which constitutively express the flavonol 3-O-galactosyltransferase from Petunia hybrida and the flavonol 3-O-rhamnosyltransferase from A. thaliana, respectively, were cultured in minimal medium with 1.5 g/L of quercetin for 16 h. TLC analysis of the supernatants of both cultures yielded two new yellow product spots. The TLC spot obtained from the sGAL1 culture, which had the same retention time as the hyperoside standard (R f = 0.5), was subsequently purified and analyzed. Both NMR and MS analysis confirmed the production of quercetin 3-O-galactoside. However, the product spot obtained from the sRHA1 culture had a different retention factor (R f = 0.55) than the quercitrin standard (R f = 0.74), and was identified as isoquercitrin (quercetin 3-O-glucoside). As opposed to other reports on wild type E. coli strains expressing RhaGT, which simultaneously produced quercitrin (quercetin 3-O-rhamnoside) and isoquercitrin [47, 48] , no rhamnoside could be detected. Examination of the E. coli W genome revealed that the gene cluster responsible for the endogenous production of dTDP-rhamnose, which functions as an alternative rhamnosyldonor for RhaGT in E. coli B and K12 derivatives [47] , was not present [46, 49] . In a follow-up experiment, sGAL1 and sRHA1 were grown on minimal medium with two different concentrations (0.15 and 1.5 g/L) of quercetin. Growth and glycoside formation were monitored during 30 h. The final titers (C p ) and specific productivities (q p ) are shown in Fig. 2 . Remarkably, an increase in quercetin concentration resulted in a two to threefold increase in productivity and titer, indicating that quercetin supply is rate-limiting and crucial for efficient in vivo glycosylation. However, while sGAL1 continuously produced hyperoside during the exponential phase, which is also reflected in the relatively high specific productivity, sRHA1 only started to accumulate significant amounts of isoquercitrin at the end of the exponential phase. This production start coincides with a reduction in specific growth rate, which dropped from 0.35 ± 0.04 to 0.06 ± 0.01 h −1 . As described in detail in the Background section, we previously metabolically engineered E. coli W to create a platform for in vivo glucosylation of small molecules [45] . In the original base glucosylation strain, sucrose phosphorylase encoded by BaSP was located on a mediumcopy plasmid and transcribed from a medium-strong constitutive promoter (P22) [50] . For reasons of comparison and flexibility, it was opted to integrate BaSP in the genome of E. coli W. In addition, chromosomal integration is advantageous because of a significant increase in gene stability. Since the level of gene expression can considerably be impacted by the genome integration site [51] due to structural differences such as supercoiling DNA regions, two different DNA sites were assessed for BaSP integration, i.e., melA and glgC, which encode an α-galactosidase and a glucose-1-phosphate adenylyltransferase, respectively. To this end, an adapted knockin-knockout procedure for large DNA fragments was applied, which is schematically shown in Additional file 1: Figure S2 . BaSP under control of promoter P22 was knocked in at the two different loci in E. coli W ΔcscAR, which resulted in the E. coli W strains ΔcscAR ΔmelA::L4-P22-BaSP-L5 and ΔcscAR ΔglgC::L4-P22-BaSP-L5. Their maximal specific growth rate (µ max ) on minimal sucrose medium, which is shown in Fig. 3 , was compared to the original strain ΔcscAR + pBaSP. The influence of the knockin locus on the maximal specific growth rate is clear. Interestingly, integration at the melA locus resulted in a strain with a µ max which was not significantly different from the reference strain ΔcscAR + pBaSP. In view of the latter and considering the aimed growth-coupled production, it was opted to integrate BaSP at the melA locus leading to the final production base strain E. coli W ΔcscAR Δpgm Δagp ΔushA ΔlacZYA::P22-lacY ΔgalETKM ΔmelA::L4-P22-BaSP-L5 (sGLYC) as shown in Table 1 . In nature, UDP-glc serves as a pivot molecule in the formation of a variety of UDP-sugars [44] . For example, using the interconverting enzymes UDP-glucose 4-epimerase (GalE) and UDP-rhamnose synthase (MUM4) UDP-glc can be converted to UDP-gal and UDP-rha, respectively. Though GalE is natively present in E. coli W an alternative homologous epimerase (GalE2) from B. bifidum was also selected and cloned due to the Fig. 2 Comparison of the specific glycoside productivities (q p ) and glycoside titers (C p ) for strains sGAL1, which produces the 3-O-galactoside, and sRHA1, which produces the 3-O-glucoside, when grown for 30 h on minimal medium containing 0.15 or 1.5 g/L of quercetin. Error bars represent standard deviations tight and complex regulation of GalE expression in E. coli W. On the other hand, UDP-rhamnose synthesis is restricted to plants. Due to lack of the rfb cluster [46] E. coli W is even unable to form endogenous dTDP-rhamnose as alternative rhamnosyl donor. Hence, the MUM4 gene from A. thaliana was expressed from plasmid pMUM4 to achieve UDP-rhamnose formation in E. coli (Fig. 1) . The constructed galactosylation (sGAL) and rhamnosylation (sRHA) strains were grown on minimal medium with two levels (0.15 and 1.5 g/L) of quercetin. Growth and production were monitored to determine the specific productivities, as shown in Fig. 4 . Again, higher extracellular quercetin concentrations resulted in a fivefold increase in q p . However, no significant difference in productivity was observed between sGAL2 and sGAL3 at 1.5 g/L quercetin, indicating that UDP-galactose formation is as efficient with both GalE homologs and not likely the rate limiting step. With sGAL3, the highest hyperoside productivity (68.7 mg/g CDW/h) and titer (0.94 g/L) were obtained, the latter being 3.5-fold higher compared to sGAL1. In contrast to sRHA1, TLC analysis of the supernatant of the cultures of sRHA2 and sRHA3 resulted in a product spot with a retention factor that corresponds to quercitrin, which was confirmed by MS analysis, thus showing in vivo activity of MUM4. A quercitrin titer of 1.18 g/L and specific productivity of 47.8 mg/g CDW/h were obtained after 30 h incubation of sRHA3 when 1.5 g/L quercetin was added to the medium, which corresponded to a 53 % conversion. Also 51 mg/L of isoquercitrin was produced extracellularly which corresponds with a quercitrin:isoquercitrin production ratio of 24:1. This suggests the preference of RhaGT for UDP-rhamnose when different UDP-sugar donors are present. Possible explanations for the significantly lower specific productivity (fivefold decrease) of sRHA2 as compared to sRHA3 are either a higher metabolic burden [52] caused by the two plasmid system or a too limited activity of the native GalU, which could be insufficient for adequate UDP-glc formation [45] . To demonstrate the scalability of the developed bioprocess, strain sGAL3 was cultured in a 1-L bioreactor, which also ensures a constant pH set at 6.80 and avoid oxygen limitation. A detailed overview of the consumption of sucrose, growth and hyperoside production is given in Fig. 5 . After a lag-phase, the strain displayed a growth rate of 0.32 ± 0.02 h −1 while simultaneously producing hyperoside. The observed specific productivity (65.9 ± 2.6 mg/g CDW/h) was comparable to the one obtained on shake flask scale. When nearly all quercetin was converted, hyperoside formation slowed down, which can be explained either by the observed correlation between quercetin concentration and q p , or by the reported reversibility of F3GT [53] . It is likely that further improvements in titer and productivity can be realized by optimizing the supply of quercetin using a fed-batch system. To the best of our knowledge, the results obtained in this study with the engineered sGAL and sRHA strains for the production of hyperoside and quercitrin are the highest reported to date both in terms of titer and production rate. The maximal production rate obtained in this contribution was 6 to 50-fold higher compared to the maximal production rates (r p,max ) of processes reported in the literature [47, 54] as is illustrated in Fig. 6 . The increased performance, in terms of titer and productivity, obtained with the developed platform can be attributed to the use of a split metabolism in combination with optimally rerouting the flux from glucose 1-phosphate towards UDP-galactose and UDP-rhamnose. The undesired conversion of the activated sugars into biomass Fig. 3 Effect of the chromosomal integration locus of the knockin of BaSP on the growth rate. Strains were grown in shake flasks and the resulting maximal growth rates (µ max ) were compared with E. coli W ΔcscAR with plasmid-based BaSP expression (+pBaSP). Error bars represent standard deviations is impeded by gene deletions, which guarantees a high product yield. In addition, since biomass formation, which is fueled by the fructose moiety of sucrose, and glycoside synthesis go hand in hand and subsequently are performed at the same time at a high rate, a high productivity is equally guaranteed (one-step fermentation process). Besides quercetin also other flavonols such as kaempferol, fisetin, morin and myricetin significantly contribute to our daily flavonoid intake, which also have extremely diverse beneficial effects [55, 56] . As the sugar moiety is a major determinant of the intestinal absorption of dietary flavonoids and their subsequent bioactivity [57, 58] , the To this end, strains sGAL3 and sRHA3 were grown in tubes with 5 mL minimal medium, each containing 1.5 g/L of either kaempferol, myricetin, morin or fisetin. Growth and production were monitored over 48 h and various spots were observed on TLC with similar retention factors as hyperoside and quercitrin. Mass spectrometry was used to identify the compounds produced, which confirmed the in vivo galactosylation of myricetin, kaempferol, morin and fisetin ( Table 2 ). All compounds occurred with an m/z of [M + 114], due to complexation with trifluoroacetic acid from the mobile phase. The galactoside of morin was produced at a slow rate, which is in accordance to the very low in vitro activity of F3GT towards this flavonol [53] . A possible explanation for this limited activity may be the presence of an unusual hydroxyl group at the 2′ position, which may sterically hinder deprotonation and consequent galactosylation of morin at hydroxyl group 3 [59] . Incubation of sRHA3 with the different flavonols investigated showed two distinct glycoside spots on TLC, which corresponded to the 3-O-rhamnoside and 3-O-glucoside. Kaempferol proved to be the best substrate for RhaGT and was predominantly rhamnosylated (8:1 ratio), with a titer exceeding 400 mg/L, which is twofold higher than previously reported [47] . Fisetin on the other hand was efficiently glucosylated, yet the formation of its rhamnoside was not as efficient, with a titer below 5 mg/L. A similar preference towards glucoside formation was also observed with myricetin and morin, which indicates that the positioning of the hydroxyl groups is the determining factor for glycosylation with RhaGT. The production of the desired rhamnosides, galactosides or glucosides may be improved considerably by using UGTs that are more specific towards certain flavonols and UDP-sugars. Transformation of the corresponding UGTs in the developed in vivo glycosylation strains presents a promising alternative for the large-scale production of various flavonol glycoforms, which are to date mainly extracted from plant material. On the other hand, due to the pivotal role of UDP-glc, various other UDP-sugars can be formed in vivo (e.g. UDP-glucuronate, UDP-xylose, UDP-arabinose). In combination with the modularity of the developed glycosylation platform, which permits rapid introduction of any UGT or UDP-sugar pathway, virtually any glycoside can be produced. Hence, this demonstrates that the proposed microbial platform is a robust, versatile and efficient microbial cell factory for the glycosylation (e.g. glucosylation, rhamnosylation, galactosylation) of small molecules. Although obtained productivities are the highest reported today and compete with the current production processes, further improvement can be limited due to solubility issues of the aglycon or of the glycoside. To this end follow-up research can focus on further metabolic engineering (e.g. introduction of the aglycon pathway allowing in vivo gradually production of the aglycon) or on process optimization [e.g. 2-phase (bilayer) fermentation which enables in situ recovery] to improve these issues. In this contribution, a biotechnological platform was developed for the galactosylation and rhamnosylation of small molecules, such as secondary metabolite natural products, starting from a previously created glucosylation host. To this end, the routes to convert UDP-glucose into UDP-galactose and UDP-rhamnose were introduced by expressing a UDP-glucose epimerase (galE) and a UDP-rhamnose synthase (MUM4), respectively. As a proof of concept, the bio-active flavonol quercetin was selected for galactosylation and rhamnosylation, yielding hyperoside (quercetin 3-O-galactoside) and quercitrin (quercetin 3-O-rhamnoside), respectively. Next, the flavonol 3-O-galactosyltransferase (F3GT) from Petunia hybrida and the flavonol 3-O-rhamnosyltransferase from Arabidopsis thaliana (RhaGT) were overexpressed in the metabolically engineered E. coli W mutants. The strains created were able to produce 940 mg/L of hyperoside and 1176 mg/L of quercitrin at specific production rates of 68.7 mg/g CDW/h and 47.8 mg/g CDW/h, respectively, which are the highest reported to date. Interestingly, both GTs showed in vivo activity towards other dietary flavonols, whereby for example over 400 mg/L of kaempferol 3-O-rhamnoside could be formed extracellularly. All plasmids used were constructed using Gibson assembly [60] or CLIVA [61] . All PCR fragments were amplified using Q5 polymerase from New England Biolabs (Ipswich, Massachusetts). Oligonucleotides were purchased from IDT (Leuven, Belgium). The plasmids and bacterial strains used in this study are listed in Table 1 . A list of primers for the creation of gene knockouts/knockins and for the cloning of the expression plasmids is given in Additional file 2: Table S1 . E. coli DH5α was used for plasmid cloning and propagation, while E. coli W was used for expression of the production plasmids and the creation of gene knockouts and knockins. Hyperoside, quercitrin, isoquercitrin, kaempferol and myricetin were purchased from Carbosynth (Berkshire, UK). All other chemicals used were purchased from Sigma Aldrich (Germany) unless otherwise indicated. The expression plasmids for the prod uction of hyperoside and quercitrin were constructed as depicted in Additional file 3: Figure S1A Figure S1D ). The galE [Genbank: JW0742] and galE2 [Genbank: KJ543703] sequences were amplified from the genomic DNA of E. coli and Bifidobacterium bifidum, respectively. CLIVA assembly resulted in the intermediary plasmid pBaSP/F3GT/UgpA ( Figure S1A ), which was subsequently used for the amplification of the F3GT/ UgpA backbone. Gibson assembly of the GalE or GalE2 inserts with this backbone resulted in the final galactosylation plasmids pGalE/F3GT/UgpA and pGalE2/F3GT/ UgpA, respectively ( Figure S1B ). Similarly, MUM4 and RhaGT were introduced using a 3-pieces Gibson assembly ( Figure S1C ), resulting in the final rhamnosylation plasmid pMUM4/RhaGT/UgpA. The overall E. coli W knockout mutants were created using the one step deletion system of Datsenko and Wanner [62] . The strategy for chromosomal integration of BaSP under control of the constitutive promoter P22 flanked by L4 and L5 at the melA and glgC loci is depicted and explained in Additional file 1: Figure S2 . Transformants were plated on minimal sucrose medium agar plates and grown overnight for screening. The in-house strain E. coli W ΔcscAR Δpgm Δagp ΔushA ΔlacZYA::P22-lacY ΔgalETKM [45] was used for the chromosomal integration of L4-P22-BaSP-L5 at the melA site, yielding the base strain sGLYC (Table 1) . This strain and the E. coli W wild type were transformed with the production plasmids described above, resulting in the galactosylation (sGAL) and rhamnosylation (sRHA) strains given in Table 1 . Composition of LB and minimal sucrose medium was described previously [45] . Minimal medium agar plates with sucrose (50 g/L) had the same composition as minimal sucrose medium, but contained additionally 15 g/L of agarose. The agarose and salts were autoclaved separately at 121 °C for 21 min. Sucrose was filter sterilized through a 0.22 µm corning filter (Fisher, Belgium) and heated for 1 min in a microwave oven at 800 W prior to mixing it with the warm agarose and salt solutions. 1 mL/L of mineral solution [45] was sterilely added prior to pouring the plates. Escherichia coli W mutant precultures were grown in 5 mL LB medium with the antibiotics (50 μg/mL kanamycin or carbenicillin) required for maintenance and selection of the plasmids. The cultures were grown for 16 h at 37 °C and 200 rpm and used for the 2 % inoculation of 100 mL minimal sucrose medium in 500 mL shake flasks. For the production of hyperoside and quercitrin, quercetin was added to the minimal medium at a concentration of 0.15 or 1.5 g/L. Growth conditions were the same as previously described [45] . Samples were taken at regular intervals from the broth and, after centrifugation, the supernatant was used for the analysis and quantification of sugars. For the analysis of quercetin and its glycosides, 200 µL of the culture was collected and extracted with 800 µL ethyl acetate. The organic layer was collected, evaporated in a SpeedVac ™ vacuum concentrator (Thermo Fisher, USA) and dissolved in 200 µL of DMSO for HPLC quantification. The bioreactor set-up and fermentation conditions used are the same as previously described [45] . Production experiments were performed on minimal sucrose medium without MOPS buffer and with the addition of quercetin as acceptor. Culture samples were primarily analyzed by TLC on Silica gel 60 F 254 precoated plates (Merck, Germany). All plates were run in a closed TLC chamber and developed using standard visualization techniques and agents: UV fluorescence (254 nm) or by staining with 10 % (v/v) H 2 SO 4 and subsequent charring. The mobile phase for detecting the various flavonols and corresponding glycosides consisted of an ethyl acetate:acetic acid:formic acid:water (100:11:11:27 v/v) mixture [63] . Product spot intensities of other flavonol glycosides were processed and quantified using ImageJ [64] . HPLC quantification of sucrose, fructose and glucose was performed using an X-bridge Amide column (35 μm, Waters, USA) as described previously [45] . Quercetin, hyperoside, quercitrin and isoquercitrin were detected with the method described by Pandey et al. [41] using a Varian HPLC system (Agilent technologies, California). Mass spectrometry for determination of the various flavonol glycosides was performed with a Micromass Quattro LC (McKinley Scientific, USA). Detection was performed in negative mode ESI-224 MS with a capillary voltage of 2.53 kV, a cone voltage of 20 V, cone and desolvation gas flows of 93 and 420 L/h, and source and cone temperatures of 150 and 350 °C, respectively. Quercetin glycosides were extracted from the broth with an equal volume of ethyl acetate after which the organic layer was evaporated to dryness. The remaining product was dissolved in the solvent system described above and run on a preparative TLC plate. The band containing hyperoside (R f 0.53) or quercitrin (R f 0.75) was scraped off, extracted with ethyl acetate and evaporated to yield a bright yellow powder. Products were confirmed by NMR. Spectra were reported elsewhere [47, 65] .

What characteristics does glycosylation have on flavonoids?

Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300492/ SHA: f0c2cd2793d71f1ea11a810442a2c06d5013e899 Authors: Yu, Haijing; Liu, Yang; Wang, Hongwu; Wan, Xiaoyang; Huang, Jiaquan; Yan, Weiming; Xi, Dong; Luo, Xiaoping; Shen, Guanxin; Ning, Qin Date: 2018-12-13 DOI: 10.3389/fimmu.2018.02935 License: cc-by Abstract: Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse, THP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | www.frontiersin.org and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10 protein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design.

What is fibrinogen-like protein 2 (FgI2)?

Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300492/ SHA: f0c2cd2793d71f1ea11a810442a2c06d5013e899 Authors: Yu, Haijing; Liu, Yang; Wang, Hongwu; Wan, Xiaoyang; Huang, Jiaquan; Yan, Weiming; Xi, Dong; Luo, Xiaoping; Shen, Guanxin; Ning, Qin Date: 2018-12-13 DOI: 10.3389/fimmu.2018.02935 License: cc-by Abstract: Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse, THP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | www.frontiersin.org and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10 protein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design.

What represses murine hepatitis virus strain 3 (MHV-3) infection?

Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300492/ SHA: f0c2cd2793d71f1ea11a810442a2c06d5013e899 Authors: Yu, Haijing; Liu, Yang; Wang, Hongwu; Wan, Xiaoyang; Huang, Jiaquan; Yan, Weiming; Xi, Dong; Luo, Xiaoping; Shen, Guanxin; Ning, Qin Date: 2018-12-13 DOI: 10.3389/fimmu.2018.02935 License: cc-by Abstract: Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse, THP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | www.frontiersin.org and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10 protein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design.

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Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300492/ SHA: f0c2cd2793d71f1ea11a810442a2c06d5013e899 Authors: Yu, Haijing; Liu, Yang; Wang, Hongwu; Wan, Xiaoyang; Huang, Jiaquan; Yan, Weiming; Xi, Dong; Luo, Xiaoping; Shen, Guanxin; Ning, Qin Date: 2018-12-13 DOI: 10.3389/fimmu.2018.02935 License: cc-by Abstract: Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse, THP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | www.frontiersin.org and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10 protein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design.

What is fulminant hepatitis?

Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300492/ SHA: f0c2cd2793d71f1ea11a810442a2c06d5013e899 Authors: Yu, Haijing; Liu, Yang; Wang, Hongwu; Wan, Xiaoyang; Huang, Jiaquan; Yan, Weiming; Xi, Dong; Luo, Xiaoping; Shen, Guanxin; Ning, Qin Date: 2018-12-13 DOI: 10.3389/fimmu.2018.02935 License: cc-by Abstract: Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse, THP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | www.frontiersin.org and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10 protein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design.

How does Prothrombinase Fgl2 affect the coagulation process?

Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300492/ SHA: f0c2cd2793d71f1ea11a810442a2c06d5013e899 Authors: Yu, Haijing; Liu, Yang; Wang, Hongwu; Wan, Xiaoyang; Huang, Jiaquan; Yan, Weiming; Xi, Dong; Luo, Xiaoping; Shen, Guanxin; Ning, Qin Date: 2018-12-13 DOI: 10.3389/fimmu.2018.02935 License: cc-by Abstract: Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse, THP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | www.frontiersin.org and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10 protein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design.

How long after MHV-3 infection were liver samples taken?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What is the relationship between urbanization and risk of emergence of flu-like diseases?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What factors and characteristics of semi-urban landscapes promote viral transmission?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What is the relationship between HIN1 viral transmission and poultry production.

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What is the principle behind infection Convergence Model ?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What is the Boosted Regression Tree method?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What is the advantage of Boosted Regression Tree method?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What is the relationship between land use and emergence of HPAI H5N1?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

Where is the highest risk of HPAI H5N1 like disease emergence?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

How does land use fragmentation increase the risk of flu-like diseases?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What is the relationship between the outbreak of HPAI H5N1 like diseases and rice cultivation?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What is the relationship between aquaculture and spread of H5N1 like diseases?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What is the relationship between proximity ofwater bodies to agricultural lands and spread of H5N1 like diseases?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What is the effect of diversity of chicken flock on H5N1 disease?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What is Compound Topological Index and how is it related to the risk of disease transmission?

Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078228/ SHA: 1cf54d1c77b7f0494ab971429d26e0e199952d09 Authors: Liu, Jia; Cao, Ruiyuan; Xu, Mingyue; Wang, Xi; Zhang, Huanyu; Hu, Hengrui; Li, Yufeng; Hu, Zhihong; Zhong, Wu; Wang, Manli Date: 2020-03-18 DOI: 10.1038/s41421-020-0156-0 License: cc-by Abstract: 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. 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. 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 . 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 (http://www.chictr.org.cn) 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. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. (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.). 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. 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 ). 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 . 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. 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 . 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. 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). 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. 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. 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. 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 . 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 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. 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. 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.

In vitro comparison of antiviral activity of Chloroquine(CQ) and Hydroxychloroquine(HCQ) against COVID-19?

Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078228/ SHA: 1cf54d1c77b7f0494ab971429d26e0e199952d09 Authors: Liu, Jia; Cao, Ruiyuan; Xu, Mingyue; Wang, Xi; Zhang, Huanyu; Hu, Hengrui; Li, Yufeng; Hu, Zhihong; Zhong, Wu; Wang, Manli Date: 2020-03-18 DOI: 10.1038/s41421-020-0156-0 License: cc-by Abstract: 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. 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. 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 . 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 (http://www.chictr.org.cn) 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. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. (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.). 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. 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 ). 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 . 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. 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 . 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. 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). 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. 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. 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. 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 . 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 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. 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. 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.

What will be the drug of choice for treating COVID-19 between Chloroquine and Remdesivir?

Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078228/ SHA: 1cf54d1c77b7f0494ab971429d26e0e199952d09 Authors: Liu, Jia; Cao, Ruiyuan; Xu, Mingyue; Wang, Xi; Zhang, Huanyu; Hu, Hengrui; Li, Yufeng; Hu, Zhihong; Zhong, Wu; Wang, Manli Date: 2020-03-18 DOI: 10.1038/s41421-020-0156-0 License: cc-by Abstract: 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. 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. 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 . 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 (http://www.chictr.org.cn) 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. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. (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.). 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. 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 ). 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 . 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. 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 . 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. 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). 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. 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. 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. 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 . 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 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. 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. 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.

Mechanism of action of Chloroquine(CQ) and Hydroxychloroquine(HCQ) against COVID-19?

Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078228/ SHA: 1cf54d1c77b7f0494ab971429d26e0e199952d09 Authors: Liu, Jia; Cao, Ruiyuan; Xu, Mingyue; Wang, Xi; Zhang, Huanyu; Hu, Hengrui; Li, Yufeng; Hu, Zhihong; Zhong, Wu; Wang, Manli Date: 2020-03-18 DOI: 10.1038/s41421-020-0156-0 License: cc-by Abstract: 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. 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. 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 . 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 (http://www.chictr.org.cn) 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. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. (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.). 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. 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 ). 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 . 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. 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 . 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. 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). 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. 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. 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. 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 . 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 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. 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. 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.

What is the effect of Chloroquine(CQ) and Hydroxychloroquine(HCQ) on endosomal maturation and endocytosis in COVID-19 treatment?

Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078228/ SHA: 1cf54d1c77b7f0494ab971429d26e0e199952d09 Authors: Liu, Jia; Cao, Ruiyuan; Xu, Mingyue; Wang, Xi; Zhang, Huanyu; Hu, Hengrui; Li, Yufeng; Hu, Zhihong; Zhong, Wu; Wang, Manli Date: 2020-03-18 DOI: 10.1038/s41421-020-0156-0 License: cc-by Abstract: 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. 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. 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 . 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 (http://www.chictr.org.cn) 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. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. (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.). 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. 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 ). 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 . 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. 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 . 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. 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). 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. 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. 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. 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 . 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 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. 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. 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.

Evidence of Hydroxychloroquine(HCQ) being anti inflammatory in SARS-CoV-2 critically ill patients with elevated plasma cytokines?

Factors Associated With Mental Health Outcomes Among Health Care Workers Exposed to Coronavirus Disease 2019 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090843/ SHA: 0a08fddd9dcee1b1254a05b49113521bbc423ccd Authors: Lai, Jianbo; Ma, Simeng; Wang, Ying; Cai, Zhongxiang; Hu, Jianbo; Wei, Ning; Wu, Jiang; Du, Hui; Chen, Tingting; Li, Ruiting; Tan, Huawei; Kang, Lijun; Yao, Lihua; Huang, Manli; Wang, Huafen; Wang, Gaohua; Liu, Zhongchun; Hu, Shaohua Date: 2020-03-23 DOI: 10.1001/jamanetworkopen.2020.3976 License: cc-by Abstract: 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. 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%]). 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. 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

What proportion of healthcare workers reported symptoms of depression?

Factors Associated With Mental Health Outcomes Among Health Care Workers Exposed to Coronavirus Disease 2019 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090843/ SHA: 0a08fddd9dcee1b1254a05b49113521bbc423ccd Authors: Lai, Jianbo; Ma, Simeng; Wang, Ying; Cai, Zhongxiang; Hu, Jianbo; Wei, Ning; Wu, Jiang; Du, Hui; Chen, Tingting; Li, Ruiting; Tan, Huawei; Kang, Lijun; Yao, Lihua; Huang, Manli; Wang, Huafen; Wang, Gaohua; Liu, Zhongchun; Hu, Shaohua Date: 2020-03-23 DOI: 10.1001/jamanetworkopen.2020.3976 License: cc-by Abstract: 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. 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%]). 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. 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

What proportion of healthcare workers reported symptoms of anxiety?

Factors Associated With Mental Health Outcomes Among Health Care Workers Exposed to Coronavirus Disease 2019 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090843/ SHA: 0a08fddd9dcee1b1254a05b49113521bbc423ccd Authors: Lai, Jianbo; Ma, Simeng; Wang, Ying; Cai, Zhongxiang; Hu, Jianbo; Wei, Ning; Wu, Jiang; Du, Hui; Chen, Tingting; Li, Ruiting; Tan, Huawei; Kang, Lijun; Yao, Lihua; Huang, Manli; Wang, Huafen; Wang, Gaohua; Liu, Zhongchun; Hu, Shaohua Date: 2020-03-23 DOI: 10.1001/jamanetworkopen.2020.3976 License: cc-by Abstract: 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. 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%]). 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. 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

What proportion of healthcare workers reported symptoms of insomnia?

Factors Associated With Mental Health Outcomes Among Health Care Workers Exposed to Coronavirus Disease 2019 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090843/ SHA: 0a08fddd9dcee1b1254a05b49113521bbc423ccd Authors: Lai, Jianbo; Ma, Simeng; Wang, Ying; Cai, Zhongxiang; Hu, Jianbo; Wei, Ning; Wu, Jiang; Du, Hui; Chen, Tingting; Li, Ruiting; Tan, Huawei; Kang, Lijun; Yao, Lihua; Huang, Manli; Wang, Huafen; Wang, Gaohua; Liu, Zhongchun; Hu, Shaohua Date: 2020-03-23 DOI: 10.1001/jamanetworkopen.2020.3976 License: cc-by Abstract: 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. 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%]). 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. 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

What proportion reported distress?

Factors Associated With Mental Health Outcomes Among Health Care Workers Exposed to Coronavirus Disease 2019 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090843/ SHA: 0a08fddd9dcee1b1254a05b49113521bbc423ccd Authors: Lai, Jianbo; Ma, Simeng; Wang, Ying; Cai, Zhongxiang; Hu, Jianbo; Wei, Ning; Wu, Jiang; Du, Hui; Chen, Tingting; Li, Ruiting; Tan, Huawei; Kang, Lijun; Yao, Lihua; Huang, Manli; Wang, Huafen; Wang, Gaohua; Liu, Zhongchun; Hu, Shaohua Date: 2020-03-23 DOI: 10.1001/jamanetworkopen.2020.3976 License: cc-by Abstract: 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. 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%]). 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. 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

What were the Generalized Anxiety Disorder scale scores?

Factors Associated With Mental Health Outcomes Among Health Care Workers Exposed to Coronavirus Disease 2019 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090843/ SHA: 0a08fddd9dcee1b1254a05b49113521bbc423ccd Authors: Lai, Jianbo; Ma, Simeng; Wang, Ying; Cai, Zhongxiang; Hu, Jianbo; Wei, Ning; Wu, Jiang; Du, Hui; Chen, Tingting; Li, Ruiting; Tan, Huawei; Kang, Lijun; Yao, Lihua; Huang, Manli; Wang, Huafen; Wang, Gaohua; Liu, Zhongchun; Hu, Shaohua Date: 2020-03-23 DOI: 10.1001/jamanetworkopen.2020.3976 License: cc-by Abstract: 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. 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%]). 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. 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

What were the Insomnia Severity Index scores ?

Factors Associated With Mental Health Outcomes Among Health Care Workers Exposed to Coronavirus Disease 2019 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090843/ SHA: 0a08fddd9dcee1b1254a05b49113521bbc423ccd Authors: Lai, Jianbo; Ma, Simeng; Wang, Ying; Cai, Zhongxiang; Hu, Jianbo; Wei, Ning; Wu, Jiang; Du, Hui; Chen, Tingting; Li, Ruiting; Tan, Huawei; Kang, Lijun; Yao, Lihua; Huang, Manli; Wang, Huafen; Wang, Gaohua; Liu, Zhongchun; Hu, Shaohua Date: 2020-03-23 DOI: 10.1001/jamanetworkopen.2020.3976 License: cc-by Abstract: 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. 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%]). 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. 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

What were the Impact of Event Scale–Revised scores?

Factors Associated With Mental Health Outcomes Among Health Care Workers Exposed to Coronavirus Disease 2019 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090843/ SHA: 0a08fddd9dcee1b1254a05b49113521bbc423ccd Authors: Lai, Jianbo; Ma, Simeng; Wang, Ying; Cai, Zhongxiang; Hu, Jianbo; Wei, Ning; Wu, Jiang; Du, Hui; Chen, Tingting; Li, Ruiting; Tan, Huawei; Kang, Lijun; Yao, Lihua; Huang, Manli; Wang, Huafen; Wang, Gaohua; Liu, Zhongchun; Hu, Shaohua Date: 2020-03-23 DOI: 10.1001/jamanetworkopen.2020.3976 License: cc-by Abstract: 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. 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%]). 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. 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

What were the results of analysis?

Factors Associated With Mental Health Outcomes Among Health Care Workers Exposed to Coronavirus Disease 2019 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090843/ SHA: 0a08fddd9dcee1b1254a05b49113521bbc423ccd Authors: Lai, Jianbo; Ma, Simeng; Wang, Ying; Cai, Zhongxiang; Hu, Jianbo; Wei, Ning; Wu, Jiang; Du, Hui; Chen, Tingting; Li, Ruiting; Tan, Huawei; Kang, Lijun; Yao, Lihua; Huang, Manli; Wang, Huafen; Wang, Gaohua; Liu, Zhongchun; Hu, Shaohua Date: 2020-03-23 DOI: 10.1001/jamanetworkopen.2020.3976 License: cc-by Abstract: 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. 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%]). 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. 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

What were the results of analysis?

Factors Associated With Mental Health Outcomes Among Health Care Workers Exposed to Coronavirus Disease 2019 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090843/ SHA: 0a08fddd9dcee1b1254a05b49113521bbc423ccd Authors: Lai, Jianbo; Ma, Simeng; Wang, Ying; Cai, Zhongxiang; Hu, Jianbo; Wei, Ning; Wu, Jiang; Du, Hui; Chen, Tingting; Li, Ruiting; Tan, Huawei; Kang, Lijun; Yao, Lihua; Huang, Manli; Wang, Huafen; Wang, Gaohua; Liu, Zhongchun; Hu, Shaohua Date: 2020-03-23 DOI: 10.1001/jamanetworkopen.2020.3976 License: cc-by Abstract: 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. 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%]). 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. 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

What are the conclusions of this study?

A missense mutation in Katnal1 underlies behavioural, neurological and ciliary anomalies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761721/ SHA: f4cebabd74b16e710fb41a737d8ef84b7d565d8d Authors: Banks, G; Lassi, G; Hoerder-Suabedissen, A; Tinarelli, F; Simon, M M; Wilcox, A; Lau, P; Lawson, T N; Johnson, S; Rutman, A; Sweeting, M; Chesham, J E; Barnard, A R; Horner, N; Westerberg, H; Smith, L B; Molnár, Z; Hastings, M H; Hirst, R A; Tucci, V; Nolan, P M Date: 2017-04-04 DOI: 10.1038/mp.2017.54 License: cc-by Abstract: 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. 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. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, [3] [4] [5] [6] 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 (http://atgu.mgh.harvard.edu/~spurcell/genebook/gene 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. 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. 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. 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. 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. 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. 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. 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). 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. 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). 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. 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. 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 . 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. 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. 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. 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). 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 . 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 ). 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). 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. 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. 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. 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). 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. 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. 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. 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. 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. 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. 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 (http://atgu.mgh.harvard.edu/~spurcell/gene 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. 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. 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. [41] [42] [43] [44] 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. 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. 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.

What are microtubule severing enzymes?

A missense mutation in Katnal1 underlies behavioural, neurological and ciliary anomalies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761721/ SHA: f4cebabd74b16e710fb41a737d8ef84b7d565d8d Authors: Banks, G; Lassi, G; Hoerder-Suabedissen, A; Tinarelli, F; Simon, M M; Wilcox, A; Lau, P; Lawson, T N; Johnson, S; Rutman, A; Sweeting, M; Chesham, J E; Barnard, A R; Horner, N; Westerberg, H; Smith, L B; Molnár, Z; Hastings, M H; Hirst, R A; Tucci, V; Nolan, P M Date: 2017-04-04 DOI: 10.1038/mp.2017.54 License: cc-by Abstract: 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. 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. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, [3] [4] [5] [6] 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 (http://atgu.mgh.harvard.edu/~spurcell/genebook/gene 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. 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. 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. 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. 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. 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. 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. 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). 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. 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). 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. 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. 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 . 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. 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. 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. 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). 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 . 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 ). 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). 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. 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. 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. 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). 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. 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. 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. 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. 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. 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. 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 (http://atgu.mgh.harvard.edu/~spurcell/gene 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. 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. 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. [41] [42] [43] [44] 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. 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. 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.

What genetic mutation is associated with hereditary spastic paraplegia?

A missense mutation in Katnal1 underlies behavioural, neurological and ciliary anomalies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761721/ SHA: f4cebabd74b16e710fb41a737d8ef84b7d565d8d Authors: Banks, G; Lassi, G; Hoerder-Suabedissen, A; Tinarelli, F; Simon, M M; Wilcox, A; Lau, P; Lawson, T N; Johnson, S; Rutman, A; Sweeting, M; Chesham, J E; Barnard, A R; Horner, N; Westerberg, H; Smith, L B; Molnár, Z; Hastings, M H; Hirst, R A; Tucci, V; Nolan, P M Date: 2017-04-04 DOI: 10.1038/mp.2017.54 License: cc-by Abstract: 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. 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. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, [3] [4] [5] [6] 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 (http://atgu.mgh.harvard.edu/~spurcell/genebook/gene 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. 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. 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. 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. 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. 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. 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. 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). 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. 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). 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. 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. 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 . 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. 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. 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. 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). 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 . 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 ). 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). 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. 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. 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. 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). 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. 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. 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. 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. 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. 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. 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 (http://atgu.mgh.harvard.edu/~spurcell/gene 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. 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. 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. [41] [42] [43] [44] 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. 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. 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.

What genetic mutation is associated with cerebral malformations?

A missense mutation in Katnal1 underlies behavioural, neurological and ciliary anomalies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761721/ SHA: f4cebabd74b16e710fb41a737d8ef84b7d565d8d Authors: Banks, G; Lassi, G; Hoerder-Suabedissen, A; Tinarelli, F; Simon, M M; Wilcox, A; Lau, P; Lawson, T N; Johnson, S; Rutman, A; Sweeting, M; Chesham, J E; Barnard, A R; Horner, N; Westerberg, H; Smith, L B; Molnár, Z; Hastings, M H; Hirst, R A; Tucci, V; Nolan, P M Date: 2017-04-04 DOI: 10.1038/mp.2017.54 License: cc-by Abstract: 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. 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. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, [3] [4] [5] [6] 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 (http://atgu.mgh.harvard.edu/~spurcell/genebook/gene 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. 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. 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. 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. 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. 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. 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. 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). 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. 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). 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. 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. 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 . 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. 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. 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. 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). 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 . 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 ). 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). 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. 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. 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. 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). 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. 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. 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. 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. 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. 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. 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 (http://atgu.mgh.harvard.edu/~spurcell/gene 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. 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. 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. [41] [42] [43] [44] 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. 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. 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.

What genetic mutation is associated with autism?

A missense mutation in Katnal1 underlies behavioural, neurological and ciliary anomalies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761721/ SHA: f4cebabd74b16e710fb41a737d8ef84b7d565d8d Authors: Banks, G; Lassi, G; Hoerder-Suabedissen, A; Tinarelli, F; Simon, M M; Wilcox, A; Lau, P; Lawson, T N; Johnson, S; Rutman, A; Sweeting, M; Chesham, J E; Barnard, A R; Horner, N; Westerberg, H; Smith, L B; Molnár, Z; Hastings, M H; Hirst, R A; Tucci, V; Nolan, P M Date: 2017-04-04 DOI: 10.1038/mp.2017.54 License: cc-by Abstract: 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. 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. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, [3] [4] [5] [6] 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 (http://atgu.mgh.harvard.edu/~spurcell/genebook/gene 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. 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. 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. 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. 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. 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. 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. 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). 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. 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). 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. 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. 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 . 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. 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. 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. 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). 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 . 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 ). 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). 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. 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. 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. 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). 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. 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. 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. 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. 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. 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. 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 (http://atgu.mgh.harvard.edu/~spurcell/gene 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. 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. 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. [41] [42] [43] [44] 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. 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. 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.

What is KATNAL1?

A missense mutation in Katnal1 underlies behavioural, neurological and ciliary anomalies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761721/ SHA: f4cebabd74b16e710fb41a737d8ef84b7d565d8d Authors: Banks, G; Lassi, G; Hoerder-Suabedissen, A; Tinarelli, F; Simon, M M; Wilcox, A; Lau, P; Lawson, T N; Johnson, S; Rutman, A; Sweeting, M; Chesham, J E; Barnard, A R; Horner, N; Westerberg, H; Smith, L B; Molnár, Z; Hastings, M H; Hirst, R A; Tucci, V; Nolan, P M Date: 2017-04-04 DOI: 10.1038/mp.2017.54 License: cc-by Abstract: 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. 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. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, [3] [4] [5] [6] 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 (http://atgu.mgh.harvard.edu/~spurcell/genebook/gene 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. 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. 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. 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. 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. 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. 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. 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). 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. 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). 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. 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. 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 . 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. 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. 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. 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). 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 . 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 ). 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). 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. 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. 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. 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). 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. 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. 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. 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. 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. 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. 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 (http://atgu.mgh.harvard.edu/~spurcell/gene 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. 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. 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. [41] [42] [43] [44] 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. 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. 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.

What organ is most associated with the KATNAL1 gene?

A missense mutation in Katnal1 underlies behavioural, neurological and ciliary anomalies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761721/ SHA: f4cebabd74b16e710fb41a737d8ef84b7d565d8d Authors: Banks, G; Lassi, G; Hoerder-Suabedissen, A; Tinarelli, F; Simon, M M; Wilcox, A; Lau, P; Lawson, T N; Johnson, S; Rutman, A; Sweeting, M; Chesham, J E; Barnard, A R; Horner, N; Westerberg, H; Smith, L B; Molnár, Z; Hastings, M H; Hirst, R A; Tucci, V; Nolan, P M Date: 2017-04-04 DOI: 10.1038/mp.2017.54 License: cc-by Abstract: 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. 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. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, [3] [4] [5] [6] 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 (http://atgu.mgh.harvard.edu/~spurcell/genebook/gene 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. 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. 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. 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. 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. 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. 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. 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). 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. 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). 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. 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. 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 . 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. 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. 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. 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). 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 . 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 ). 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). 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. 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. 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. 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). 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. 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. 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. 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. 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. 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. 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 (http://atgu.mgh.harvard.edu/~spurcell/gene 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. 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. 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. [41] [42] [43] [44] 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. 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. 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.

What CNS functions can be measured by studying the movement of mice in a T-maze?

A missense mutation in Katnal1 underlies behavioural, neurological and ciliary anomalies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761721/ SHA: f4cebabd74b16e710fb41a737d8ef84b7d565d8d Authors: Banks, G; Lassi, G; Hoerder-Suabedissen, A; Tinarelli, F; Simon, M M; Wilcox, A; Lau, P; Lawson, T N; Johnson, S; Rutman, A; Sweeting, M; Chesham, J E; Barnard, A R; Horner, N; Westerberg, H; Smith, L B; Molnár, Z; Hastings, M H; Hirst, R A; Tucci, V; Nolan, P M Date: 2017-04-04 DOI: 10.1038/mp.2017.54 License: cc-by Abstract: 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. 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. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively, [3] [4] [5] [6] 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 (http://atgu.mgh.harvard.edu/~spurcell/genebook/gene 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. 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. 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. 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. 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. 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. 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. 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). 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. 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). 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. 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. 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 . 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. 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. 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. 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). 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 . 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 ). 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). 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. 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. 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. 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). 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. 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. 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. 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. 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. 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. 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 (http://atgu.mgh.harvard.edu/~spurcell/gene 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. 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. 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. [41] [42] [43] [44] 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. 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. 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.

What CNS functions are changed by mutations in the KATNAL1 gene?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

How is Japanese encephalitis transmitted?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

What element is essential to promoting JEV infection?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

Where is Q130 located in the NS4B protein?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

What is the mechanism of action for manidipine?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

How many different pathogens are members of the Flaviviridae family of virus?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

What is the size of a flavivirus?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

How many open reading frames are in the flavivirus genome?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

What are the structural protein elements of a flavivirus?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

What is the function of the nonstructural protein elements of the flavivirus?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

What are RVPs?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

What is HTS?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

What is the selective index in high throughput screening?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

What is the structure of a recombiant viral particle?

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/ SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964 Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei Date: 2017-10-13 DOI: 10.1128/jvi.01055-17 License: cc-by Abstract: 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. 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. 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. 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 (1) . 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 (2) ; 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) (3) . 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. 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 (4, 5) . 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. 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. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . 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. 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) . 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. 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. 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. 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 (5, 14) (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. 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) . 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. 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. 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 (15) , 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 (16) (17) (18) (19) , 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 ). 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 ). 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). 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. 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. 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. 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. 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. 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) . 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 (20, 21) . 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. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . 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. 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. 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 (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . 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. 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 (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , 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 (37) (38) (39) . 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 (40) , 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 (41, 42) . 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. 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 (4, 5) . 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 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 (43) . 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. 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. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . 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. 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 (43) . 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. 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. 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 (4) . 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). 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. 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. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).

What measure is used in high-throughput screening to identify potential antiviral compounds?

Rotavirus A in wild and domestic animals from areas with environmental degradation in the Brazilian Amazon https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298726/ SHA: f3c309c596c20f48f493b77e714ce957d877bdcb Authors: de Barros, Bruno de Cássio Veloso; Chagas, Elaine Nunes; Bezerra, Luna Wanessa; Ribeiro, Laila Graziela; Duarte Júnior, Jose Wandilson Barboza; Pereira, Diego; da Penha Junior, Edvaldo Tavares; Silva, Julia Rezende; Bezerra, Delana Andreza Melo; Bandeira, Renato Silva; Pinheiro, Helder Henrique Costa; Guerra, Sylvia de Fátima dos Santos; Guimarães, Ricardo José de Paula Souza e; Mascarenhas, Joana D'Arc Pereira Date: 2018-12-18 DOI: 10.1371/journal.pone.0209005 License: cc-by Abstract: Acute gastroenteritis is one of the main causes of mortality in humans and young animals. Domestic and mainly wild animals such as bats, small rodents and birds are highly diversified animals in relation to their habitats and ecological niches and are widely distributed geographically in environments of forest fragmentation in some areas of the Amazon, being considered important sources for viruses that affect humans and other animals. Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health. Although the knowledge of the global diversity of enteric viruses is scarce, there are reports demonstrating the detection of rotavirus in domestic animals and animals of productive systems, such as bovines and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments. The fecal specimens were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas. Text: Emerging and reemerging infectious diseases are increasing each year in several countries, with an impact both on human populations and on domestic and wild animals living in areas with considerable forest remnants [1] . Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] . The populations of wild animals that inhabit forest fragments are strategic groups for studies of public health and the transmission of zoonosis, given that they act as indicators in the assistance and intervention in the human populations, aiming at the prevention of outbreaks and epidemics [3] . Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] . They represent one of the main causes of mortality in humans, and in young animals, counting for about 25% of mortality [8] . Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] . The rotavirus (RV) belongs to the Reoviridae family and comprises nine species known as Rotavirus group A to I, with a recent proposal of the J species [13, 14] . Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] . They have a double-stranded ribonucleic acid (dsRNA) genome, divided into 11 segments coding for structural proteins (VP1-VP4, VP6 and VP7) and nonstructural (NSP1-NSP5/NSP6) proteins [16, 17] . There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] . Although the results of RVA have already been described globally [12, [23] [24] [25] [26] [27] [28] [29] [30] , in Brazil, the occurrence, diversity and role of rotavirus in these animals are still poorly studied, considering the large number of present species [4, [31] [32] [33] [34] . In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] . Therefore, it is important to monitor the health of domestic and wild animals in their natural habitat, especially in areas with anthropic alterations that have an interface with rural communities and enterprises, in order to investigate the occurrence of RVA in this population. These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] . The qualitative real-time polymerase chain reaction (qRT-PCR) used the NSP3 gene and the TaqMan probe from a highly conserved region of the rotavirus non-structural protein 3 (NSP3), which was previously used in samples from human origin and with low viral loads Precipitation data were obtained from The Brazilian National Institute of Meteorology (Inmethttp://www.inmet.gov.br/) for the years of capture in the Expedito Ribeiro Settlement (2014) and Açailândia (2015) of the Data Collection Platforms (PCDs) of Belém, located 50 km from Santa Bárbara do Pará, and Tracuateua, located 50 km from Peixe-Boi and 100 km from Viseu. Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field. The municipal boundaries were obtained on the website of the Brazilian Institute of Geography and Statistics (IBGE) (http://www.ibge.gov.br/) and data on deforestation and land use were obtained from the PRODES [43] and TerraClass [44] Projects. PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004. The satellite image was generated using the sensor Sentinel 2 of the European Space Agency (ESA) (https://sentinel.esa.int/ web/sentinel/user-guides/sentinel-2-msi) with Open Access CC-BY License (http://open.esa.int/) from the years of 2017 and 2018. All the data obtained was stored in a Geographic Database (BDG). The BDG was imported/ stored in a GIS for the editing of the graphic elements, establishment of topological relations between the elements and their respective attributes, spatial analysis and visualization of the result through thematic maps. For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use. The selected fragments were distributed within the mesoregions studied, and in each selected fragment fecal samples were randomly collected from domestic and wild animals [45] . Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed). The data processing, interpretation, visualization and spatial analysis were performed in ArcGIS software (http://www.arcgis.com/). For the analysis of data related to the determination of the richness, composition and abundance of the fauna of the animals studied in the study area, considering the collection methods adopted and the species available in each city, each sample was considered as an independent sample. The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] . For the calculation of the Test T, the Statistica software was used, and the indices of infected animals in the two environments (forest fragment and peridomicile) were calculated for each treatment sampled by collection area, using the software Past 1.92. Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] . The data obtained for the occurrence of RVA and the questionnaires was inserted into a database for a descriptive analysis of the epidemiological profile of the animal population in the three forest ecosystems studied. In this analysis, descriptive statistical treatments were carried out, using customized "row-columns" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T. Population study, collection of clinical specimens and laboratory methodology. The flying animals (wild birds and chiroptera) were captured using mist nets which were opened at dawn (4:00 a.m.) and closed in the morning (9:00 a.m.) and were inspected every one hour until the closing, with a sampling effort of 15 days. This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site. The fecal specimens were collected by stimulation of the rectal ampulla with the use of a "Zaragatoa", packed in cryogenic vials, identified, stored in liquid nitrogen, and later sent to the Laboratory. Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm). In each sample plot, 61 traps were distributed, 20 Shermans and 41 Tomahawks being baited with a mixture made with peanut paste, sardines, cod liver oil and corn meal, as well as fruit like banana, apple and pineapple. All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material. The wild animals were sedated with a combination of ketamine 20mg/kg and xylazine 2mg/kg intramuscularly and subsequently, euthanized with anesthetic overdose of 2% lidocaine in the foramen magnum, according to the recommendation of the National Council for the Control of Animal Experimentation (CONCEA). From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals. Amongthese, 648 (50.5%) samples were randomly selected for RVA research and handled in Level Three Biosafety Laboratory (NB3). The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data. The qRT-PCR was conducted according to Zeng et al. [40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence. The assay was conducted in a mixture containing: RNAse-free H 2 O, TaqMan RT-PCR Mix (2x), TAqMan RT Enzyme Mix (40x), primers for the NSP3 gene, Primer NSP3 Forward (20mM), Primer NSP3 Reverse (20mM), probe NSP3 S (10nm), Template (RNA) 3μL, having a total reaction volume of 17μL and reverse transcription cycling of 50˚C, 30 minutes, denaturation of 95˚C, 10 minutes, annealing of 45 cycles of 95˚C, 15 seconds and extension of 60˚C, 1 minute. The analyzes were considered positive when presenting the cycle threshold (CT) � 40. In order to guarantee a reliable test result, the measurements of contamination control were performed with the use of positive animal control (SA11 prototype) and a negative control (ultrapure water). All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples. First round was performed with consensus primers N-VP4F1/N-VP4R1 and the Nested-PCR was conducted with N-VP4F2/N-VP4R2 primers to amplify VP4 gene. Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR. The sequences were collected from an automated ABI Prism 3130xl DNA sequencer (Applied Biosystems). The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7. Posteriorly, the data were compared with othersequences from the National Center for Biotechnology Information GenBank database using BLAST alignment tool to elucidate the RVA genotype of the samples. From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%). The CT interval ranged from 28. 47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea. Rotavirus A (RVA) detected in the present study of wild and domestic animals belonging to the three areas of forest fragment, according to Fig 2. In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively. In addition, most of the animals were young with ages varying from 1 day to 8 yearsold, history of deficient vaccination, lack of technical assistance and raised in the form of subsistence. The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status. In relation to chiroptera, 32 (17.98%) positive samples for RVA were distributed among Carollia perspicillata species, with 12 (37.5%) being all adults, 9 (28.12%) Desmodus rotundus samples (4 young and 5 adults), 5 (15.6%) of Uroderma bilobata (15.62%), 3 (9.37%) of Artibeus lituratus and the species Artibeus Planirostus, Diaemus iyoug and Glossophagine with 1 (3.12%) each. These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms. Fig 3 shows the results obtained for all the species of animals investigated in the forest fragment as well as in the peridomicillus area. The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) . Considering the factors related to the anthropic activities in the three studied areas within the three cities of the present study, it was observed that the city of Santa Bárbara is the one that has a better area of preserved forest and the city of Viseu a smaller area. However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment. It was observed in this chosen area of the city, the presence of different families living in a rural settlement, surviving from the exploitation of forest resources and the creation of small animals for subsistence, such as poultry and fish farming, as well as family farming products. The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu. Extensive livestock farming was practiced with beef cattle, equines for work and small animals (swine and goats). In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation. When comparing the climates of the three areas it was observed that the predominant climate is megathermal and humid with average annual temperature around 27˚C. The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C. Annual rainfall is quite high, generally around 2,350 mm, but strongly concentrated from January to June (80%). From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months. The relative humidity of the average air oscillates around 85%, as shown in Fig 6 [48] . The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region. The average deforestation index in the three study areas was calculated from data obtained from INPE information systems. It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi. In relation to the period of 2016, great changes were observed in Peixe Boi (79%) and in the city of Viseu (70%), thus demonstrating that changes in the natural ecosystem may be associated with the frequencies for RVA in the studied areas, according to Fig 7. When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained. Applying the selected statistical analysis, a Pearson x2 Chi-square value was obtained: 16.7159, df = 1 and p <0.001, meaning that the hypothesis was corroborated, that is, the greater the degradation of the environment, the more likely it will be the search for food by wild animals in adjacent areas, or in the edge of the forest or even in the peri domiciliary region. In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment. It is important to point out that the animals detected in this study are important sources of viral strains. A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene. Eight strains (10%) were positive for VP4 gene, being 2 strains bellowed to P [6] genotype and 6 to P[4]-type, according to In the present study, RVA was detected circulating in 27.5% of the animals; 36% in domestic animals and 64% in wild animals, providing a unique dataset with qRT-PCR detecting a low viral load of RVA in different species, which further correlates with the deforestation index. These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] . With the advent of real-time PCR (qPCR), there was an exponential growth, compared to conventional PCR essays, since its superior accuracy, sensitivity and specificity is remarkable, and it is Rotavirus A in wild and domestic animals possible to detect RVA in a variety of animal species using NSP3 gene [49] . The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon. Currently, no data have been described in the literature regarding the RVA detection using real-time qPCR technique in a wide variety of wild animal species. However, a study by Soltan et al. [50] conducted with horses and cattle detected RVA by RT-PCR, commercial RT-PCR and RT-qPCR in 36.7%, 51.4% and 56.9% respectively, differently from the present study that showed higher positivity for chiropterans (17.98%), canines (21.35%), birds (23.6%) and cattle (14.6%). The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] . Afterwards, several strains of RVA were detected by different molecular techniques involving chiroptera, in several countries, including Kenya (E. helvum), China (Rhinolophus hipposideros and Aselliscus stoliczkanus), France (Myotis mystacinus), Cameroon (E. helvum) and Brazil [31, [51] [52] [53] [54] [55] . The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%). Barquez et al. [56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico. Of the positive chiroptera for RVA in the present study, a prevalence of 28.12% was of Desmodus rotundus. This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study. These findings show the importance of epidemiological data on the studied species due to the lack of studies involving species of neotropical chiroptera, and it is not possible to establish comparative parameters for these animals. Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively. Although in the Amazon region there are records of RVA, RVD, RVF and RVG that infect birds [57] [58] and RVD in migratory birds [59] , all were detected by RT-PCR assays differently from the present study which detected the RVA by RT-qPCR involving a variety of animal species. On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations. The study detected the presence of RVA in different species of animals both in areas near the home and in areas located in fragments of forest, characterized as forest remnants, since they were located in cities that suffered high environmental impacts due to vegetal extractivism, pasture formation for cattle breeding, exploitation of natural resources, and direct reflexes on the habitats of wild animals that can serve as virus sources, thus facilitating the dispersion of RVA among communities of coexisting animals. It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study. However, it is noteworthy that only in the communities of Santa Bárbara and Viseu were collected fecal specimens from asymptomatic humans for diarrhea and tested for RVA, but all were negative. It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data. The fragmentation of the forest generates many consequences on the Amazonian biota, being able to alter the diversity and the composition of the animal communities in the fragments and even to interfere in the ecological processes, without considering that the fragments of forest in the Amazon are influenced by the climate, possibly facilitating the dispersion of pathogens by the environment, since the wild animals detected in the present study are asymptomatic and have low viral load for RVA. The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea. Wild birds have unlimited flight capacity, were captured in an interface region between the peridomicile and forest fragments and it is believed that this region has not been influenced by anthropic activities such as those observed in the area of the present study. On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment. The high rates of increase and the analysis of land use in the researched areas may be important indicators of how these animals interact, since with deforestation, the populations of wild animals seek refuge in nearby communities facilitating the dispersal of infectious agents and the possible occurrence of carrier animals by direct contact or contamination of the local environment. To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities. The detection of positive animals was useful to monitor the infection of the agent in the animal population and to provide an early warning signal to predict an impending epidemic and a favorable risk for the human population, given the evidence of RVA circulation in the different forest fragments. In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al. [60] and Waruhiu et al. [61] . Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe. The most prevalent genotypes isolated from feces of Belgian diarrheal and non-diarrheal piglets in 2012 [62] demonstrate a wide range of combinations of genotypes G / P including; G3P [6] , G4P [6] , G5P [6] , G4P [7] , G5P [7] , G9P [7] , G9P [13] and G9P [23] . On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea. Finding shows that different P genotypes of RVA strains interact with distinct blood group histological antigens (HBGA, ABOH, Lewis) and sialic acids via VP4 providing insight into the regional prevalence and increased zoonotic potential of some RVA of origin swine [63] . The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] . In the population of animals studied the zoonotic transmission can be frequent, since the animals live in contact with humans and in precarious sanitary conditions. In Brazil, this genotype was described in animal and human populations in studies of Luchs et al. [32] ; Honma et al. [67] ; Araújo et al. [68] ; Mascarenhas et al. [69] and Lorenzetti et al. [70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions. Regarding the genotype P [4] , itwas most detected in our samples in bats, dogs, swine and feline. This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] . It is important to emphasize that the indicators of environmental contamination in Brazil are significant and contribute to the possibility of human-animal transmission [71] . Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible. Our findings may be useful for tracking fecal contamination in the environment using animals as possible sources thus minimizing the risk of infection by exposure to susceptible individuals, in this case different animal species or even human populations. RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA. Although the samples are asymptomatic for diarrhea, it is necessary to conduct strategies for the monitoring and control of the animals in the areas studied in the human population as well as in other species of animals, as well as the implementation of preventive measures aimed at future outbreaks in communities animals in the resident population in these impacted areas. Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals. It is noteworthy that, although the quality of the analyzed samples is characterized as low detectable viral load, the technique presented a good analytical response in the detection of the source animals for RVA, facilitating the selection of the samples for future genetic characterization tests.

How many known species of Rotavirus exist?

Rotavirus A in wild and domestic animals from areas with environmental degradation in the Brazilian Amazon https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298726/ SHA: f3c309c596c20f48f493b77e714ce957d877bdcb Authors: de Barros, Bruno de Cássio Veloso; Chagas, Elaine Nunes; Bezerra, Luna Wanessa; Ribeiro, Laila Graziela; Duarte Júnior, Jose Wandilson Barboza; Pereira, Diego; da Penha Junior, Edvaldo Tavares; Silva, Julia Rezende; Bezerra, Delana Andreza Melo; Bandeira, Renato Silva; Pinheiro, Helder Henrique Costa; Guerra, Sylvia de Fátima dos Santos; Guimarães, Ricardo José de Paula Souza e; Mascarenhas, Joana D'Arc Pereira Date: 2018-12-18 DOI: 10.1371/journal.pone.0209005 License: cc-by Abstract: Acute gastroenteritis is one of the main causes of mortality in humans and young animals. Domestic and mainly wild animals such as bats, small rodents and birds are highly diversified animals in relation to their habitats and ecological niches and are widely distributed geographically in environments of forest fragmentation in some areas of the Amazon, being considered important sources for viruses that affect humans and other animals. Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health. Although the knowledge of the global diversity of enteric viruses is scarce, there are reports demonstrating the detection of rotavirus in domestic animals and animals of productive systems, such as bovines and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments. The fecal specimens were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas. Text: Emerging and reemerging infectious diseases are increasing each year in several countries, with an impact both on human populations and on domestic and wild animals living in areas with considerable forest remnants [1] . Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] . The populations of wild animals that inhabit forest fragments are strategic groups for studies of public health and the transmission of zoonosis, given that they act as indicators in the assistance and intervention in the human populations, aiming at the prevention of outbreaks and epidemics [3] . Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] . They represent one of the main causes of mortality in humans, and in young animals, counting for about 25% of mortality [8] . Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] . The rotavirus (RV) belongs to the Reoviridae family and comprises nine species known as Rotavirus group A to I, with a recent proposal of the J species [13, 14] . Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] . They have a double-stranded ribonucleic acid (dsRNA) genome, divided into 11 segments coding for structural proteins (VP1-VP4, VP6 and VP7) and nonstructural (NSP1-NSP5/NSP6) proteins [16, 17] . There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] . Although the results of RVA have already been described globally [12, [23] [24] [25] [26] [27] [28] [29] [30] , in Brazil, the occurrence, diversity and role of rotavirus in these animals are still poorly studied, considering the large number of present species [4, [31] [32] [33] [34] . In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] . Therefore, it is important to monitor the health of domestic and wild animals in their natural habitat, especially in areas with anthropic alterations that have an interface with rural communities and enterprises, in order to investigate the occurrence of RVA in this population. These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] . The qualitative real-time polymerase chain reaction (qRT-PCR) used the NSP3 gene and the TaqMan probe from a highly conserved region of the rotavirus non-structural protein 3 (NSP3), which was previously used in samples from human origin and with low viral loads Precipitation data were obtained from The Brazilian National Institute of Meteorology (Inmethttp://www.inmet.gov.br/) for the years of capture in the Expedito Ribeiro Settlement (2014) and Açailândia (2015) of the Data Collection Platforms (PCDs) of Belém, located 50 km from Santa Bárbara do Pará, and Tracuateua, located 50 km from Peixe-Boi and 100 km from Viseu. Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field. The municipal boundaries were obtained on the website of the Brazilian Institute of Geography and Statistics (IBGE) (http://www.ibge.gov.br/) and data on deforestation and land use were obtained from the PRODES [43] and TerraClass [44] Projects. PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004. The satellite image was generated using the sensor Sentinel 2 of the European Space Agency (ESA) (https://sentinel.esa.int/ web/sentinel/user-guides/sentinel-2-msi) with Open Access CC-BY License (http://open.esa.int/) from the years of 2017 and 2018. All the data obtained was stored in a Geographic Database (BDG). The BDG was imported/ stored in a GIS for the editing of the graphic elements, establishment of topological relations between the elements and their respective attributes, spatial analysis and visualization of the result through thematic maps. For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use. The selected fragments were distributed within the mesoregions studied, and in each selected fragment fecal samples were randomly collected from domestic and wild animals [45] . Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed). The data processing, interpretation, visualization and spatial analysis were performed in ArcGIS software (http://www.arcgis.com/). For the analysis of data related to the determination of the richness, composition and abundance of the fauna of the animals studied in the study area, considering the collection methods adopted and the species available in each city, each sample was considered as an independent sample. The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] . For the calculation of the Test T, the Statistica software was used, and the indices of infected animals in the two environments (forest fragment and peridomicile) were calculated for each treatment sampled by collection area, using the software Past 1.92. Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] . The data obtained for the occurrence of RVA and the questionnaires was inserted into a database for a descriptive analysis of the epidemiological profile of the animal population in the three forest ecosystems studied. In this analysis, descriptive statistical treatments were carried out, using customized "row-columns" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T. Population study, collection of clinical specimens and laboratory methodology. The flying animals (wild birds and chiroptera) were captured using mist nets which were opened at dawn (4:00 a.m.) and closed in the morning (9:00 a.m.) and were inspected every one hour until the closing, with a sampling effort of 15 days. This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site. The fecal specimens were collected by stimulation of the rectal ampulla with the use of a "Zaragatoa", packed in cryogenic vials, identified, stored in liquid nitrogen, and later sent to the Laboratory. Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm). In each sample plot, 61 traps were distributed, 20 Shermans and 41 Tomahawks being baited with a mixture made with peanut paste, sardines, cod liver oil and corn meal, as well as fruit like banana, apple and pineapple. All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material. The wild animals were sedated with a combination of ketamine 20mg/kg and xylazine 2mg/kg intramuscularly and subsequently, euthanized with anesthetic overdose of 2% lidocaine in the foramen magnum, according to the recommendation of the National Council for the Control of Animal Experimentation (CONCEA). From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals. Amongthese, 648 (50.5%) samples were randomly selected for RVA research and handled in Level Three Biosafety Laboratory (NB3). The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data. The qRT-PCR was conducted according to Zeng et al. [40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence. The assay was conducted in a mixture containing: RNAse-free H 2 O, TaqMan RT-PCR Mix (2x), TAqMan RT Enzyme Mix (40x), primers for the NSP3 gene, Primer NSP3 Forward (20mM), Primer NSP3 Reverse (20mM), probe NSP3 S (10nm), Template (RNA) 3μL, having a total reaction volume of 17μL and reverse transcription cycling of 50˚C, 30 minutes, denaturation of 95˚C, 10 minutes, annealing of 45 cycles of 95˚C, 15 seconds and extension of 60˚C, 1 minute. The analyzes were considered positive when presenting the cycle threshold (CT) � 40. In order to guarantee a reliable test result, the measurements of contamination control were performed with the use of positive animal control (SA11 prototype) and a negative control (ultrapure water). All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples. First round was performed with consensus primers N-VP4F1/N-VP4R1 and the Nested-PCR was conducted with N-VP4F2/N-VP4R2 primers to amplify VP4 gene. Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR. The sequences were collected from an automated ABI Prism 3130xl DNA sequencer (Applied Biosystems). The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7. Posteriorly, the data were compared with othersequences from the National Center for Biotechnology Information GenBank database using BLAST alignment tool to elucidate the RVA genotype of the samples. From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%). The CT interval ranged from 28. 47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea. Rotavirus A (RVA) detected in the present study of wild and domestic animals belonging to the three areas of forest fragment, according to Fig 2. In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively. In addition, most of the animals were young with ages varying from 1 day to 8 yearsold, history of deficient vaccination, lack of technical assistance and raised in the form of subsistence. The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status. In relation to chiroptera, 32 (17.98%) positive samples for RVA were distributed among Carollia perspicillata species, with 12 (37.5%) being all adults, 9 (28.12%) Desmodus rotundus samples (4 young and 5 adults), 5 (15.6%) of Uroderma bilobata (15.62%), 3 (9.37%) of Artibeus lituratus and the species Artibeus Planirostus, Diaemus iyoug and Glossophagine with 1 (3.12%) each. These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms. Fig 3 shows the results obtained for all the species of animals investigated in the forest fragment as well as in the peridomicillus area. The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) . Considering the factors related to the anthropic activities in the three studied areas within the three cities of the present study, it was observed that the city of Santa Bárbara is the one that has a better area of preserved forest and the city of Viseu a smaller area. However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment. It was observed in this chosen area of the city, the presence of different families living in a rural settlement, surviving from the exploitation of forest resources and the creation of small animals for subsistence, such as poultry and fish farming, as well as family farming products. The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu. Extensive livestock farming was practiced with beef cattle, equines for work and small animals (swine and goats). In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation. When comparing the climates of the three areas it was observed that the predominant climate is megathermal and humid with average annual temperature around 27˚C. The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C. Annual rainfall is quite high, generally around 2,350 mm, but strongly concentrated from January to June (80%). From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months. The relative humidity of the average air oscillates around 85%, as shown in Fig 6 [48] . The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region. The average deforestation index in the three study areas was calculated from data obtained from INPE information systems. It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi. In relation to the period of 2016, great changes were observed in Peixe Boi (79%) and in the city of Viseu (70%), thus demonstrating that changes in the natural ecosystem may be associated with the frequencies for RVA in the studied areas, according to Fig 7. When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained. Applying the selected statistical analysis, a Pearson x2 Chi-square value was obtained: 16.7159, df = 1 and p <0.001, meaning that the hypothesis was corroborated, that is, the greater the degradation of the environment, the more likely it will be the search for food by wild animals in adjacent areas, or in the edge of the forest or even in the peri domiciliary region. In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment. It is important to point out that the animals detected in this study are important sources of viral strains. A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene. Eight strains (10%) were positive for VP4 gene, being 2 strains bellowed to P [6] genotype and 6 to P[4]-type, according to In the present study, RVA was detected circulating in 27.5% of the animals; 36% in domestic animals and 64% in wild animals, providing a unique dataset with qRT-PCR detecting a low viral load of RVA in different species, which further correlates with the deforestation index. These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] . With the advent of real-time PCR (qPCR), there was an exponential growth, compared to conventional PCR essays, since its superior accuracy, sensitivity and specificity is remarkable, and it is Rotavirus A in wild and domestic animals possible to detect RVA in a variety of animal species using NSP3 gene [49] . The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon. Currently, no data have been described in the literature regarding the RVA detection using real-time qPCR technique in a wide variety of wild animal species. However, a study by Soltan et al. [50] conducted with horses and cattle detected RVA by RT-PCR, commercial RT-PCR and RT-qPCR in 36.7%, 51.4% and 56.9% respectively, differently from the present study that showed higher positivity for chiropterans (17.98%), canines (21.35%), birds (23.6%) and cattle (14.6%). The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] . Afterwards, several strains of RVA were detected by different molecular techniques involving chiroptera, in several countries, including Kenya (E. helvum), China (Rhinolophus hipposideros and Aselliscus stoliczkanus), France (Myotis mystacinus), Cameroon (E. helvum) and Brazil [31, [51] [52] [53] [54] [55] . The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%). Barquez et al. [56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico. Of the positive chiroptera for RVA in the present study, a prevalence of 28.12% was of Desmodus rotundus. This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study. These findings show the importance of epidemiological data on the studied species due to the lack of studies involving species of neotropical chiroptera, and it is not possible to establish comparative parameters for these animals. Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively. Although in the Amazon region there are records of RVA, RVD, RVF and RVG that infect birds [57] [58] and RVD in migratory birds [59] , all were detected by RT-PCR assays differently from the present study which detected the RVA by RT-qPCR involving a variety of animal species. On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations. The study detected the presence of RVA in different species of animals both in areas near the home and in areas located in fragments of forest, characterized as forest remnants, since they were located in cities that suffered high environmental impacts due to vegetal extractivism, pasture formation for cattle breeding, exploitation of natural resources, and direct reflexes on the habitats of wild animals that can serve as virus sources, thus facilitating the dispersion of RVA among communities of coexisting animals. It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study. However, it is noteworthy that only in the communities of Santa Bárbara and Viseu were collected fecal specimens from asymptomatic humans for diarrhea and tested for RVA, but all were negative. It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data. The fragmentation of the forest generates many consequences on the Amazonian biota, being able to alter the diversity and the composition of the animal communities in the fragments and even to interfere in the ecological processes, without considering that the fragments of forest in the Amazon are influenced by the climate, possibly facilitating the dispersion of pathogens by the environment, since the wild animals detected in the present study are asymptomatic and have low viral load for RVA. The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea. Wild birds have unlimited flight capacity, were captured in an interface region between the peridomicile and forest fragments and it is believed that this region has not been influenced by anthropic activities such as those observed in the area of the present study. On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment. The high rates of increase and the analysis of land use in the researched areas may be important indicators of how these animals interact, since with deforestation, the populations of wild animals seek refuge in nearby communities facilitating the dispersal of infectious agents and the possible occurrence of carrier animals by direct contact or contamination of the local environment. To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities. The detection of positive animals was useful to monitor the infection of the agent in the animal population and to provide an early warning signal to predict an impending epidemic and a favorable risk for the human population, given the evidence of RVA circulation in the different forest fragments. In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al. [60] and Waruhiu et al. [61] . Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe. The most prevalent genotypes isolated from feces of Belgian diarrheal and non-diarrheal piglets in 2012 [62] demonstrate a wide range of combinations of genotypes G / P including; G3P [6] , G4P [6] , G5P [6] , G4P [7] , G5P [7] , G9P [7] , G9P [13] and G9P [23] . On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea. Finding shows that different P genotypes of RVA strains interact with distinct blood group histological antigens (HBGA, ABOH, Lewis) and sialic acids via VP4 providing insight into the regional prevalence and increased zoonotic potential of some RVA of origin swine [63] . The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] . In the population of animals studied the zoonotic transmission can be frequent, since the animals live in contact with humans and in precarious sanitary conditions. In Brazil, this genotype was described in animal and human populations in studies of Luchs et al. [32] ; Honma et al. [67] ; Araújo et al. [68] ; Mascarenhas et al. [69] and Lorenzetti et al. [70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions. Regarding the genotype P [4] , itwas most detected in our samples in bats, dogs, swine and feline. This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] . It is important to emphasize that the indicators of environmental contamination in Brazil are significant and contribute to the possibility of human-animal transmission [71] . Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible. Our findings may be useful for tracking fecal contamination in the environment using animals as possible sources thus minimizing the risk of infection by exposure to susceptible individuals, in this case different animal species or even human populations. RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA. Although the samples are asymptomatic for diarrhea, it is necessary to conduct strategies for the monitoring and control of the animals in the areas studied in the human population as well as in other species of animals, as well as the implementation of preventive measures aimed at future outbreaks in communities animals in the resident population in these impacted areas. Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals. It is noteworthy that, although the quality of the analyzed samples is characterized as low detectable viral load, the technique presented a good analytical response in the detection of the source animals for RVA, facilitating the selection of the samples for future genetic characterization tests.

Is Rotavirus single or double-stranded?

Rotavirus A in wild and domestic animals from areas with environmental degradation in the Brazilian Amazon https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298726/ SHA: f3c309c596c20f48f493b77e714ce957d877bdcb Authors: de Barros, Bruno de Cássio Veloso; Chagas, Elaine Nunes; Bezerra, Luna Wanessa; Ribeiro, Laila Graziela; Duarte Júnior, Jose Wandilson Barboza; Pereira, Diego; da Penha Junior, Edvaldo Tavares; Silva, Julia Rezende; Bezerra, Delana Andreza Melo; Bandeira, Renato Silva; Pinheiro, Helder Henrique Costa; Guerra, Sylvia de Fátima dos Santos; Guimarães, Ricardo José de Paula Souza e; Mascarenhas, Joana D'Arc Pereira Date: 2018-12-18 DOI: 10.1371/journal.pone.0209005 License: cc-by Abstract: Acute gastroenteritis is one of the main causes of mortality in humans and young animals. Domestic and mainly wild animals such as bats, small rodents and birds are highly diversified animals in relation to their habitats and ecological niches and are widely distributed geographically in environments of forest fragmentation in some areas of the Amazon, being considered important sources for viruses that affect humans and other animals. Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health. Although the knowledge of the global diversity of enteric viruses is scarce, there are reports demonstrating the detection of rotavirus in domestic animals and animals of productive systems, such as bovines and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments. The fecal specimens were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas. Text: Emerging and reemerging infectious diseases are increasing each year in several countries, with an impact both on human populations and on domestic and wild animals living in areas with considerable forest remnants [1] . Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] . The populations of wild animals that inhabit forest fragments are strategic groups for studies of public health and the transmission of zoonosis, given that they act as indicators in the assistance and intervention in the human populations, aiming at the prevention of outbreaks and epidemics [3] . Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] . They represent one of the main causes of mortality in humans, and in young animals, counting for about 25% of mortality [8] . Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] . The rotavirus (RV) belongs to the Reoviridae family and comprises nine species known as Rotavirus group A to I, with a recent proposal of the J species [13, 14] . Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] . They have a double-stranded ribonucleic acid (dsRNA) genome, divided into 11 segments coding for structural proteins (VP1-VP4, VP6 and VP7) and nonstructural (NSP1-NSP5/NSP6) proteins [16, 17] . There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] . Although the results of RVA have already been described globally [12, [23] [24] [25] [26] [27] [28] [29] [30] , in Brazil, the occurrence, diversity and role of rotavirus in these animals are still poorly studied, considering the large number of present species [4, [31] [32] [33] [34] . In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] . Therefore, it is important to monitor the health of domestic and wild animals in their natural habitat, especially in areas with anthropic alterations that have an interface with rural communities and enterprises, in order to investigate the occurrence of RVA in this population. These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] . The qualitative real-time polymerase chain reaction (qRT-PCR) used the NSP3 gene and the TaqMan probe from a highly conserved region of the rotavirus non-structural protein 3 (NSP3), which was previously used in samples from human origin and with low viral loads Precipitation data were obtained from The Brazilian National Institute of Meteorology (Inmethttp://www.inmet.gov.br/) for the years of capture in the Expedito Ribeiro Settlement (2014) and Açailândia (2015) of the Data Collection Platforms (PCDs) of Belém, located 50 km from Santa Bárbara do Pará, and Tracuateua, located 50 km from Peixe-Boi and 100 km from Viseu. Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field. The municipal boundaries were obtained on the website of the Brazilian Institute of Geography and Statistics (IBGE) (http://www.ibge.gov.br/) and data on deforestation and land use were obtained from the PRODES [43] and TerraClass [44] Projects. PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004. The satellite image was generated using the sensor Sentinel 2 of the European Space Agency (ESA) (https://sentinel.esa.int/ web/sentinel/user-guides/sentinel-2-msi) with Open Access CC-BY License (http://open.esa.int/) from the years of 2017 and 2018. All the data obtained was stored in a Geographic Database (BDG). The BDG was imported/ stored in a GIS for the editing of the graphic elements, establishment of topological relations between the elements and their respective attributes, spatial analysis and visualization of the result through thematic maps. For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use. The selected fragments were distributed within the mesoregions studied, and in each selected fragment fecal samples were randomly collected from domestic and wild animals [45] . Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed). The data processing, interpretation, visualization and spatial analysis were performed in ArcGIS software (http://www.arcgis.com/). For the analysis of data related to the determination of the richness, composition and abundance of the fauna of the animals studied in the study area, considering the collection methods adopted and the species available in each city, each sample was considered as an independent sample. The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] . For the calculation of the Test T, the Statistica software was used, and the indices of infected animals in the two environments (forest fragment and peridomicile) were calculated for each treatment sampled by collection area, using the software Past 1.92. Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] . The data obtained for the occurrence of RVA and the questionnaires was inserted into a database for a descriptive analysis of the epidemiological profile of the animal population in the three forest ecosystems studied. In this analysis, descriptive statistical treatments were carried out, using customized "row-columns" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T. Population study, collection of clinical specimens and laboratory methodology. The flying animals (wild birds and chiroptera) were captured using mist nets which were opened at dawn (4:00 a.m.) and closed in the morning (9:00 a.m.) and were inspected every one hour until the closing, with a sampling effort of 15 days. This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site. The fecal specimens were collected by stimulation of the rectal ampulla with the use of a "Zaragatoa", packed in cryogenic vials, identified, stored in liquid nitrogen, and later sent to the Laboratory. Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm). In each sample plot, 61 traps were distributed, 20 Shermans and 41 Tomahawks being baited with a mixture made with peanut paste, sardines, cod liver oil and corn meal, as well as fruit like banana, apple and pineapple. All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material. The wild animals were sedated with a combination of ketamine 20mg/kg and xylazine 2mg/kg intramuscularly and subsequently, euthanized with anesthetic overdose of 2% lidocaine in the foramen magnum, according to the recommendation of the National Council for the Control of Animal Experimentation (CONCEA). From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals. Amongthese, 648 (50.5%) samples were randomly selected for RVA research and handled in Level Three Biosafety Laboratory (NB3). The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data. The qRT-PCR was conducted according to Zeng et al. [40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence. The assay was conducted in a mixture containing: RNAse-free H 2 O, TaqMan RT-PCR Mix (2x), TAqMan RT Enzyme Mix (40x), primers for the NSP3 gene, Primer NSP3 Forward (20mM), Primer NSP3 Reverse (20mM), probe NSP3 S (10nm), Template (RNA) 3μL, having a total reaction volume of 17μL and reverse transcription cycling of 50˚C, 30 minutes, denaturation of 95˚C, 10 minutes, annealing of 45 cycles of 95˚C, 15 seconds and extension of 60˚C, 1 minute. The analyzes were considered positive when presenting the cycle threshold (CT) � 40. In order to guarantee a reliable test result, the measurements of contamination control were performed with the use of positive animal control (SA11 prototype) and a negative control (ultrapure water). All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples. First round was performed with consensus primers N-VP4F1/N-VP4R1 and the Nested-PCR was conducted with N-VP4F2/N-VP4R2 primers to amplify VP4 gene. Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR. The sequences were collected from an automated ABI Prism 3130xl DNA sequencer (Applied Biosystems). The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7. Posteriorly, the data were compared with othersequences from the National Center for Biotechnology Information GenBank database using BLAST alignment tool to elucidate the RVA genotype of the samples. From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%). The CT interval ranged from 28. 47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea. Rotavirus A (RVA) detected in the present study of wild and domestic animals belonging to the three areas of forest fragment, according to Fig 2. In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively. In addition, most of the animals were young with ages varying from 1 day to 8 yearsold, history of deficient vaccination, lack of technical assistance and raised in the form of subsistence. The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status. In relation to chiroptera, 32 (17.98%) positive samples for RVA were distributed among Carollia perspicillata species, with 12 (37.5%) being all adults, 9 (28.12%) Desmodus rotundus samples (4 young and 5 adults), 5 (15.6%) of Uroderma bilobata (15.62%), 3 (9.37%) of Artibeus lituratus and the species Artibeus Planirostus, Diaemus iyoug and Glossophagine with 1 (3.12%) each. These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms. Fig 3 shows the results obtained for all the species of animals investigated in the forest fragment as well as in the peridomicillus area. The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) . Considering the factors related to the anthropic activities in the three studied areas within the three cities of the present study, it was observed that the city of Santa Bárbara is the one that has a better area of preserved forest and the city of Viseu a smaller area. However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment. It was observed in this chosen area of the city, the presence of different families living in a rural settlement, surviving from the exploitation of forest resources and the creation of small animals for subsistence, such as poultry and fish farming, as well as family farming products. The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu. Extensive livestock farming was practiced with beef cattle, equines for work and small animals (swine and goats). In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation. When comparing the climates of the three areas it was observed that the predominant climate is megathermal and humid with average annual temperature around 27˚C. The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C. Annual rainfall is quite high, generally around 2,350 mm, but strongly concentrated from January to June (80%). From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months. The relative humidity of the average air oscillates around 85%, as shown in Fig 6 [48] . The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region. The average deforestation index in the three study areas was calculated from data obtained from INPE information systems. It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi. In relation to the period of 2016, great changes were observed in Peixe Boi (79%) and in the city of Viseu (70%), thus demonstrating that changes in the natural ecosystem may be associated with the frequencies for RVA in the studied areas, according to Fig 7. When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained. Applying the selected statistical analysis, a Pearson x2 Chi-square value was obtained: 16.7159, df = 1 and p <0.001, meaning that the hypothesis was corroborated, that is, the greater the degradation of the environment, the more likely it will be the search for food by wild animals in adjacent areas, or in the edge of the forest or even in the peri domiciliary region. In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment. It is important to point out that the animals detected in this study are important sources of viral strains. A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene. Eight strains (10%) were positive for VP4 gene, being 2 strains bellowed to P [6] genotype and 6 to P[4]-type, according to In the present study, RVA was detected circulating in 27.5% of the animals; 36% in domestic animals and 64% in wild animals, providing a unique dataset with qRT-PCR detecting a low viral load of RVA in different species, which further correlates with the deforestation index. These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] . With the advent of real-time PCR (qPCR), there was an exponential growth, compared to conventional PCR essays, since its superior accuracy, sensitivity and specificity is remarkable, and it is Rotavirus A in wild and domestic animals possible to detect RVA in a variety of animal species using NSP3 gene [49] . The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon. Currently, no data have been described in the literature regarding the RVA detection using real-time qPCR technique in a wide variety of wild animal species. However, a study by Soltan et al. [50] conducted with horses and cattle detected RVA by RT-PCR, commercial RT-PCR and RT-qPCR in 36.7%, 51.4% and 56.9% respectively, differently from the present study that showed higher positivity for chiropterans (17.98%), canines (21.35%), birds (23.6%) and cattle (14.6%). The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] . Afterwards, several strains of RVA were detected by different molecular techniques involving chiroptera, in several countries, including Kenya (E. helvum), China (Rhinolophus hipposideros and Aselliscus stoliczkanus), France (Myotis mystacinus), Cameroon (E. helvum) and Brazil [31, [51] [52] [53] [54] [55] . The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%). Barquez et al. [56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico. Of the positive chiroptera for RVA in the present study, a prevalence of 28.12% was of Desmodus rotundus. This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study. These findings show the importance of epidemiological data on the studied species due to the lack of studies involving species of neotropical chiroptera, and it is not possible to establish comparative parameters for these animals. Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively. Although in the Amazon region there are records of RVA, RVD, RVF and RVG that infect birds [57] [58] and RVD in migratory birds [59] , all were detected by RT-PCR assays differently from the present study which detected the RVA by RT-qPCR involving a variety of animal species. On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations. The study detected the presence of RVA in different species of animals both in areas near the home and in areas located in fragments of forest, characterized as forest remnants, since they were located in cities that suffered high environmental impacts due to vegetal extractivism, pasture formation for cattle breeding, exploitation of natural resources, and direct reflexes on the habitats of wild animals that can serve as virus sources, thus facilitating the dispersion of RVA among communities of coexisting animals. It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study. However, it is noteworthy that only in the communities of Santa Bárbara and Viseu were collected fecal specimens from asymptomatic humans for diarrhea and tested for RVA, but all were negative. It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data. The fragmentation of the forest generates many consequences on the Amazonian biota, being able to alter the diversity and the composition of the animal communities in the fragments and even to interfere in the ecological processes, without considering that the fragments of forest in the Amazon are influenced by the climate, possibly facilitating the dispersion of pathogens by the environment, since the wild animals detected in the present study are asymptomatic and have low viral load for RVA. The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea. Wild birds have unlimited flight capacity, were captured in an interface region between the peridomicile and forest fragments and it is believed that this region has not been influenced by anthropic activities such as those observed in the area of the present study. On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment. The high rates of increase and the analysis of land use in the researched areas may be important indicators of how these animals interact, since with deforestation, the populations of wild animals seek refuge in nearby communities facilitating the dispersal of infectious agents and the possible occurrence of carrier animals by direct contact or contamination of the local environment. To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities. The detection of positive animals was useful to monitor the infection of the agent in the animal population and to provide an early warning signal to predict an impending epidemic and a favorable risk for the human population, given the evidence of RVA circulation in the different forest fragments. In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al. [60] and Waruhiu et al. [61] . Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe. The most prevalent genotypes isolated from feces of Belgian diarrheal and non-diarrheal piglets in 2012 [62] demonstrate a wide range of combinations of genotypes G / P including; G3P [6] , G4P [6] , G5P [6] , G4P [7] , G5P [7] , G9P [7] , G9P [13] and G9P [23] . On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea. Finding shows that different P genotypes of RVA strains interact with distinct blood group histological antigens (HBGA, ABOH, Lewis) and sialic acids via VP4 providing insight into the regional prevalence and increased zoonotic potential of some RVA of origin swine [63] . The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] . In the population of animals studied the zoonotic transmission can be frequent, since the animals live in contact with humans and in precarious sanitary conditions. In Brazil, this genotype was described in animal and human populations in studies of Luchs et al. [32] ; Honma et al. [67] ; Araújo et al. [68] ; Mascarenhas et al. [69] and Lorenzetti et al. [70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions. Regarding the genotype P [4] , itwas most detected in our samples in bats, dogs, swine and feline. This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] . It is important to emphasize that the indicators of environmental contamination in Brazil are significant and contribute to the possibility of human-animal transmission [71] . Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible. Our findings may be useful for tracking fecal contamination in the environment using animals as possible sources thus minimizing the risk of infection by exposure to susceptible individuals, in this case different animal species or even human populations. RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA. Although the samples are asymptomatic for diarrhea, it is necessary to conduct strategies for the monitoring and control of the animals in the areas studied in the human population as well as in other species of animals, as well as the implementation of preventive measures aimed at future outbreaks in communities animals in the resident population in these impacted areas. Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals. It is noteworthy that, although the quality of the analyzed samples is characterized as low detectable viral load, the technique presented a good analytical response in the detection of the source animals for RVA, facilitating the selection of the samples for future genetic characterization tests.

What structural proteins are coded by Rotavirus?

Rotavirus A in wild and domestic animals from areas with environmental degradation in the Brazilian Amazon https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298726/ SHA: f3c309c596c20f48f493b77e714ce957d877bdcb Authors: de Barros, Bruno de Cássio Veloso; Chagas, Elaine Nunes; Bezerra, Luna Wanessa; Ribeiro, Laila Graziela; Duarte Júnior, Jose Wandilson Barboza; Pereira, Diego; da Penha Junior, Edvaldo Tavares; Silva, Julia Rezende; Bezerra, Delana Andreza Melo; Bandeira, Renato Silva; Pinheiro, Helder Henrique Costa; Guerra, Sylvia de Fátima dos Santos; Guimarães, Ricardo José de Paula Souza e; Mascarenhas, Joana D'Arc Pereira Date: 2018-12-18 DOI: 10.1371/journal.pone.0209005 License: cc-by Abstract: Acute gastroenteritis is one of the main causes of mortality in humans and young animals. Domestic and mainly wild animals such as bats, small rodents and birds are highly diversified animals in relation to their habitats and ecological niches and are widely distributed geographically in environments of forest fragmentation in some areas of the Amazon, being considered important sources for viruses that affect humans and other animals. Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health. Although the knowledge of the global diversity of enteric viruses is scarce, there are reports demonstrating the detection of rotavirus in domestic animals and animals of productive systems, such as bovines and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments. The fecal specimens were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas. Text: Emerging and reemerging infectious diseases are increasing each year in several countries, with an impact both on human populations and on domestic and wild animals living in areas with considerable forest remnants [1] . Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] . The populations of wild animals that inhabit forest fragments are strategic groups for studies of public health and the transmission of zoonosis, given that they act as indicators in the assistance and intervention in the human populations, aiming at the prevention of outbreaks and epidemics [3] . Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] . They represent one of the main causes of mortality in humans, and in young animals, counting for about 25% of mortality [8] . Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] . The rotavirus (RV) belongs to the Reoviridae family and comprises nine species known as Rotavirus group A to I, with a recent proposal of the J species [13, 14] . Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] . They have a double-stranded ribonucleic acid (dsRNA) genome, divided into 11 segments coding for structural proteins (VP1-VP4, VP6 and VP7) and nonstructural (NSP1-NSP5/NSP6) proteins [16, 17] . There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] . Although the results of RVA have already been described globally [12, [23] [24] [25] [26] [27] [28] [29] [30] , in Brazil, the occurrence, diversity and role of rotavirus in these animals are still poorly studied, considering the large number of present species [4, [31] [32] [33] [34] . In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] . Therefore, it is important to monitor the health of domestic and wild animals in their natural habitat, especially in areas with anthropic alterations that have an interface with rural communities and enterprises, in order to investigate the occurrence of RVA in this population. These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] . The qualitative real-time polymerase chain reaction (qRT-PCR) used the NSP3 gene and the TaqMan probe from a highly conserved region of the rotavirus non-structural protein 3 (NSP3), which was previously used in samples from human origin and with low viral loads Precipitation data were obtained from The Brazilian National Institute of Meteorology (Inmethttp://www.inmet.gov.br/) for the years of capture in the Expedito Ribeiro Settlement (2014) and Açailândia (2015) of the Data Collection Platforms (PCDs) of Belém, located 50 km from Santa Bárbara do Pará, and Tracuateua, located 50 km from Peixe-Boi and 100 km from Viseu. Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field. The municipal boundaries were obtained on the website of the Brazilian Institute of Geography and Statistics (IBGE) (http://www.ibge.gov.br/) and data on deforestation and land use were obtained from the PRODES [43] and TerraClass [44] Projects. PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004. The satellite image was generated using the sensor Sentinel 2 of the European Space Agency (ESA) (https://sentinel.esa.int/ web/sentinel/user-guides/sentinel-2-msi) with Open Access CC-BY License (http://open.esa.int/) from the years of 2017 and 2018. All the data obtained was stored in a Geographic Database (BDG). The BDG was imported/ stored in a GIS for the editing of the graphic elements, establishment of topological relations between the elements and their respective attributes, spatial analysis and visualization of the result through thematic maps. For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use. The selected fragments were distributed within the mesoregions studied, and in each selected fragment fecal samples were randomly collected from domestic and wild animals [45] . Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed). The data processing, interpretation, visualization and spatial analysis were performed in ArcGIS software (http://www.arcgis.com/). For the analysis of data related to the determination of the richness, composition and abundance of the fauna of the animals studied in the study area, considering the collection methods adopted and the species available in each city, each sample was considered as an independent sample. The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] . For the calculation of the Test T, the Statistica software was used, and the indices of infected animals in the two environments (forest fragment and peridomicile) were calculated for each treatment sampled by collection area, using the software Past 1.92. Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] . The data obtained for the occurrence of RVA and the questionnaires was inserted into a database for a descriptive analysis of the epidemiological profile of the animal population in the three forest ecosystems studied. In this analysis, descriptive statistical treatments were carried out, using customized "row-columns" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T. Population study, collection of clinical specimens and laboratory methodology. The flying animals (wild birds and chiroptera) were captured using mist nets which were opened at dawn (4:00 a.m.) and closed in the morning (9:00 a.m.) and were inspected every one hour until the closing, with a sampling effort of 15 days. This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site. The fecal specimens were collected by stimulation of the rectal ampulla with the use of a "Zaragatoa", packed in cryogenic vials, identified, stored in liquid nitrogen, and later sent to the Laboratory. Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm). In each sample plot, 61 traps were distributed, 20 Shermans and 41 Tomahawks being baited with a mixture made with peanut paste, sardines, cod liver oil and corn meal, as well as fruit like banana, apple and pineapple. All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material. The wild animals were sedated with a combination of ketamine 20mg/kg and xylazine 2mg/kg intramuscularly and subsequently, euthanized with anesthetic overdose of 2% lidocaine in the foramen magnum, according to the recommendation of the National Council for the Control of Animal Experimentation (CONCEA). From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals. Amongthese, 648 (50.5%) samples were randomly selected for RVA research and handled in Level Three Biosafety Laboratory (NB3). The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data. The qRT-PCR was conducted according to Zeng et al. [40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence. The assay was conducted in a mixture containing: RNAse-free H 2 O, TaqMan RT-PCR Mix (2x), TAqMan RT Enzyme Mix (40x), primers for the NSP3 gene, Primer NSP3 Forward (20mM), Primer NSP3 Reverse (20mM), probe NSP3 S (10nm), Template (RNA) 3μL, having a total reaction volume of 17μL and reverse transcription cycling of 50˚C, 30 minutes, denaturation of 95˚C, 10 minutes, annealing of 45 cycles of 95˚C, 15 seconds and extension of 60˚C, 1 minute. The analyzes were considered positive when presenting the cycle threshold (CT) � 40. In order to guarantee a reliable test result, the measurements of contamination control were performed with the use of positive animal control (SA11 prototype) and a negative control (ultrapure water). All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples. First round was performed with consensus primers N-VP4F1/N-VP4R1 and the Nested-PCR was conducted with N-VP4F2/N-VP4R2 primers to amplify VP4 gene. Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR. The sequences were collected from an automated ABI Prism 3130xl DNA sequencer (Applied Biosystems). The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7. Posteriorly, the data were compared with othersequences from the National Center for Biotechnology Information GenBank database using BLAST alignment tool to elucidate the RVA genotype of the samples. From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%). The CT interval ranged from 28. 47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea. Rotavirus A (RVA) detected in the present study of wild and domestic animals belonging to the three areas of forest fragment, according to Fig 2. In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively. In addition, most of the animals were young with ages varying from 1 day to 8 yearsold, history of deficient vaccination, lack of technical assistance and raised in the form of subsistence. The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status. In relation to chiroptera, 32 (17.98%) positive samples for RVA were distributed among Carollia perspicillata species, with 12 (37.5%) being all adults, 9 (28.12%) Desmodus rotundus samples (4 young and 5 adults), 5 (15.6%) of Uroderma bilobata (15.62%), 3 (9.37%) of Artibeus lituratus and the species Artibeus Planirostus, Diaemus iyoug and Glossophagine with 1 (3.12%) each. These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms. Fig 3 shows the results obtained for all the species of animals investigated in the forest fragment as well as in the peridomicillus area. The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) . Considering the factors related to the anthropic activities in the three studied areas within the three cities of the present study, it was observed that the city of Santa Bárbara is the one that has a better area of preserved forest and the city of Viseu a smaller area. However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment. It was observed in this chosen area of the city, the presence of different families living in a rural settlement, surviving from the exploitation of forest resources and the creation of small animals for subsistence, such as poultry and fish farming, as well as family farming products. The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu. Extensive livestock farming was practiced with beef cattle, equines for work and small animals (swine and goats). In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation. When comparing the climates of the three areas it was observed that the predominant climate is megathermal and humid with average annual temperature around 27˚C. The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C. Annual rainfall is quite high, generally around 2,350 mm, but strongly concentrated from January to June (80%). From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months. The relative humidity of the average air oscillates around 85%, as shown in Fig 6 [48] . The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region. The average deforestation index in the three study areas was calculated from data obtained from INPE information systems. It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi. In relation to the period of 2016, great changes were observed in Peixe Boi (79%) and in the city of Viseu (70%), thus demonstrating that changes in the natural ecosystem may be associated with the frequencies for RVA in the studied areas, according to Fig 7. When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained. Applying the selected statistical analysis, a Pearson x2 Chi-square value was obtained: 16.7159, df = 1 and p <0.001, meaning that the hypothesis was corroborated, that is, the greater the degradation of the environment, the more likely it will be the search for food by wild animals in adjacent areas, or in the edge of the forest or even in the peri domiciliary region. In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment. It is important to point out that the animals detected in this study are important sources of viral strains. A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene. Eight strains (10%) were positive for VP4 gene, being 2 strains bellowed to P [6] genotype and 6 to P[4]-type, according to In the present study, RVA was detected circulating in 27.5% of the animals; 36% in domestic animals and 64% in wild animals, providing a unique dataset with qRT-PCR detecting a low viral load of RVA in different species, which further correlates with the deforestation index. These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] . With the advent of real-time PCR (qPCR), there was an exponential growth, compared to conventional PCR essays, since its superior accuracy, sensitivity and specificity is remarkable, and it is Rotavirus A in wild and domestic animals possible to detect RVA in a variety of animal species using NSP3 gene [49] . The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon. Currently, no data have been described in the literature regarding the RVA detection using real-time qPCR technique in a wide variety of wild animal species. However, a study by Soltan et al. [50] conducted with horses and cattle detected RVA by RT-PCR, commercial RT-PCR and RT-qPCR in 36.7%, 51.4% and 56.9% respectively, differently from the present study that showed higher positivity for chiropterans (17.98%), canines (21.35%), birds (23.6%) and cattle (14.6%). The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] . Afterwards, several strains of RVA were detected by different molecular techniques involving chiroptera, in several countries, including Kenya (E. helvum), China (Rhinolophus hipposideros and Aselliscus stoliczkanus), France (Myotis mystacinus), Cameroon (E. helvum) and Brazil [31, [51] [52] [53] [54] [55] . The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%). Barquez et al. [56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico. Of the positive chiroptera for RVA in the present study, a prevalence of 28.12% was of Desmodus rotundus. This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study. These findings show the importance of epidemiological data on the studied species due to the lack of studies involving species of neotropical chiroptera, and it is not possible to establish comparative parameters for these animals. Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively. Although in the Amazon region there are records of RVA, RVD, RVF and RVG that infect birds [57] [58] and RVD in migratory birds [59] , all were detected by RT-PCR assays differently from the present study which detected the RVA by RT-qPCR involving a variety of animal species. On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations. The study detected the presence of RVA in different species of animals both in areas near the home and in areas located in fragments of forest, characterized as forest remnants, since they were located in cities that suffered high environmental impacts due to vegetal extractivism, pasture formation for cattle breeding, exploitation of natural resources, and direct reflexes on the habitats of wild animals that can serve as virus sources, thus facilitating the dispersion of RVA among communities of coexisting animals. It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study. However, it is noteworthy that only in the communities of Santa Bárbara and Viseu were collected fecal specimens from asymptomatic humans for diarrhea and tested for RVA, but all were negative. It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data. The fragmentation of the forest generates many consequences on the Amazonian biota, being able to alter the diversity and the composition of the animal communities in the fragments and even to interfere in the ecological processes, without considering that the fragments of forest in the Amazon are influenced by the climate, possibly facilitating the dispersion of pathogens by the environment, since the wild animals detected in the present study are asymptomatic and have low viral load for RVA. The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea. Wild birds have unlimited flight capacity, were captured in an interface region between the peridomicile and forest fragments and it is believed that this region has not been influenced by anthropic activities such as those observed in the area of the present study. On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment. The high rates of increase and the analysis of land use in the researched areas may be important indicators of how these animals interact, since with deforestation, the populations of wild animals seek refuge in nearby communities facilitating the dispersal of infectious agents and the possible occurrence of carrier animals by direct contact or contamination of the local environment. To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities. The detection of positive animals was useful to monitor the infection of the agent in the animal population and to provide an early warning signal to predict an impending epidemic and a favorable risk for the human population, given the evidence of RVA circulation in the different forest fragments. In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al. [60] and Waruhiu et al. [61] . Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe. The most prevalent genotypes isolated from feces of Belgian diarrheal and non-diarrheal piglets in 2012 [62] demonstrate a wide range of combinations of genotypes G / P including; G3P [6] , G4P [6] , G5P [6] , G4P [7] , G5P [7] , G9P [7] , G9P [13] and G9P [23] . On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea. Finding shows that different P genotypes of RVA strains interact with distinct blood group histological antigens (HBGA, ABOH, Lewis) and sialic acids via VP4 providing insight into the regional prevalence and increased zoonotic potential of some RVA of origin swine [63] . The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] . In the population of animals studied the zoonotic transmission can be frequent, since the animals live in contact with humans and in precarious sanitary conditions. In Brazil, this genotype was described in animal and human populations in studies of Luchs et al. [32] ; Honma et al. [67] ; Araújo et al. [68] ; Mascarenhas et al. [69] and Lorenzetti et al. [70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions. Regarding the genotype P [4] , itwas most detected in our samples in bats, dogs, swine and feline. This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] . It is important to emphasize that the indicators of environmental contamination in Brazil are significant and contribute to the possibility of human-animal transmission [71] . Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible. Our findings may be useful for tracking fecal contamination in the environment using animals as possible sources thus minimizing the risk of infection by exposure to susceptible individuals, in this case different animal species or even human populations. RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA. Although the samples are asymptomatic for diarrhea, it is necessary to conduct strategies for the monitoring and control of the animals in the areas studied in the human population as well as in other species of animals, as well as the implementation of preventive measures aimed at future outbreaks in communities animals in the resident population in these impacted areas. Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals. It is noteworthy that, although the quality of the analyzed samples is characterized as low detectable viral load, the technique presented a good analytical response in the detection of the source animals for RVA, facilitating the selection of the samples for future genetic characterization tests.

What non-structural proteins are coded by Rotavirus?

Rotavirus A in wild and domestic animals from areas with environmental degradation in the Brazilian Amazon https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298726/ SHA: f3c309c596c20f48f493b77e714ce957d877bdcb Authors: de Barros, Bruno de Cássio Veloso; Chagas, Elaine Nunes; Bezerra, Luna Wanessa; Ribeiro, Laila Graziela; Duarte Júnior, Jose Wandilson Barboza; Pereira, Diego; da Penha Junior, Edvaldo Tavares; Silva, Julia Rezende; Bezerra, Delana Andreza Melo; Bandeira, Renato Silva; Pinheiro, Helder Henrique Costa; Guerra, Sylvia de Fátima dos Santos; Guimarães, Ricardo José de Paula Souza e; Mascarenhas, Joana D'Arc Pereira Date: 2018-12-18 DOI: 10.1371/journal.pone.0209005 License: cc-by Abstract: Acute gastroenteritis is one of the main causes of mortality in humans and young animals. Domestic and mainly wild animals such as bats, small rodents and birds are highly diversified animals in relation to their habitats and ecological niches and are widely distributed geographically in environments of forest fragmentation in some areas of the Amazon, being considered important sources for viruses that affect humans and other animals. Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health. Although the knowledge of the global diversity of enteric viruses is scarce, there are reports demonstrating the detection of rotavirus in domestic animals and animals of productive systems, such as bovines and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments. The fecal specimens were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas. Text: Emerging and reemerging infectious diseases are increasing each year in several countries, with an impact both on human populations and on domestic and wild animals living in areas with considerable forest remnants [1] . Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] . The populations of wild animals that inhabit forest fragments are strategic groups for studies of public health and the transmission of zoonosis, given that they act as indicators in the assistance and intervention in the human populations, aiming at the prevention of outbreaks and epidemics [3] . Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] . They represent one of the main causes of mortality in humans, and in young animals, counting for about 25% of mortality [8] . Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] . The rotavirus (RV) belongs to the Reoviridae family and comprises nine species known as Rotavirus group A to I, with a recent proposal of the J species [13, 14] . Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] . They have a double-stranded ribonucleic acid (dsRNA) genome, divided into 11 segments coding for structural proteins (VP1-VP4, VP6 and VP7) and nonstructural (NSP1-NSP5/NSP6) proteins [16, 17] . There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] . Although the results of RVA have already been described globally [12, [23] [24] [25] [26] [27] [28] [29] [30] , in Brazil, the occurrence, diversity and role of rotavirus in these animals are still poorly studied, considering the large number of present species [4, [31] [32] [33] [34] . In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] . Therefore, it is important to monitor the health of domestic and wild animals in their natural habitat, especially in areas with anthropic alterations that have an interface with rural communities and enterprises, in order to investigate the occurrence of RVA in this population. These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] . The qualitative real-time polymerase chain reaction (qRT-PCR) used the NSP3 gene and the TaqMan probe from a highly conserved region of the rotavirus non-structural protein 3 (NSP3), which was previously used in samples from human origin and with low viral loads Precipitation data were obtained from The Brazilian National Institute of Meteorology (Inmethttp://www.inmet.gov.br/) for the years of capture in the Expedito Ribeiro Settlement (2014) and Açailândia (2015) of the Data Collection Platforms (PCDs) of Belém, located 50 km from Santa Bárbara do Pará, and Tracuateua, located 50 km from Peixe-Boi and 100 km from Viseu. Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field. The municipal boundaries were obtained on the website of the Brazilian Institute of Geography and Statistics (IBGE) (http://www.ibge.gov.br/) and data on deforestation and land use were obtained from the PRODES [43] and TerraClass [44] Projects. PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004. The satellite image was generated using the sensor Sentinel 2 of the European Space Agency (ESA) (https://sentinel.esa.int/ web/sentinel/user-guides/sentinel-2-msi) with Open Access CC-BY License (http://open.esa.int/) from the years of 2017 and 2018. All the data obtained was stored in a Geographic Database (BDG). The BDG was imported/ stored in a GIS for the editing of the graphic elements, establishment of topological relations between the elements and their respective attributes, spatial analysis and visualization of the result through thematic maps. For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use. The selected fragments were distributed within the mesoregions studied, and in each selected fragment fecal samples were randomly collected from domestic and wild animals [45] . Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed). The data processing, interpretation, visualization and spatial analysis were performed in ArcGIS software (http://www.arcgis.com/). For the analysis of data related to the determination of the richness, composition and abundance of the fauna of the animals studied in the study area, considering the collection methods adopted and the species available in each city, each sample was considered as an independent sample. The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] . For the calculation of the Test T, the Statistica software was used, and the indices of infected animals in the two environments (forest fragment and peridomicile) were calculated for each treatment sampled by collection area, using the software Past 1.92. Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] . The data obtained for the occurrence of RVA and the questionnaires was inserted into a database for a descriptive analysis of the epidemiological profile of the animal population in the three forest ecosystems studied. In this analysis, descriptive statistical treatments were carried out, using customized "row-columns" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T. Population study, collection of clinical specimens and laboratory methodology. The flying animals (wild birds and chiroptera) were captured using mist nets which were opened at dawn (4:00 a.m.) and closed in the morning (9:00 a.m.) and were inspected every one hour until the closing, with a sampling effort of 15 days. This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site. The fecal specimens were collected by stimulation of the rectal ampulla with the use of a "Zaragatoa", packed in cryogenic vials, identified, stored in liquid nitrogen, and later sent to the Laboratory. Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm). In each sample plot, 61 traps were distributed, 20 Shermans and 41 Tomahawks being baited with a mixture made with peanut paste, sardines, cod liver oil and corn meal, as well as fruit like banana, apple and pineapple. All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material. The wild animals were sedated with a combination of ketamine 20mg/kg and xylazine 2mg/kg intramuscularly and subsequently, euthanized with anesthetic overdose of 2% lidocaine in the foramen magnum, according to the recommendation of the National Council for the Control of Animal Experimentation (CONCEA). From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals. Amongthese, 648 (50.5%) samples were randomly selected for RVA research and handled in Level Three Biosafety Laboratory (NB3). The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data. The qRT-PCR was conducted according to Zeng et al. [40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence. The assay was conducted in a mixture containing: RNAse-free H 2 O, TaqMan RT-PCR Mix (2x), TAqMan RT Enzyme Mix (40x), primers for the NSP3 gene, Primer NSP3 Forward (20mM), Primer NSP3 Reverse (20mM), probe NSP3 S (10nm), Template (RNA) 3μL, having a total reaction volume of 17μL and reverse transcription cycling of 50˚C, 30 minutes, denaturation of 95˚C, 10 minutes, annealing of 45 cycles of 95˚C, 15 seconds and extension of 60˚C, 1 minute. The analyzes were considered positive when presenting the cycle threshold (CT) � 40. In order to guarantee a reliable test result, the measurements of contamination control were performed with the use of positive animal control (SA11 prototype) and a negative control (ultrapure water). All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples. First round was performed with consensus primers N-VP4F1/N-VP4R1 and the Nested-PCR was conducted with N-VP4F2/N-VP4R2 primers to amplify VP4 gene. Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR. The sequences were collected from an automated ABI Prism 3130xl DNA sequencer (Applied Biosystems). The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7. Posteriorly, the data were compared with othersequences from the National Center for Biotechnology Information GenBank database using BLAST alignment tool to elucidate the RVA genotype of the samples. From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%). The CT interval ranged from 28. 47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea. Rotavirus A (RVA) detected in the present study of wild and domestic animals belonging to the three areas of forest fragment, according to Fig 2. In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively. In addition, most of the animals were young with ages varying from 1 day to 8 yearsold, history of deficient vaccination, lack of technical assistance and raised in the form of subsistence. The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status. In relation to chiroptera, 32 (17.98%) positive samples for RVA were distributed among Carollia perspicillata species, with 12 (37.5%) being all adults, 9 (28.12%) Desmodus rotundus samples (4 young and 5 adults), 5 (15.6%) of Uroderma bilobata (15.62%), 3 (9.37%) of Artibeus lituratus and the species Artibeus Planirostus, Diaemus iyoug and Glossophagine with 1 (3.12%) each. These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms. Fig 3 shows the results obtained for all the species of animals investigated in the forest fragment as well as in the peridomicillus area. The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) . Considering the factors related to the anthropic activities in the three studied areas within the three cities of the present study, it was observed that the city of Santa Bárbara is the one that has a better area of preserved forest and the city of Viseu a smaller area. However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment. It was observed in this chosen area of the city, the presence of different families living in a rural settlement, surviving from the exploitation of forest resources and the creation of small animals for subsistence, such as poultry and fish farming, as well as family farming products. The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu. Extensive livestock farming was practiced with beef cattle, equines for work and small animals (swine and goats). In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation. When comparing the climates of the three areas it was observed that the predominant climate is megathermal and humid with average annual temperature around 27˚C. The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C. Annual rainfall is quite high, generally around 2,350 mm, but strongly concentrated from January to June (80%). From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months. The relative humidity of the average air oscillates around 85%, as shown in Fig 6 [48] . The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region. The average deforestation index in the three study areas was calculated from data obtained from INPE information systems. It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi. In relation to the period of 2016, great changes were observed in Peixe Boi (79%) and in the city of Viseu (70%), thus demonstrating that changes in the natural ecosystem may be associated with the frequencies for RVA in the studied areas, according to Fig 7. When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained. Applying the selected statistical analysis, a Pearson x2 Chi-square value was obtained: 16.7159, df = 1 and p <0.001, meaning that the hypothesis was corroborated, that is, the greater the degradation of the environment, the more likely it will be the search for food by wild animals in adjacent areas, or in the edge of the forest or even in the peri domiciliary region. In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment. It is important to point out that the animals detected in this study are important sources of viral strains. A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene. Eight strains (10%) were positive for VP4 gene, being 2 strains bellowed to P [6] genotype and 6 to P[4]-type, according to In the present study, RVA was detected circulating in 27.5% of the animals; 36% in domestic animals and 64% in wild animals, providing a unique dataset with qRT-PCR detecting a low viral load of RVA in different species, which further correlates with the deforestation index. These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] . With the advent of real-time PCR (qPCR), there was an exponential growth, compared to conventional PCR essays, since its superior accuracy, sensitivity and specificity is remarkable, and it is Rotavirus A in wild and domestic animals possible to detect RVA in a variety of animal species using NSP3 gene [49] . The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon. Currently, no data have been described in the literature regarding the RVA detection using real-time qPCR technique in a wide variety of wild animal species. However, a study by Soltan et al. [50] conducted with horses and cattle detected RVA by RT-PCR, commercial RT-PCR and RT-qPCR in 36.7%, 51.4% and 56.9% respectively, differently from the present study that showed higher positivity for chiropterans (17.98%), canines (21.35%), birds (23.6%) and cattle (14.6%). The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] . Afterwards, several strains of RVA were detected by different molecular techniques involving chiroptera, in several countries, including Kenya (E. helvum), China (Rhinolophus hipposideros and Aselliscus stoliczkanus), France (Myotis mystacinus), Cameroon (E. helvum) and Brazil [31, [51] [52] [53] [54] [55] . The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%). Barquez et al. [56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico. Of the positive chiroptera for RVA in the present study, a prevalence of 28.12% was of Desmodus rotundus. This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study. These findings show the importance of epidemiological data on the studied species due to the lack of studies involving species of neotropical chiroptera, and it is not possible to establish comparative parameters for these animals. Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively. Although in the Amazon region there are records of RVA, RVD, RVF and RVG that infect birds [57] [58] and RVD in migratory birds [59] , all were detected by RT-PCR assays differently from the present study which detected the RVA by RT-qPCR involving a variety of animal species. On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations. The study detected the presence of RVA in different species of animals both in areas near the home and in areas located in fragments of forest, characterized as forest remnants, since they were located in cities that suffered high environmental impacts due to vegetal extractivism, pasture formation for cattle breeding, exploitation of natural resources, and direct reflexes on the habitats of wild animals that can serve as virus sources, thus facilitating the dispersion of RVA among communities of coexisting animals. It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study. However, it is noteworthy that only in the communities of Santa Bárbara and Viseu were collected fecal specimens from asymptomatic humans for diarrhea and tested for RVA, but all were negative. It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data. The fragmentation of the forest generates many consequences on the Amazonian biota, being able to alter the diversity and the composition of the animal communities in the fragments and even to interfere in the ecological processes, without considering that the fragments of forest in the Amazon are influenced by the climate, possibly facilitating the dispersion of pathogens by the environment, since the wild animals detected in the present study are asymptomatic and have low viral load for RVA. The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea. Wild birds have unlimited flight capacity, were captured in an interface region between the peridomicile and forest fragments and it is believed that this region has not been influenced by anthropic activities such as those observed in the area of the present study. On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment. The high rates of increase and the analysis of land use in the researched areas may be important indicators of how these animals interact, since with deforestation, the populations of wild animals seek refuge in nearby communities facilitating the dispersal of infectious agents and the possible occurrence of carrier animals by direct contact or contamination of the local environment. To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities. The detection of positive animals was useful to monitor the infection of the agent in the animal population and to provide an early warning signal to predict an impending epidemic and a favorable risk for the human population, given the evidence of RVA circulation in the different forest fragments. In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al. [60] and Waruhiu et al. [61] . Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe. The most prevalent genotypes isolated from feces of Belgian diarrheal and non-diarrheal piglets in 2012 [62] demonstrate a wide range of combinations of genotypes G / P including; G3P [6] , G4P [6] , G5P [6] , G4P [7] , G5P [7] , G9P [7] , G9P [13] and G9P [23] . On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea. Finding shows that different P genotypes of RVA strains interact with distinct blood group histological antigens (HBGA, ABOH, Lewis) and sialic acids via VP4 providing insight into the regional prevalence and increased zoonotic potential of some RVA of origin swine [63] . The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] . In the population of animals studied the zoonotic transmission can be frequent, since the animals live in contact with humans and in precarious sanitary conditions. In Brazil, this genotype was described in animal and human populations in studies of Luchs et al. [32] ; Honma et al. [67] ; Araújo et al. [68] ; Mascarenhas et al. [69] and Lorenzetti et al. [70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions. Regarding the genotype P [4] , itwas most detected in our samples in bats, dogs, swine and feline. This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] . It is important to emphasize that the indicators of environmental contamination in Brazil are significant and contribute to the possibility of human-animal transmission [71] . Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible. Our findings may be useful for tracking fecal contamination in the environment using animals as possible sources thus minimizing the risk of infection by exposure to susceptible individuals, in this case different animal species or even human populations. RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA. Although the samples are asymptomatic for diarrhea, it is necessary to conduct strategies for the monitoring and control of the animals in the areas studied in the human population as well as in other species of animals, as well as the implementation of preventive measures aimed at future outbreaks in communities animals in the resident population in these impacted areas. Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals. It is noteworthy that, although the quality of the analyzed samples is characterized as low detectable viral load, the technique presented a good analytical response in the detection of the source animals for RVA, facilitating the selection of the samples for future genetic characterization tests.

What is qRT-PCR?

Regulation and Maintenance of an Adoptive T-Cell Dependent Memory B Cell Pool https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120830/ SHA: f4a82ad66962355ffb09e4d1b57fde3e94f0ec53 Authors: Anson, Marie; Amado, Inês; Mailhé, Marie-Pierre; Donnadieu, Emmanuel; Garcia, Sylvie; Huetz, François; Freitas, Antonio A. Date: 2016-11-23 DOI: 10.1371/journal.pone.0167003 License: cc-by Abstract: We investigated the ability of monoclonal B cells to restore primary and secondary T-cell dependent antibody responses in adoptive immune-deficient hosts. Priming induced B cell activation and expansion, AID expression, antibody production and the generation of IgM(+)IgG(-) and IgM(-)IgG(+) antigen-experienced B-cell subsets that persisted in the lymphopenic environment by cell division. Upon secondary transfer and recall the IgM(-)IgG(+) cells responded by the production of antigen-specific IgG while the IgM(+) memory cells secreted mainly IgM and little IgG, but generated new B cells expressing germinal center markers. The recall responses were more efficient if the antigenic boost was delayed suggesting that a period of adaptation is necessary before the transferred cells are able to respond. Overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced B cell subsets. We also found that the size of the memory B cell pool did not rely on the number of the responding naïve B cells, suggesting autonomous homeostatic controls for naïve and memory B cells. By reconstituting a stable memory B cell pool in immune-deficient hosts using a monoclonal high-affinity B cell population we demonstrate the potential value of B cell adoptive immunotherapy. Text: Immune responses to infectious agents have different out-comes that can either protect or fail to control disease. Protection from re-infection relies on the establishment of efficient secondary immune responses that require the generation of antigen-specific "memory" B and T lymphocytes. The generation and selection of T-cell dependent "memory" B cells involves distinct molecular mechanisms: immunoglobulin isotype recombination and somatic hyper mutation, both dependent on the expression of AID [1] . Therefore, a long-standing paradigm defined memory B cells as IgM -IgG + isotype switched cells [2] . Different lines of evidence indicate that this is not always the case. In humans, it has been shown that some IgM + B cells bear the phenotype of other memory cells, being CD27 + , and carry frequent point mutations in the V region of the Ig genes, suggesting that they must represent highly selected B cell populations [3] . In mice, populations of CD19 + IgM + able to mount secondary responses have been identified [4] [5] [6] [7] . Overall these findings suggest that the T-cell dependent memory B cell pool comprises distinct subsets of memory B cells with different properties and effector functions [4] [5] [6] . The biological properties that ensure the long-term persistence of memory and efficient secondary antibody responses have not been yet completely established. While initial studies proposed that after transfer memory B cells faded rapidly [8, 9] suggesting that long-lasting memory required the continuous recruitment of new cells [8] and/or antigen persistence [9, 10] , others suggested that memory B cells were able of extended survival without cell division [11] in the absence of antigen [2] . Long-term persistence of antibody responses has also been attributed to populations of long-lived plasma cells mainly resident in the bone marrow following immunization [12, 13] . The demonstration of the compartmentalization of "antibody memory" into different cellular layers suggested that the separate subsets of memory B cells behave differently. Accordingly, it has been reported that IgG + cells that could rapidly respond upon challenge did not persist long, while IgM + cells could generate a second wave of germinal center responses allowing persistence of memory [4] [5] [6] 14] . Currently, immunotherapy approaches using passive antibody transfer [15, 16] ) is limited by the short half-life of immunoglobulin. Therefore new therapy strategies may require the adoptive transfer of high-affinity memory B cells, ready to respond and able to persist. The development of these new strategies requires a profound understanding of the mechanisms that regulate memory B cell numbers and ensure long persistence upon adoptive transfer. Moreover, knowledge of the mechanisms that determine the size of the memory B cell pool may be also critical to device new reconstitution strategies. So far, studies comparing populations of naïve and memory B cells have been hindered both by the vast clonal heterogeneity of the cells involved and by our inability to generate significant numbers of antigen specific memory B cells. Indeed in a normal laboratory mouse the population of B cells bearing a "memory IgG + phenotype' represent a small fraction of the total B cell pool (<0.5%) and upon immunization the number of the clonal diverse antigen-specific memory B cells generated is generally very limited (<10 3 ) [1, 6] . To circumvent these limits, we decided to compare the properties of homogeneous populations of naïve and memory B cells of known antigen specificity, belonging to the same clone. We used SW HEL transgenic mice where B cells bear a high-affinity BCR specific for HEL and are capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify "memory B cells" the SW HEL mice were crossed with mice where AID transcription provokes the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . These mice were in a Rag2-deficient background and therefore contain a pure population of monoclonal HEL-specific B cells. To generate memory cells, purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into adoptive hosts together with monoclonal OVA-specific CD4 + T cells from OTII.Rag2 -/-TCR transgenic mice. Upon immunization with OVA-HEL complexes, we obtained a significant number of persisting HEL-specific IgM + Ig-G -YFP + and IgM -IgG + YFP + memory B cells, number that did not correlate to the number of precursor naïve cells initially injected suggesting that the memory B cell pool is regulated independently. We characterized the functional capacity of these two memory cell types in immune deficient hosts. Mice B6 and B6.Rag2-/- [20] mice were kept at the Centre Des Techniques Avancées (CDTA), Centre National de la Recherche Scientifique (CNRS), Orleans, France; SWHEL.AID/YFP.Rag-/-mice, obtained by crossing SWHEL (18)(a gift of Dr. Robert Brink) and AID/YFP [19] (a gift of Dr. Rafael Casellas) with B6.Rag2-/-mice. OTII.Rag-/-mice were kept in our animal facilities at the Pasteur Institute. Experiments were preformed according to Pasteur Institute Safety Committee in accordance with French and European guidelines and the ethics Committee of Paris 1 (permits 2010-0002, -0003 and -0004). Euthanasia of the mice was performed by cervical dislocation. This specific study was approved by the European Research Council (ERC) committee related to the grant AdG09 249740-QSIS. The general status of the mice was controlled daily by monitoring the appearence of obvious pain, distress or suffering (prostration, respiratory issues, loss of weight). The end-point of the experiment was determined by a loss of more than 20% of the weight or as soon as the distress signs appeared. In this case, experiment was stopped and the animals were euthanized. Single-cell suspensions of B cells from spleens and lymph nodes of SW HEL .AID/YFP.Rag -/mice together with CD4 + T cells from spleens and lymph nodes of OTII.Rag -/mice were transferred intravenously into the retro-orbital sinus of B6.Ly5 a IgH a or B6.Rag2 -/recipient mice. Mice received 10 6 HEL + B cells and 10 6 CD4 + T cells unless stated otherwise. Mice were immunized 24H later with 1 mg of Ovalbumin coupled to Hen Egg Lysozyme (OVA-HEL) in 50μg of Alu-S-Gel (Serva) we determined as the optimal dose of Ag (data not shown). Naive cells from SW HEL .AID/YFP.Rag -/mice and memory B cells subsets from immunized B6.Rag -/hosts mice were purified from spleens and lymph nodes by flow cytometry sorting. Single-cell suspensions containing 5×10 4 B cells and 10 6 T cells were transferred intravenously into B6.Rag2 -/recipient hosts. The purity of sorted cells was above 98%. 24 h after transfer, mice were immunized with 1 mg of OVA-HEL. Spleen, bone marrow, inguinal and mesenteric lymph nodes single-cell suspensions were stained for cell surface or intracellular proteins with appropriate combinations of the following monoclonal antibodies conjugated to pacific blue, Qdot-655, Brillant Violet 605, allophycocyanin, peridinin chlorophyll protein-cyanine 5.5, phycoerythrin, phycoerythrin-cyanine7: anti-CD19 (6D5), anti-IgM (R6-60.2), anti-IgG1 (X56), anti-CD138 (281-2), anti-Gl7 (Gl7), anti-CD95 (Jo2), anti-CD62L (MEL-14), anti-CD69 (H1-2F3), anti-BAFFR (7H22-E16), anti-CXCR5 (L138D7), anti-IA b (AF6-120.1), anti-CD80 (16-10A1), anti-CD73 (TY-11-8) and anti-PDL2 (TY25) and anti-Ki-67 (mm1) purchased from Becton Dickinson Pharmingen, Biolegend, Invitrogen and eBioscience. Cells were also stained with HEL (Sigma) coupled with AF594 using Alexa Fluor1 594 Protein Labeling Kit from Life technologies. Before staining, cells were treated with Fc-Block (CD16/CD32, Becton Dickinson Pharmingen). Dead cells were excluded during analysis according to their light-scattering characteristics. For intracellular stainings, cells were first stained with antibodies specific for cell surface antigens. Then, cells were fixed and permeabilized according the manufacturer's recommendations (BD Bisciences). For proliferation assay, mice were injected i.p. with 50 mg/kg of BrdU (Sigma-Aldrich) and were killed 24 or 72 hours later. Incorporated BrdU was detected intracellularly using anti-BrdU APC-conjugated antibodies according to the manufacturer's recommendations (BD Biosciences). All data acquisitions and analyses were performed with LSRFortessa (Becton Dickinson) interfaced with BD FACSDiva (Becton Dickinson) and FlowJo (Tree Star) software. Subsets of memory B cells were sorted as CD19 + HEL + YFP + IgM + or IgG + and naive cells as CD19 + HEL + YFP -IgM + using a FACSAriaIII flow cytometer. The purity of the sorted populations varied from 90-95%. Sera HEL-specific Ig concentrations were quantified by ELISA. Plates were coated with HEL and saturated with PBS-5% Milk. Dilutions of sera were added. After incubation (2 hours, 37˚C) and washing, HRP-labeled anti-mouse IgM or IgG antibodies were added. After incubation and washing, bound antibodies were revealed with the substrate O-phenylenediamine and H2O2. The reaction was stopped after 10 min. by addition of 10% SDS and the absorbance read at 492nm in a multiscan spectrometer. Ig concentrations were determined by comparing the displacement of the dilution curves in the linear interval between standards at a concentration of 1 mg/ml and the serum samples. The quantification of IgG or IgM secreting cells was assayed by ELISpot technique. Briefly, plates were coated with HEL. After saturating, the cells were distributed into the micro wells in RPMI1640-2%FCS. The plates were incubated for 12 h at 37˚C, 5% CO2 atmosphere. After extensive wash, plates were incubated with goat anti-mouse IgM or anti-IgG labeled with alkaline phosphatase. After washing, the revealing substrate was added (2,3 mM 5-bromo-4-chloro-3-indolyl phosphate diluted in 2-amino-2-methyl-1-proprenolol buffer). Spleens from 14 day-immunized mice were initially fixed with paraformaldehyde and embedded in 4% low-gelling-temperature agarose (type VII-A; Sigma-Aldrich) prepared in PBS. 150μm slices were cut with a vibratome (VT 1000S; Leica) in a bath of ice-cold PBS. For immunolabeling, samples were saturated with PBS supplemented with 10% of fetal calf serum, then were labeled with primary antibodies anti-B220-APC (clone RA3-6B2) and anti-IgD-PE (clone 11-26c.2a) and analyzed with a spinning disk confocal microscope equipped with a CoolSnap HQ2 camera (Photometrics) and a 20x objective. Images were acquired and analyzed with MetaMorph 7 imaging software Molecular Devices). Sample means were compared using the Student's t test. Sample means were considered significantly different at p < 0.05. During an immune response the complexity of determinants expressed by immunizing antigen and the degeneracy of antigen-specific recognition results in a vast heterogeneity of responding cells rendering impossible the direct comparison of the properties of naïve and memory B cells belonging to the same clone. We have devised an experimental system that permits the comparison between naïve and memory B cells expressing the same antigen receptor and allows marking permanently memory B cells. For that purpose we used SW HEL transgenic mice in a Rag2-deficient background holding a single population of monoclonal B cells, all bearing a high-affinity BCR specific for HEL and capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify antigen-experienced B cells the SW HEL . Rag2 -/mice were crossed with mice where AID transcription induces the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . Since in intact Tg mice immune responses were not traceable, probably because of the presence of low level pre-existing anti-HEL antibodies that neutralize the immunizing protein, we used an adoptive cell transfer strategy to study the ability of the high affinity monoclonal B cell to reconstitute response in immune-deficient hosts and generate antibody memory. Purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into Rag2-deficient mice together with monoclonal OVA-specific CD4 + T helper cells from OTII.Rag2 -/-TCR transgenic mice. The day after, host mice were immunized with OVA-HEL complexes (Fig 1A) . In these conditions, antigenic challenge resulted in B cell activation and the development of significant numbers of CD19 + HEL + AID/YFP + B cells, which were not detected in non-immunized mice or in mice immunized in absence of helper T cells (Fig 1B) . We followed the early kinetics of this response. The number of HEL-specific B cells increased from the initial 2x10 6 transferred to about 15x10 6 at day 14 (Fig 1C left) the B cells expressing AID/YFP being the dominant population (Fig 1C right) . A fraction of the HEL-specific B cells underwent class switch recombination and at day 14 we recovered both IgM + IgG -AID/YFP + and IgM -IgG + AID/YFP + cell populations (Fig 1B) . B cell expansion and phenotypic changes were accompanied by the production of IgM and IgG HEL-specific antibodies ( Fig 1D) . Two weeks after antigenic challenge we observed the formation of germinal centers in the spleen of the host mice ( Fig 1E) . Coherently we found that while upon adoptive transfer all B cells expressed CD95, only after antigenic challenge most YFP + B cells expressed the germinal center specific marker GL7 (Fig 1F) . In conclusion, the adoptive cell transfer strategy allowed the development of a primary immune response with B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production and germinal center formation. We studied the evolution of the B cell response. From two weeks onwards the total number of B cells contracted and at four weeks we recovered about 2-4x10 6 cells, number that remained stable up to week 20 (Fig 2A) . High titers of HEL-specific IgG were kept from week 3 to 8, declined thereafter, but were still significantly elevated 20 weeks later (Fig 2B) . A population of cells secreting HEL-specific Igs was present in the spleen (Fig 2C) , but not in the BM (not shown) even at the late time points. About 60% of the recovered cells exhibited the phenotype of antigen-experienced ("memory") CD19 + HEL + AID/YFP + expressing either IgM or IgG ( Fig 2D and 2E) . We compared the phenotype of the two AID/YFP + IgM + and AID/YFP + IgM -IgG + memory cell populations recovered with that of the naïve B cells (Fig 2F) . We found that antigen-experience and naïve B cells expressed similar levels of CD62L, CD69 and BAFFR (not shown). Antigen-experienced cells presented sustained expression of CD95 and increased levels of PNA, but the vast majority lost expression of the germinal center marker GL7 present at earlier times post-immunization ( Fig 2F compare to Fig 1F) . Compared to naïve B cells, AID/ YFP + cells expressed higher levels of CD80 and MHC class II and down-regulated expression of CXCR5 (Fig 2F) . These findings indicate that the post-germinal center AID/YFP + B cells express an activated phenotype [5, 21] , have increased antigen-presenting capacity [22] , but may loose the ability to re-enter primary follicles being CXCR5 low [23] . We have also compared the patterns of gene expression (RNAseq) by naïve, activated (YFPcells of immunized mice) and both populations of YFP + memory cells. The data shows a clear discrimination of naïve and activated/memory cells while indicating only minor differences between both subsets of YFP + memory cells (Fig 3) . mRNA was isolated from sort-purified Naïve (CD19 + HEL + YFP -IgM + ) IgM + IgGor IgM -IgG + HEL + CD19 + YFP + memory B cells from spleen of different recipient mice. Total recommended by the manufacturer. The validated libraries were then subjected to DNA sequencing. The analysis is performed using the R software, Bioconductor packages including DESeq2 and the PF2tools package (version 1.2.9) developed at PF2 (Institut Pasteur). Normalization and differential analysis are carried out according to the DESeq2 model and package (version 1.8.1). Fig 3A shows a representative heat map of the different cells populations. Fig 3B shows Late in the immune response persistent B cell numbers were kept by active cell division as a significant fraction of the cells were Ki67 + (Fig 2G left) and incorporated BrdU (Fig 2G middle) . The frequency of BrdU + cells was higher among the AID/YFPcells (15%) than in the major AID/YFP + memory population (3%) and similar between the IgM + and IgM -AID/ YFP + populations ( Fig 2G middle and not shown) . Three days after BrdU pulse populations were clear of BrdU + cells (Fig 2G right) attesting their high division rate. In spite of their increased proliferation rate, memory cells numbers were stable indicating that proliferation may be compensated by cell death as suggested by the frequency of caspase3 + cells (Fig 2H) . The frequency of Caspase3 + cells was higher among the AID/YFP + cells suggesting that a fraction of these cells may represent cells undergoing terminal differentiation. Importantly, these findings demonstrate that the transfer strategy allowed the generation of significant numbers of persisting antigen-experienced YFP + cells. It is not yet known whether the number of antigen-experienced memory B cells correlated to the number of naïve B cells or if it is controlled independently of the initial number of antigenspecific B cells present. To approach this question we transferred different numbers of mature naïve B cells from SW HEL .AID/YFP.Rag2 -/donors (ranging from 10 5 to 5.10 6 ) into Rag2-deficient mice together with an excess of CD4 + T helper cells (10 6 ) and immunize the hosts the day after cell transfer with OVA-HEL in optimal non-limiting quantities. To directly compare the results obtained after the transfer of different all numbers we allowed the responses to reach steady-state eight weeks after antigenic challenge. We studied the amplitude of the immune response by measuring the serum titers of HEL-specific IgG antibodies and enumerating the number of HEL-specific B cells recovered. We found that in the presence of excess T cell help, the levels of the HEL-specific IgGs (Fig 4C) , and both the total number of HEL-specific ( Fig 4A) and of memory YFP + B cells recovered (Fig 4B) , did not correlate to the number of antigen specific naïve B cells initially transferred. Memory B cells are defined functionally by their ability to induce secondary IgG antibody responses upon secondary antigenic challenge. We investigated whether the subsets of AID/ YFP + IgM + and AID/YFP + IgM -IgG + antigen-experienced (memory) B cells persisting at late time points could mount secondary IgG responses and persist after secondary transfer. For this purpose we followed two different experimental strategies. In the first, 5x10 4 cells of either IgM + or IgM -IgG + memory B cells, were transferred with an excess helper OTII CD4 + T cells into secondary Rag-deficient hosts that were boosted with OVA-HEL the day after cell transfer. In the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory B cell subsets was about 10-20% of the initial cell input, exceeding naïve B cell recovery (Fig 5A) , supporting the notion that memory B cells may not require specific ligand recognition to survive (2). One cannot exclude, however, that cross-reactivity of the BCR transgene with environmental antigens may allow signaling sufficient to maintain naïve and memory cell survival in the absence of HEL [24] . Following immunization, the secondarily transferred AID/YFP + IgM -IgG + cells responded promptly with the exclusive production of significant levels HEL-specific IgG thus confirming their memory statute (11) . The AID/YFP + IgM + B cells in response to antigenic boost produced only limited amounts of IgM antibodies (Fig 4B) , little IgG antibodies, but did generate GL7 + B cells more efficiently than the IgG + memory B cell population (Fig 5D) . Thus the IgM + subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of IgG + effectors (4). With time antibody levels decayed rapidly suggesting that the number of transferred memory B cells declined in the secondary hosts after antigenic boost. Indeed, IgM + and IgG + memory B cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare Fig 5E and 5A) . In similar experimental conditions, naïve B cells following immunization expanded, acquired AID/ YFP expression and their numbers more than doubled the number initially injected (Figs 5F and 2A). These data suggest that a significant fraction of the memory B cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions. Besides long-term survival memory B cells must maintain functional activity in the absence of nominal antigen to be fully effective. To test this we used an alternative approach where memory cells were parked in secondary Rag-deficient hosts for 30 days before re-immunization. We found that under these conditions antigenic challenge resulted in the production of HEL-specific IgG antibodies and in a 100 fold increase in the number of cells recovered, expansion that largely exceed that observed after immediate challenge (Fig 5G) . The aim of this study was to characterize the fate of activated B cells and the generation of memory B cells. To do this, we adoptively transferred monoclonal B cells into immune deficient hosts followed by immunization in presence of T cell help. This strategy resulted in the development of different B cell memory subsets, namely IgM + and IgG + , as described for in situ generated memory cells [4, 6, 14] . These findings indicate that distinct memory B cell subsets are not the result of the heterogeneity of initially responding naive cells, but originate from the differentiation of a single B cell clone. While studying the respective rate of proliferation of both types of memory B cells, we found the same high rate of proliferation for IgM + and IgG + memory B cells. These results contrast with previous published data. First it was been reported that "in situ" memory B cells persist as resting non-dividing cells [11, 25] . However, we have shown that upon adoptive transfer and in absence of competing cells, B cells increase their division rate to occupy the available empty niche [26] , which may explain the higher division rate observed here using this adoptive cell transfer strategy. Secondly, comparing life spans among heterogeneous memory B cell populations it was previously reported a lower division rate among the IgM + subset compared to the IgG + polyclonal subset [6] . Differences in BCR affinity between IgM + and IgG + memory clones may explain the higher division rate previously observed among the IgG + cells [6] . In contrast we compared memory B cell subsets belonging to the same clone bearing the same high affinity BCR. Overall these observations support the notion that lymphocyte division rates and life spans are not an intrinsic cell property, but rather determined by the environment and the presence of competing populations [27] . They demonstrate that upon the correct conditions memory B cells can persist by cell division. An important question was whether the number of memory B cells depends on the number of initial naïve B cells. We found that, in the presence of an excess of T cell help, that was not the case. However, it was previously reported during polyclonal responses that serum titers of anti-HSA was proportional to the number of cells transferred into irradiated mice [28] . It is possible that limited antigen-specific T-B cell encounters may constraint the number of responding B cells and thus determine linear precursor-progeny between naïve and memory B cells. Our findings indicate that within a single clone the number of precursor naive B cells present in the peripheral B cell pool does determine neither the intensity nor the final number of memory B cells in response to an optimal dose of antigen. They suggest that the size of memory B cell pool may be controlled independently of the number of naïve B cell precursors and that in the absence of clonal competition the memory niche can be filled with a single monoclonal population. Considering diverse polyclonal populations, the limited niche for memory cells will imply strong competition among clones resulting in the selection of best fit (high affinity) cells: rare mutated clones being able to out compete more frequent but less avid clones. In our settings, the transgenic memory B cells are likely to counter select any new mutant clones since they express a very high affinity BCR selected in the course of a secondary immune response [29] . Thus, notwithstanding the expression of AID and proliferation we did not detect any BCR VH and VL Ig-chain nucleotide mutations among the recovered memory B cells (not shown). These findings may have implication for vaccination protocols as they indicate that each new antigenic exposure or unrelated immunization would add extra competing clones supporting the need for repeated antigenic boosts to prevent memory B cell attrition. They also demonstrate that the memory B cell pool can be reconstituted from a relatively small number of antigen-specific cells. It is likely that the relatively poor memory B cell expansion observed after immediate boost after adoptive transfer could be due to the lack in Rag-deficient hosts of the appropriate environment required for memory B cell survival and function. It should be pointed out that B cell transfer into transgenic ML5 Rag-deficient hosts expressing low levels of HEL [29] resulted in rapid cell loss and recovery suggesting that in these hosts, B cells are trapped by antigen in locations were they are unable to survive (not shown). Nevertheless, it has been shown that B cells can drive the maturation of follicular dendritic cells and the organization of lymphoid follicles [30] . Similarly, transferred helper cells may also modify their immediate environment. Thus, by allowing lymphocytes to adapt and modify their immediate environment we improved their response and more important, we recovered the memory B cell pool size present in the original donor mice. In this study we show that it is possible to fully reconstitute a primary response and the establishment of antibody memory in immune deficient mice after adoptive transfer of antigen-specific monoclonal B cells together with a population of monoclonal helper T cells. Indeed, it is generally believed that in immune deficiencies, B cell therapy has restricted application due to intrinsic defects of host's lymphoid organs structure that may prevent development of immune responses, germinal center formation, establishment of antibody memory and limit cell survival. In contrast we showed that after adoptive transfer in immune deficient hosts antigen immunization induced B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production, germinal center formation and the generation of two subsets of AID/YFP + IgM + IgGand AID/YFP + Ig-M -IgG + antigen-experienced B cell subsets able to persist in a lymphopenic environment by cell division mimicking responses obtained in intact non-Tg mice [4] . Upon challenge the AID/YFP + IgM -IgG + cells responded promptly with the production of HEL-specific IgG while the AID/YFP + IgM + B cells secreted only limited amounts of IgM antibodies and fail to produce IgG. In contrast the AID/YFP + IgM + B cells could give rise to new GL7 + B cells, suggesting that full reconstitution of the memory B cell pool may require transfer of the different antigen-experienced B cell subsets. Importantly, we found that the recall responses were more efficient if the transferred memory cells were given the required time to adapt to their new environment, suggesting that a period of accommodation is necessary before the transferred cells are fully capable to respond. Our findings also show that different processes can modify the survival conditions of memory B cells. Finally, we found that the generation of the memory B cell pool in response to an optimal dose of Ag did not rely on the number of the initially responding B cells, suggesting autonomous homeostatic controls for naïve and memory B cells a property that may allow reconstitution of the memory pool in immune-deficient hosts using a limited number of precursor naïve B cells. An autonomous control of the memory B cell pool where each antigenic exposure adds new competing clones supports the notion of vaccination strategies using antigenic boosting to prevent memory B cell attrition. Overall the findings reported demonstrate that it is possible to reconstitute the memory B cell pool of an immune deficient host with an artificially induced population of monoclonal high affinity memory B cells.

What is required to establish a secondary immune response to a viral infection?

Regulation and Maintenance of an Adoptive T-Cell Dependent Memory B Cell Pool https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120830/ SHA: f4a82ad66962355ffb09e4d1b57fde3e94f0ec53 Authors: Anson, Marie; Amado, Inês; Mailhé, Marie-Pierre; Donnadieu, Emmanuel; Garcia, Sylvie; Huetz, François; Freitas, Antonio A. Date: 2016-11-23 DOI: 10.1371/journal.pone.0167003 License: cc-by Abstract: We investigated the ability of monoclonal B cells to restore primary and secondary T-cell dependent antibody responses in adoptive immune-deficient hosts. Priming induced B cell activation and expansion, AID expression, antibody production and the generation of IgM(+)IgG(-) and IgM(-)IgG(+) antigen-experienced B-cell subsets that persisted in the lymphopenic environment by cell division. Upon secondary transfer and recall the IgM(-)IgG(+) cells responded by the production of antigen-specific IgG while the IgM(+) memory cells secreted mainly IgM and little IgG, but generated new B cells expressing germinal center markers. The recall responses were more efficient if the antigenic boost was delayed suggesting that a period of adaptation is necessary before the transferred cells are able to respond. Overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced B cell subsets. We also found that the size of the memory B cell pool did not rely on the number of the responding naïve B cells, suggesting autonomous homeostatic controls for naïve and memory B cells. By reconstituting a stable memory B cell pool in immune-deficient hosts using a monoclonal high-affinity B cell population we demonstrate the potential value of B cell adoptive immunotherapy. Text: Immune responses to infectious agents have different out-comes that can either protect or fail to control disease. Protection from re-infection relies on the establishment of efficient secondary immune responses that require the generation of antigen-specific "memory" B and T lymphocytes. The generation and selection of T-cell dependent "memory" B cells involves distinct molecular mechanisms: immunoglobulin isotype recombination and somatic hyper mutation, both dependent on the expression of AID [1] . Therefore, a long-standing paradigm defined memory B cells as IgM -IgG + isotype switched cells [2] . Different lines of evidence indicate that this is not always the case. In humans, it has been shown that some IgM + B cells bear the phenotype of other memory cells, being CD27 + , and carry frequent point mutations in the V region of the Ig genes, suggesting that they must represent highly selected B cell populations [3] . In mice, populations of CD19 + IgM + able to mount secondary responses have been identified [4] [5] [6] [7] . Overall these findings suggest that the T-cell dependent memory B cell pool comprises distinct subsets of memory B cells with different properties and effector functions [4] [5] [6] . The biological properties that ensure the long-term persistence of memory and efficient secondary antibody responses have not been yet completely established. While initial studies proposed that after transfer memory B cells faded rapidly [8, 9] suggesting that long-lasting memory required the continuous recruitment of new cells [8] and/or antigen persistence [9, 10] , others suggested that memory B cells were able of extended survival without cell division [11] in the absence of antigen [2] . Long-term persistence of antibody responses has also been attributed to populations of long-lived plasma cells mainly resident in the bone marrow following immunization [12, 13] . The demonstration of the compartmentalization of "antibody memory" into different cellular layers suggested that the separate subsets of memory B cells behave differently. Accordingly, it has been reported that IgG + cells that could rapidly respond upon challenge did not persist long, while IgM + cells could generate a second wave of germinal center responses allowing persistence of memory [4] [5] [6] 14] . Currently, immunotherapy approaches using passive antibody transfer [15, 16] ) is limited by the short half-life of immunoglobulin. Therefore new therapy strategies may require the adoptive transfer of high-affinity memory B cells, ready to respond and able to persist. The development of these new strategies requires a profound understanding of the mechanisms that regulate memory B cell numbers and ensure long persistence upon adoptive transfer. Moreover, knowledge of the mechanisms that determine the size of the memory B cell pool may be also critical to device new reconstitution strategies. So far, studies comparing populations of naïve and memory B cells have been hindered both by the vast clonal heterogeneity of the cells involved and by our inability to generate significant numbers of antigen specific memory B cells. Indeed in a normal laboratory mouse the population of B cells bearing a "memory IgG + phenotype' represent a small fraction of the total B cell pool (<0.5%) and upon immunization the number of the clonal diverse antigen-specific memory B cells generated is generally very limited (<10 3 ) [1, 6] . To circumvent these limits, we decided to compare the properties of homogeneous populations of naïve and memory B cells of known antigen specificity, belonging to the same clone. We used SW HEL transgenic mice where B cells bear a high-affinity BCR specific for HEL and are capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify "memory B cells" the SW HEL mice were crossed with mice where AID transcription provokes the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . These mice were in a Rag2-deficient background and therefore contain a pure population of monoclonal HEL-specific B cells. To generate memory cells, purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into adoptive hosts together with monoclonal OVA-specific CD4 + T cells from OTII.Rag2 -/-TCR transgenic mice. Upon immunization with OVA-HEL complexes, we obtained a significant number of persisting HEL-specific IgM + Ig-G -YFP + and IgM -IgG + YFP + memory B cells, number that did not correlate to the number of precursor naïve cells initially injected suggesting that the memory B cell pool is regulated independently. We characterized the functional capacity of these two memory cell types in immune deficient hosts. Mice B6 and B6.Rag2-/- [20] mice were kept at the Centre Des Techniques Avancées (CDTA), Centre National de la Recherche Scientifique (CNRS), Orleans, France; SWHEL.AID/YFP.Rag-/-mice, obtained by crossing SWHEL (18)(a gift of Dr. Robert Brink) and AID/YFP [19] (a gift of Dr. Rafael Casellas) with B6.Rag2-/-mice. OTII.Rag-/-mice were kept in our animal facilities at the Pasteur Institute. Experiments were preformed according to Pasteur Institute Safety Committee in accordance with French and European guidelines and the ethics Committee of Paris 1 (permits 2010-0002, -0003 and -0004). Euthanasia of the mice was performed by cervical dislocation. This specific study was approved by the European Research Council (ERC) committee related to the grant AdG09 249740-QSIS. The general status of the mice was controlled daily by monitoring the appearence of obvious pain, distress or suffering (prostration, respiratory issues, loss of weight). The end-point of the experiment was determined by a loss of more than 20% of the weight or as soon as the distress signs appeared. In this case, experiment was stopped and the animals were euthanized. Single-cell suspensions of B cells from spleens and lymph nodes of SW HEL .AID/YFP.Rag -/mice together with CD4 + T cells from spleens and lymph nodes of OTII.Rag -/mice were transferred intravenously into the retro-orbital sinus of B6.Ly5 a IgH a or B6.Rag2 -/recipient mice. Mice received 10 6 HEL + B cells and 10 6 CD4 + T cells unless stated otherwise. Mice were immunized 24H later with 1 mg of Ovalbumin coupled to Hen Egg Lysozyme (OVA-HEL) in 50μg of Alu-S-Gel (Serva) we determined as the optimal dose of Ag (data not shown). Naive cells from SW HEL .AID/YFP.Rag -/mice and memory B cells subsets from immunized B6.Rag -/hosts mice were purified from spleens and lymph nodes by flow cytometry sorting. Single-cell suspensions containing 5×10 4 B cells and 10 6 T cells were transferred intravenously into B6.Rag2 -/recipient hosts. The purity of sorted cells was above 98%. 24 h after transfer, mice were immunized with 1 mg of OVA-HEL. Spleen, bone marrow, inguinal and mesenteric lymph nodes single-cell suspensions were stained for cell surface or intracellular proteins with appropriate combinations of the following monoclonal antibodies conjugated to pacific blue, Qdot-655, Brillant Violet 605, allophycocyanin, peridinin chlorophyll protein-cyanine 5.5, phycoerythrin, phycoerythrin-cyanine7: anti-CD19 (6D5), anti-IgM (R6-60.2), anti-IgG1 (X56), anti-CD138 (281-2), anti-Gl7 (Gl7), anti-CD95 (Jo2), anti-CD62L (MEL-14), anti-CD69 (H1-2F3), anti-BAFFR (7H22-E16), anti-CXCR5 (L138D7), anti-IA b (AF6-120.1), anti-CD80 (16-10A1), anti-CD73 (TY-11-8) and anti-PDL2 (TY25) and anti-Ki-67 (mm1) purchased from Becton Dickinson Pharmingen, Biolegend, Invitrogen and eBioscience. Cells were also stained with HEL (Sigma) coupled with AF594 using Alexa Fluor1 594 Protein Labeling Kit from Life technologies. Before staining, cells were treated with Fc-Block (CD16/CD32, Becton Dickinson Pharmingen). Dead cells were excluded during analysis according to their light-scattering characteristics. For intracellular stainings, cells were first stained with antibodies specific for cell surface antigens. Then, cells were fixed and permeabilized according the manufacturer's recommendations (BD Bisciences). For proliferation assay, mice were injected i.p. with 50 mg/kg of BrdU (Sigma-Aldrich) and were killed 24 or 72 hours later. Incorporated BrdU was detected intracellularly using anti-BrdU APC-conjugated antibodies according to the manufacturer's recommendations (BD Biosciences). All data acquisitions and analyses were performed with LSRFortessa (Becton Dickinson) interfaced with BD FACSDiva (Becton Dickinson) and FlowJo (Tree Star) software. Subsets of memory B cells were sorted as CD19 + HEL + YFP + IgM + or IgG + and naive cells as CD19 + HEL + YFP -IgM + using a FACSAriaIII flow cytometer. The purity of the sorted populations varied from 90-95%. Sera HEL-specific Ig concentrations were quantified by ELISA. Plates were coated with HEL and saturated with PBS-5% Milk. Dilutions of sera were added. After incubation (2 hours, 37˚C) and washing, HRP-labeled anti-mouse IgM or IgG antibodies were added. After incubation and washing, bound antibodies were revealed with the substrate O-phenylenediamine and H2O2. The reaction was stopped after 10 min. by addition of 10% SDS and the absorbance read at 492nm in a multiscan spectrometer. Ig concentrations were determined by comparing the displacement of the dilution curves in the linear interval between standards at a concentration of 1 mg/ml and the serum samples. The quantification of IgG or IgM secreting cells was assayed by ELISpot technique. Briefly, plates were coated with HEL. After saturating, the cells were distributed into the micro wells in RPMI1640-2%FCS. The plates were incubated for 12 h at 37˚C, 5% CO2 atmosphere. After extensive wash, plates were incubated with goat anti-mouse IgM or anti-IgG labeled with alkaline phosphatase. After washing, the revealing substrate was added (2,3 mM 5-bromo-4-chloro-3-indolyl phosphate diluted in 2-amino-2-methyl-1-proprenolol buffer). Spleens from 14 day-immunized mice were initially fixed with paraformaldehyde and embedded in 4% low-gelling-temperature agarose (type VII-A; Sigma-Aldrich) prepared in PBS. 150μm slices were cut with a vibratome (VT 1000S; Leica) in a bath of ice-cold PBS. For immunolabeling, samples were saturated with PBS supplemented with 10% of fetal calf serum, then were labeled with primary antibodies anti-B220-APC (clone RA3-6B2) and anti-IgD-PE (clone 11-26c.2a) and analyzed with a spinning disk confocal microscope equipped with a CoolSnap HQ2 camera (Photometrics) and a 20x objective. Images were acquired and analyzed with MetaMorph 7 imaging software Molecular Devices). Sample means were compared using the Student's t test. Sample means were considered significantly different at p < 0.05. During an immune response the complexity of determinants expressed by immunizing antigen and the degeneracy of antigen-specific recognition results in a vast heterogeneity of responding cells rendering impossible the direct comparison of the properties of naïve and memory B cells belonging to the same clone. We have devised an experimental system that permits the comparison between naïve and memory B cells expressing the same antigen receptor and allows marking permanently memory B cells. For that purpose we used SW HEL transgenic mice in a Rag2-deficient background holding a single population of monoclonal B cells, all bearing a high-affinity BCR specific for HEL and capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify antigen-experienced B cells the SW HEL . Rag2 -/mice were crossed with mice where AID transcription induces the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . Since in intact Tg mice immune responses were not traceable, probably because of the presence of low level pre-existing anti-HEL antibodies that neutralize the immunizing protein, we used an adoptive cell transfer strategy to study the ability of the high affinity monoclonal B cell to reconstitute response in immune-deficient hosts and generate antibody memory. Purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into Rag2-deficient mice together with monoclonal OVA-specific CD4 + T helper cells from OTII.Rag2 -/-TCR transgenic mice. The day after, host mice were immunized with OVA-HEL complexes (Fig 1A) . In these conditions, antigenic challenge resulted in B cell activation and the development of significant numbers of CD19 + HEL + AID/YFP + B cells, which were not detected in non-immunized mice or in mice immunized in absence of helper T cells (Fig 1B) . We followed the early kinetics of this response. The number of HEL-specific B cells increased from the initial 2x10 6 transferred to about 15x10 6 at day 14 (Fig 1C left) the B cells expressing AID/YFP being the dominant population (Fig 1C right) . A fraction of the HEL-specific B cells underwent class switch recombination and at day 14 we recovered both IgM + IgG -AID/YFP + and IgM -IgG + AID/YFP + cell populations (Fig 1B) . B cell expansion and phenotypic changes were accompanied by the production of IgM and IgG HEL-specific antibodies ( Fig 1D) . Two weeks after antigenic challenge we observed the formation of germinal centers in the spleen of the host mice ( Fig 1E) . Coherently we found that while upon adoptive transfer all B cells expressed CD95, only after antigenic challenge most YFP + B cells expressed the germinal center specific marker GL7 (Fig 1F) . In conclusion, the adoptive cell transfer strategy allowed the development of a primary immune response with B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production and germinal center formation. We studied the evolution of the B cell response. From two weeks onwards the total number of B cells contracted and at four weeks we recovered about 2-4x10 6 cells, number that remained stable up to week 20 (Fig 2A) . High titers of HEL-specific IgG were kept from week 3 to 8, declined thereafter, but were still significantly elevated 20 weeks later (Fig 2B) . A population of cells secreting HEL-specific Igs was present in the spleen (Fig 2C) , but not in the BM (not shown) even at the late time points. About 60% of the recovered cells exhibited the phenotype of antigen-experienced ("memory") CD19 + HEL + AID/YFP + expressing either IgM or IgG ( Fig 2D and 2E) . We compared the phenotype of the two AID/YFP + IgM + and AID/YFP + IgM -IgG + memory cell populations recovered with that of the naïve B cells (Fig 2F) . We found that antigen-experience and naïve B cells expressed similar levels of CD62L, CD69 and BAFFR (not shown). Antigen-experienced cells presented sustained expression of CD95 and increased levels of PNA, but the vast majority lost expression of the germinal center marker GL7 present at earlier times post-immunization ( Fig 2F compare to Fig 1F) . Compared to naïve B cells, AID/ YFP + cells expressed higher levels of CD80 and MHC class II and down-regulated expression of CXCR5 (Fig 2F) . These findings indicate that the post-germinal center AID/YFP + B cells express an activated phenotype [5, 21] , have increased antigen-presenting capacity [22] , but may loose the ability to re-enter primary follicles being CXCR5 low [23] . We have also compared the patterns of gene expression (RNAseq) by naïve, activated (YFPcells of immunized mice) and both populations of YFP + memory cells. The data shows a clear discrimination of naïve and activated/memory cells while indicating only minor differences between both subsets of YFP + memory cells (Fig 3) . mRNA was isolated from sort-purified Naïve (CD19 + HEL + YFP -IgM + ) IgM + IgGor IgM -IgG + HEL + CD19 + YFP + memory B cells from spleen of different recipient mice. Total recommended by the manufacturer. The validated libraries were then subjected to DNA sequencing. The analysis is performed using the R software, Bioconductor packages including DESeq2 and the PF2tools package (version 1.2.9) developed at PF2 (Institut Pasteur). Normalization and differential analysis are carried out according to the DESeq2 model and package (version 1.8.1). Fig 3A shows a representative heat map of the different cells populations. Fig 3B shows Late in the immune response persistent B cell numbers were kept by active cell division as a significant fraction of the cells were Ki67 + (Fig 2G left) and incorporated BrdU (Fig 2G middle) . The frequency of BrdU + cells was higher among the AID/YFPcells (15%) than in the major AID/YFP + memory population (3%) and similar between the IgM + and IgM -AID/ YFP + populations ( Fig 2G middle and not shown) . Three days after BrdU pulse populations were clear of BrdU + cells (Fig 2G right) attesting their high division rate. In spite of their increased proliferation rate, memory cells numbers were stable indicating that proliferation may be compensated by cell death as suggested by the frequency of caspase3 + cells (Fig 2H) . The frequency of Caspase3 + cells was higher among the AID/YFP + cells suggesting that a fraction of these cells may represent cells undergoing terminal differentiation. Importantly, these findings demonstrate that the transfer strategy allowed the generation of significant numbers of persisting antigen-experienced YFP + cells. It is not yet known whether the number of antigen-experienced memory B cells correlated to the number of naïve B cells or if it is controlled independently of the initial number of antigenspecific B cells present. To approach this question we transferred different numbers of mature naïve B cells from SW HEL .AID/YFP.Rag2 -/donors (ranging from 10 5 to 5.10 6 ) into Rag2-deficient mice together with an excess of CD4 + T helper cells (10 6 ) and immunize the hosts the day after cell transfer with OVA-HEL in optimal non-limiting quantities. To directly compare the results obtained after the transfer of different all numbers we allowed the responses to reach steady-state eight weeks after antigenic challenge. We studied the amplitude of the immune response by measuring the serum titers of HEL-specific IgG antibodies and enumerating the number of HEL-specific B cells recovered. We found that in the presence of excess T cell help, the levels of the HEL-specific IgGs (Fig 4C) , and both the total number of HEL-specific ( Fig 4A) and of memory YFP + B cells recovered (Fig 4B) , did not correlate to the number of antigen specific naïve B cells initially transferred. Memory B cells are defined functionally by their ability to induce secondary IgG antibody responses upon secondary antigenic challenge. We investigated whether the subsets of AID/ YFP + IgM + and AID/YFP + IgM -IgG + antigen-experienced (memory) B cells persisting at late time points could mount secondary IgG responses and persist after secondary transfer. For this purpose we followed two different experimental strategies. In the first, 5x10 4 cells of either IgM + or IgM -IgG + memory B cells, were transferred with an excess helper OTII CD4 + T cells into secondary Rag-deficient hosts that were boosted with OVA-HEL the day after cell transfer. In the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory B cell subsets was about 10-20% of the initial cell input, exceeding naïve B cell recovery (Fig 5A) , supporting the notion that memory B cells may not require specific ligand recognition to survive (2). One cannot exclude, however, that cross-reactivity of the BCR transgene with environmental antigens may allow signaling sufficient to maintain naïve and memory cell survival in the absence of HEL [24] . Following immunization, the secondarily transferred AID/YFP + IgM -IgG + cells responded promptly with the exclusive production of significant levels HEL-specific IgG thus confirming their memory statute (11) . The AID/YFP + IgM + B cells in response to antigenic boost produced only limited amounts of IgM antibodies (Fig 4B) , little IgG antibodies, but did generate GL7 + B cells more efficiently than the IgG + memory B cell population (Fig 5D) . Thus the IgM + subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of IgG + effectors (4). With time antibody levels decayed rapidly suggesting that the number of transferred memory B cells declined in the secondary hosts after antigenic boost. Indeed, IgM + and IgG + memory B cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare Fig 5E and 5A) . In similar experimental conditions, naïve B cells following immunization expanded, acquired AID/ YFP expression and their numbers more than doubled the number initially injected (Figs 5F and 2A). These data suggest that a significant fraction of the memory B cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions. Besides long-term survival memory B cells must maintain functional activity in the absence of nominal antigen to be fully effective. To test this we used an alternative approach where memory cells were parked in secondary Rag-deficient hosts for 30 days before re-immunization. We found that under these conditions antigenic challenge resulted in the production of HEL-specific IgG antibodies and in a 100 fold increase in the number of cells recovered, expansion that largely exceed that observed after immediate challenge (Fig 5G) . The aim of this study was to characterize the fate of activated B cells and the generation of memory B cells. To do this, we adoptively transferred monoclonal B cells into immune deficient hosts followed by immunization in presence of T cell help. This strategy resulted in the development of different B cell memory subsets, namely IgM + and IgG + , as described for in situ generated memory cells [4, 6, 14] . These findings indicate that distinct memory B cell subsets are not the result of the heterogeneity of initially responding naive cells, but originate from the differentiation of a single B cell clone. While studying the respective rate of proliferation of both types of memory B cells, we found the same high rate of proliferation for IgM + and IgG + memory B cells. These results contrast with previous published data. First it was been reported that "in situ" memory B cells persist as resting non-dividing cells [11, 25] . However, we have shown that upon adoptive transfer and in absence of competing cells, B cells increase their division rate to occupy the available empty niche [26] , which may explain the higher division rate observed here using this adoptive cell transfer strategy. Secondly, comparing life spans among heterogeneous memory B cell populations it was previously reported a lower division rate among the IgM + subset compared to the IgG + polyclonal subset [6] . Differences in BCR affinity between IgM + and IgG + memory clones may explain the higher division rate previously observed among the IgG + cells [6] . In contrast we compared memory B cell subsets belonging to the same clone bearing the same high affinity BCR. Overall these observations support the notion that lymphocyte division rates and life spans are not an intrinsic cell property, but rather determined by the environment and the presence of competing populations [27] . They demonstrate that upon the correct conditions memory B cells can persist by cell division. An important question was whether the number of memory B cells depends on the number of initial naïve B cells. We found that, in the presence of an excess of T cell help, that was not the case. However, it was previously reported during polyclonal responses that serum titers of anti-HSA was proportional to the number of cells transferred into irradiated mice [28] . It is possible that limited antigen-specific T-B cell encounters may constraint the number of responding B cells and thus determine linear precursor-progeny between naïve and memory B cells. Our findings indicate that within a single clone the number of precursor naive B cells present in the peripheral B cell pool does determine neither the intensity nor the final number of memory B cells in response to an optimal dose of antigen. They suggest that the size of memory B cell pool may be controlled independently of the number of naïve B cell precursors and that in the absence of clonal competition the memory niche can be filled with a single monoclonal population. Considering diverse polyclonal populations, the limited niche for memory cells will imply strong competition among clones resulting in the selection of best fit (high affinity) cells: rare mutated clones being able to out compete more frequent but less avid clones. In our settings, the transgenic memory B cells are likely to counter select any new mutant clones since they express a very high affinity BCR selected in the course of a secondary immune response [29] . Thus, notwithstanding the expression of AID and proliferation we did not detect any BCR VH and VL Ig-chain nucleotide mutations among the recovered memory B cells (not shown). These findings may have implication for vaccination protocols as they indicate that each new antigenic exposure or unrelated immunization would add extra competing clones supporting the need for repeated antigenic boosts to prevent memory B cell attrition. They also demonstrate that the memory B cell pool can be reconstituted from a relatively small number of antigen-specific cells. It is likely that the relatively poor memory B cell expansion observed after immediate boost after adoptive transfer could be due to the lack in Rag-deficient hosts of the appropriate environment required for memory B cell survival and function. It should be pointed out that B cell transfer into transgenic ML5 Rag-deficient hosts expressing low levels of HEL [29] resulted in rapid cell loss and recovery suggesting that in these hosts, B cells are trapped by antigen in locations were they are unable to survive (not shown). Nevertheless, it has been shown that B cells can drive the maturation of follicular dendritic cells and the organization of lymphoid follicles [30] . Similarly, transferred helper cells may also modify their immediate environment. Thus, by allowing lymphocytes to adapt and modify their immediate environment we improved their response and more important, we recovered the memory B cell pool size present in the original donor mice. In this study we show that it is possible to fully reconstitute a primary response and the establishment of antibody memory in immune deficient mice after adoptive transfer of antigen-specific monoclonal B cells together with a population of monoclonal helper T cells. Indeed, it is generally believed that in immune deficiencies, B cell therapy has restricted application due to intrinsic defects of host's lymphoid organs structure that may prevent development of immune responses, germinal center formation, establishment of antibody memory and limit cell survival. In contrast we showed that after adoptive transfer in immune deficient hosts antigen immunization induced B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production, germinal center formation and the generation of two subsets of AID/YFP + IgM + IgGand AID/YFP + Ig-M -IgG + antigen-experienced B cell subsets able to persist in a lymphopenic environment by cell division mimicking responses obtained in intact non-Tg mice [4] . Upon challenge the AID/YFP + IgM -IgG + cells responded promptly with the production of HEL-specific IgG while the AID/YFP + IgM + B cells secreted only limited amounts of IgM antibodies and fail to produce IgG. In contrast the AID/YFP + IgM + B cells could give rise to new GL7 + B cells, suggesting that full reconstitution of the memory B cell pool may require transfer of the different antigen-experienced B cell subsets. Importantly, we found that the recall responses were more efficient if the transferred memory cells were given the required time to adapt to their new environment, suggesting that a period of accommodation is necessary before the transferred cells are fully capable to respond. Our findings also show that different processes can modify the survival conditions of memory B cells. Finally, we found that the generation of the memory B cell pool in response to an optimal dose of Ag did not rely on the number of the initially responding B cells, suggesting autonomous homeostatic controls for naïve and memory B cells a property that may allow reconstitution of the memory pool in immune-deficient hosts using a limited number of precursor naïve B cells. An autonomous control of the memory B cell pool where each antigenic exposure adds new competing clones supports the notion of vaccination strategies using antigenic boosting to prevent memory B cell attrition. Overall the findings reported demonstrate that it is possible to reconstitute the memory B cell pool of an immune deficient host with an artificially induced population of monoclonal high affinity memory B cells.

What do immunoglobulin isotype recombination and somatic hyper mutation depend on?

Regulation and Maintenance of an Adoptive T-Cell Dependent Memory B Cell Pool https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120830/ SHA: f4a82ad66962355ffb09e4d1b57fde3e94f0ec53 Authors: Anson, Marie; Amado, Inês; Mailhé, Marie-Pierre; Donnadieu, Emmanuel; Garcia, Sylvie; Huetz, François; Freitas, Antonio A. Date: 2016-11-23 DOI: 10.1371/journal.pone.0167003 License: cc-by Abstract: We investigated the ability of monoclonal B cells to restore primary and secondary T-cell dependent antibody responses in adoptive immune-deficient hosts. Priming induced B cell activation and expansion, AID expression, antibody production and the generation of IgM(+)IgG(-) and IgM(-)IgG(+) antigen-experienced B-cell subsets that persisted in the lymphopenic environment by cell division. Upon secondary transfer and recall the IgM(-)IgG(+) cells responded by the production of antigen-specific IgG while the IgM(+) memory cells secreted mainly IgM and little IgG, but generated new B cells expressing germinal center markers. The recall responses were more efficient if the antigenic boost was delayed suggesting that a period of adaptation is necessary before the transferred cells are able to respond. Overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced B cell subsets. We also found that the size of the memory B cell pool did not rely on the number of the responding naïve B cells, suggesting autonomous homeostatic controls for naïve and memory B cells. By reconstituting a stable memory B cell pool in immune-deficient hosts using a monoclonal high-affinity B cell population we demonstrate the potential value of B cell adoptive immunotherapy. Text: Immune responses to infectious agents have different out-comes that can either protect or fail to control disease. Protection from re-infection relies on the establishment of efficient secondary immune responses that require the generation of antigen-specific "memory" B and T lymphocytes. The generation and selection of T-cell dependent "memory" B cells involves distinct molecular mechanisms: immunoglobulin isotype recombination and somatic hyper mutation, both dependent on the expression of AID [1] . Therefore, a long-standing paradigm defined memory B cells as IgM -IgG + isotype switched cells [2] . Different lines of evidence indicate that this is not always the case. In humans, it has been shown that some IgM + B cells bear the phenotype of other memory cells, being CD27 + , and carry frequent point mutations in the V region of the Ig genes, suggesting that they must represent highly selected B cell populations [3] . In mice, populations of CD19 + IgM + able to mount secondary responses have been identified [4] [5] [6] [7] . Overall these findings suggest that the T-cell dependent memory B cell pool comprises distinct subsets of memory B cells with different properties and effector functions [4] [5] [6] . The biological properties that ensure the long-term persistence of memory and efficient secondary antibody responses have not been yet completely established. While initial studies proposed that after transfer memory B cells faded rapidly [8, 9] suggesting that long-lasting memory required the continuous recruitment of new cells [8] and/or antigen persistence [9, 10] , others suggested that memory B cells were able of extended survival without cell division [11] in the absence of antigen [2] . Long-term persistence of antibody responses has also been attributed to populations of long-lived plasma cells mainly resident in the bone marrow following immunization [12, 13] . The demonstration of the compartmentalization of "antibody memory" into different cellular layers suggested that the separate subsets of memory B cells behave differently. Accordingly, it has been reported that IgG + cells that could rapidly respond upon challenge did not persist long, while IgM + cells could generate a second wave of germinal center responses allowing persistence of memory [4] [5] [6] 14] . Currently, immunotherapy approaches using passive antibody transfer [15, 16] ) is limited by the short half-life of immunoglobulin. Therefore new therapy strategies may require the adoptive transfer of high-affinity memory B cells, ready to respond and able to persist. The development of these new strategies requires a profound understanding of the mechanisms that regulate memory B cell numbers and ensure long persistence upon adoptive transfer. Moreover, knowledge of the mechanisms that determine the size of the memory B cell pool may be also critical to device new reconstitution strategies. So far, studies comparing populations of naïve and memory B cells have been hindered both by the vast clonal heterogeneity of the cells involved and by our inability to generate significant numbers of antigen specific memory B cells. Indeed in a normal laboratory mouse the population of B cells bearing a "memory IgG + phenotype' represent a small fraction of the total B cell pool (<0.5%) and upon immunization the number of the clonal diverse antigen-specific memory B cells generated is generally very limited (<10 3 ) [1, 6] . To circumvent these limits, we decided to compare the properties of homogeneous populations of naïve and memory B cells of known antigen specificity, belonging to the same clone. We used SW HEL transgenic mice where B cells bear a high-affinity BCR specific for HEL and are capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify "memory B cells" the SW HEL mice were crossed with mice where AID transcription provokes the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . These mice were in a Rag2-deficient background and therefore contain a pure population of monoclonal HEL-specific B cells. To generate memory cells, purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into adoptive hosts together with monoclonal OVA-specific CD4 + T cells from OTII.Rag2 -/-TCR transgenic mice. Upon immunization with OVA-HEL complexes, we obtained a significant number of persisting HEL-specific IgM + Ig-G -YFP + and IgM -IgG + YFP + memory B cells, number that did not correlate to the number of precursor naïve cells initially injected suggesting that the memory B cell pool is regulated independently. We characterized the functional capacity of these two memory cell types in immune deficient hosts. Mice B6 and B6.Rag2-/- [20] mice were kept at the Centre Des Techniques Avancées (CDTA), Centre National de la Recherche Scientifique (CNRS), Orleans, France; SWHEL.AID/YFP.Rag-/-mice, obtained by crossing SWHEL (18)(a gift of Dr. Robert Brink) and AID/YFP [19] (a gift of Dr. Rafael Casellas) with B6.Rag2-/-mice. OTII.Rag-/-mice were kept in our animal facilities at the Pasteur Institute. Experiments were preformed according to Pasteur Institute Safety Committee in accordance with French and European guidelines and the ethics Committee of Paris 1 (permits 2010-0002, -0003 and -0004). Euthanasia of the mice was performed by cervical dislocation. This specific study was approved by the European Research Council (ERC) committee related to the grant AdG09 249740-QSIS. The general status of the mice was controlled daily by monitoring the appearence of obvious pain, distress or suffering (prostration, respiratory issues, loss of weight). The end-point of the experiment was determined by a loss of more than 20% of the weight or as soon as the distress signs appeared. In this case, experiment was stopped and the animals were euthanized. Single-cell suspensions of B cells from spleens and lymph nodes of SW HEL .AID/YFP.Rag -/mice together with CD4 + T cells from spleens and lymph nodes of OTII.Rag -/mice were transferred intravenously into the retro-orbital sinus of B6.Ly5 a IgH a or B6.Rag2 -/recipient mice. Mice received 10 6 HEL + B cells and 10 6 CD4 + T cells unless stated otherwise. Mice were immunized 24H later with 1 mg of Ovalbumin coupled to Hen Egg Lysozyme (OVA-HEL) in 50μg of Alu-S-Gel (Serva) we determined as the optimal dose of Ag (data not shown). Naive cells from SW HEL .AID/YFP.Rag -/mice and memory B cells subsets from immunized B6.Rag -/hosts mice were purified from spleens and lymph nodes by flow cytometry sorting. Single-cell suspensions containing 5×10 4 B cells and 10 6 T cells were transferred intravenously into B6.Rag2 -/recipient hosts. The purity of sorted cells was above 98%. 24 h after transfer, mice were immunized with 1 mg of OVA-HEL. Spleen, bone marrow, inguinal and mesenteric lymph nodes single-cell suspensions were stained for cell surface or intracellular proteins with appropriate combinations of the following monoclonal antibodies conjugated to pacific blue, Qdot-655, Brillant Violet 605, allophycocyanin, peridinin chlorophyll protein-cyanine 5.5, phycoerythrin, phycoerythrin-cyanine7: anti-CD19 (6D5), anti-IgM (R6-60.2), anti-IgG1 (X56), anti-CD138 (281-2), anti-Gl7 (Gl7), anti-CD95 (Jo2), anti-CD62L (MEL-14), anti-CD69 (H1-2F3), anti-BAFFR (7H22-E16), anti-CXCR5 (L138D7), anti-IA b (AF6-120.1), anti-CD80 (16-10A1), anti-CD73 (TY-11-8) and anti-PDL2 (TY25) and anti-Ki-67 (mm1) purchased from Becton Dickinson Pharmingen, Biolegend, Invitrogen and eBioscience. Cells were also stained with HEL (Sigma) coupled with AF594 using Alexa Fluor1 594 Protein Labeling Kit from Life technologies. Before staining, cells were treated with Fc-Block (CD16/CD32, Becton Dickinson Pharmingen). Dead cells were excluded during analysis according to their light-scattering characteristics. For intracellular stainings, cells were first stained with antibodies specific for cell surface antigens. Then, cells were fixed and permeabilized according the manufacturer's recommendations (BD Bisciences). For proliferation assay, mice were injected i.p. with 50 mg/kg of BrdU (Sigma-Aldrich) and were killed 24 or 72 hours later. Incorporated BrdU was detected intracellularly using anti-BrdU APC-conjugated antibodies according to the manufacturer's recommendations (BD Biosciences). All data acquisitions and analyses were performed with LSRFortessa (Becton Dickinson) interfaced with BD FACSDiva (Becton Dickinson) and FlowJo (Tree Star) software. Subsets of memory B cells were sorted as CD19 + HEL + YFP + IgM + or IgG + and naive cells as CD19 + HEL + YFP -IgM + using a FACSAriaIII flow cytometer. The purity of the sorted populations varied from 90-95%. Sera HEL-specific Ig concentrations were quantified by ELISA. Plates were coated with HEL and saturated with PBS-5% Milk. Dilutions of sera were added. After incubation (2 hours, 37˚C) and washing, HRP-labeled anti-mouse IgM or IgG antibodies were added. After incubation and washing, bound antibodies were revealed with the substrate O-phenylenediamine and H2O2. The reaction was stopped after 10 min. by addition of 10% SDS and the absorbance read at 492nm in a multiscan spectrometer. Ig concentrations were determined by comparing the displacement of the dilution curves in the linear interval between standards at a concentration of 1 mg/ml and the serum samples. The quantification of IgG or IgM secreting cells was assayed by ELISpot technique. Briefly, plates were coated with HEL. After saturating, the cells were distributed into the micro wells in RPMI1640-2%FCS. The plates were incubated for 12 h at 37˚C, 5% CO2 atmosphere. After extensive wash, plates were incubated with goat anti-mouse IgM or anti-IgG labeled with alkaline phosphatase. After washing, the revealing substrate was added (2,3 mM 5-bromo-4-chloro-3-indolyl phosphate diluted in 2-amino-2-methyl-1-proprenolol buffer). Spleens from 14 day-immunized mice were initially fixed with paraformaldehyde and embedded in 4% low-gelling-temperature agarose (type VII-A; Sigma-Aldrich) prepared in PBS. 150μm slices were cut with a vibratome (VT 1000S; Leica) in a bath of ice-cold PBS. For immunolabeling, samples were saturated with PBS supplemented with 10% of fetal calf serum, then were labeled with primary antibodies anti-B220-APC (clone RA3-6B2) and anti-IgD-PE (clone 11-26c.2a) and analyzed with a spinning disk confocal microscope equipped with a CoolSnap HQ2 camera (Photometrics) and a 20x objective. Images were acquired and analyzed with MetaMorph 7 imaging software Molecular Devices). Sample means were compared using the Student's t test. Sample means were considered significantly different at p < 0.05. During an immune response the complexity of determinants expressed by immunizing antigen and the degeneracy of antigen-specific recognition results in a vast heterogeneity of responding cells rendering impossible the direct comparison of the properties of naïve and memory B cells belonging to the same clone. We have devised an experimental system that permits the comparison between naïve and memory B cells expressing the same antigen receptor and allows marking permanently memory B cells. For that purpose we used SW HEL transgenic mice in a Rag2-deficient background holding a single population of monoclonal B cells, all bearing a high-affinity BCR specific for HEL and capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify antigen-experienced B cells the SW HEL . Rag2 -/mice were crossed with mice where AID transcription induces the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . Since in intact Tg mice immune responses were not traceable, probably because of the presence of low level pre-existing anti-HEL antibodies that neutralize the immunizing protein, we used an adoptive cell transfer strategy to study the ability of the high affinity monoclonal B cell to reconstitute response in immune-deficient hosts and generate antibody memory. Purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into Rag2-deficient mice together with monoclonal OVA-specific CD4 + T helper cells from OTII.Rag2 -/-TCR transgenic mice. The day after, host mice were immunized with OVA-HEL complexes (Fig 1A) . In these conditions, antigenic challenge resulted in B cell activation and the development of significant numbers of CD19 + HEL + AID/YFP + B cells, which were not detected in non-immunized mice or in mice immunized in absence of helper T cells (Fig 1B) . We followed the early kinetics of this response. The number of HEL-specific B cells increased from the initial 2x10 6 transferred to about 15x10 6 at day 14 (Fig 1C left) the B cells expressing AID/YFP being the dominant population (Fig 1C right) . A fraction of the HEL-specific B cells underwent class switch recombination and at day 14 we recovered both IgM + IgG -AID/YFP + and IgM -IgG + AID/YFP + cell populations (Fig 1B) . B cell expansion and phenotypic changes were accompanied by the production of IgM and IgG HEL-specific antibodies ( Fig 1D) . Two weeks after antigenic challenge we observed the formation of germinal centers in the spleen of the host mice ( Fig 1E) . Coherently we found that while upon adoptive transfer all B cells expressed CD95, only after antigenic challenge most YFP + B cells expressed the germinal center specific marker GL7 (Fig 1F) . In conclusion, the adoptive cell transfer strategy allowed the development of a primary immune response with B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production and germinal center formation. We studied the evolution of the B cell response. From two weeks onwards the total number of B cells contracted and at four weeks we recovered about 2-4x10 6 cells, number that remained stable up to week 20 (Fig 2A) . High titers of HEL-specific IgG were kept from week 3 to 8, declined thereafter, but were still significantly elevated 20 weeks later (Fig 2B) . A population of cells secreting HEL-specific Igs was present in the spleen (Fig 2C) , but not in the BM (not shown) even at the late time points. About 60% of the recovered cells exhibited the phenotype of antigen-experienced ("memory") CD19 + HEL + AID/YFP + expressing either IgM or IgG ( Fig 2D and 2E) . We compared the phenotype of the two AID/YFP + IgM + and AID/YFP + IgM -IgG + memory cell populations recovered with that of the naïve B cells (Fig 2F) . We found that antigen-experience and naïve B cells expressed similar levels of CD62L, CD69 and BAFFR (not shown). Antigen-experienced cells presented sustained expression of CD95 and increased levels of PNA, but the vast majority lost expression of the germinal center marker GL7 present at earlier times post-immunization ( Fig 2F compare to Fig 1F) . Compared to naïve B cells, AID/ YFP + cells expressed higher levels of CD80 and MHC class II and down-regulated expression of CXCR5 (Fig 2F) . These findings indicate that the post-germinal center AID/YFP + B cells express an activated phenotype [5, 21] , have increased antigen-presenting capacity [22] , but may loose the ability to re-enter primary follicles being CXCR5 low [23] . We have also compared the patterns of gene expression (RNAseq) by naïve, activated (YFPcells of immunized mice) and both populations of YFP + memory cells. The data shows a clear discrimination of naïve and activated/memory cells while indicating only minor differences between both subsets of YFP + memory cells (Fig 3) . mRNA was isolated from sort-purified Naïve (CD19 + HEL + YFP -IgM + ) IgM + IgGor IgM -IgG + HEL + CD19 + YFP + memory B cells from spleen of different recipient mice. Total recommended by the manufacturer. The validated libraries were then subjected to DNA sequencing. The analysis is performed using the R software, Bioconductor packages including DESeq2 and the PF2tools package (version 1.2.9) developed at PF2 (Institut Pasteur). Normalization and differential analysis are carried out according to the DESeq2 model and package (version 1.8.1). Fig 3A shows a representative heat map of the different cells populations. Fig 3B shows Late in the immune response persistent B cell numbers were kept by active cell division as a significant fraction of the cells were Ki67 + (Fig 2G left) and incorporated BrdU (Fig 2G middle) . The frequency of BrdU + cells was higher among the AID/YFPcells (15%) than in the major AID/YFP + memory population (3%) and similar between the IgM + and IgM -AID/ YFP + populations ( Fig 2G middle and not shown) . Three days after BrdU pulse populations were clear of BrdU + cells (Fig 2G right) attesting their high division rate. In spite of their increased proliferation rate, memory cells numbers were stable indicating that proliferation may be compensated by cell death as suggested by the frequency of caspase3 + cells (Fig 2H) . The frequency of Caspase3 + cells was higher among the AID/YFP + cells suggesting that a fraction of these cells may represent cells undergoing terminal differentiation. Importantly, these findings demonstrate that the transfer strategy allowed the generation of significant numbers of persisting antigen-experienced YFP + cells. It is not yet known whether the number of antigen-experienced memory B cells correlated to the number of naïve B cells or if it is controlled independently of the initial number of antigenspecific B cells present. To approach this question we transferred different numbers of mature naïve B cells from SW HEL .AID/YFP.Rag2 -/donors (ranging from 10 5 to 5.10 6 ) into Rag2-deficient mice together with an excess of CD4 + T helper cells (10 6 ) and immunize the hosts the day after cell transfer with OVA-HEL in optimal non-limiting quantities. To directly compare the results obtained after the transfer of different all numbers we allowed the responses to reach steady-state eight weeks after antigenic challenge. We studied the amplitude of the immune response by measuring the serum titers of HEL-specific IgG antibodies and enumerating the number of HEL-specific B cells recovered. We found that in the presence of excess T cell help, the levels of the HEL-specific IgGs (Fig 4C) , and both the total number of HEL-specific ( Fig 4A) and of memory YFP + B cells recovered (Fig 4B) , did not correlate to the number of antigen specific naïve B cells initially transferred. Memory B cells are defined functionally by their ability to induce secondary IgG antibody responses upon secondary antigenic challenge. We investigated whether the subsets of AID/ YFP + IgM + and AID/YFP + IgM -IgG + antigen-experienced (memory) B cells persisting at late time points could mount secondary IgG responses and persist after secondary transfer. For this purpose we followed two different experimental strategies. In the first, 5x10 4 cells of either IgM + or IgM -IgG + memory B cells, were transferred with an excess helper OTII CD4 + T cells into secondary Rag-deficient hosts that were boosted with OVA-HEL the day after cell transfer. In the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory B cell subsets was about 10-20% of the initial cell input, exceeding naïve B cell recovery (Fig 5A) , supporting the notion that memory B cells may not require specific ligand recognition to survive (2). One cannot exclude, however, that cross-reactivity of the BCR transgene with environmental antigens may allow signaling sufficient to maintain naïve and memory cell survival in the absence of HEL [24] . Following immunization, the secondarily transferred AID/YFP + IgM -IgG + cells responded promptly with the exclusive production of significant levels HEL-specific IgG thus confirming their memory statute (11) . The AID/YFP + IgM + B cells in response to antigenic boost produced only limited amounts of IgM antibodies (Fig 4B) , little IgG antibodies, but did generate GL7 + B cells more efficiently than the IgG + memory B cell population (Fig 5D) . Thus the IgM + subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of IgG + effectors (4). With time antibody levels decayed rapidly suggesting that the number of transferred memory B cells declined in the secondary hosts after antigenic boost. Indeed, IgM + and IgG + memory B cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare Fig 5E and 5A) . In similar experimental conditions, naïve B cells following immunization expanded, acquired AID/ YFP expression and their numbers more than doubled the number initially injected (Figs 5F and 2A). These data suggest that a significant fraction of the memory B cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions. Besides long-term survival memory B cells must maintain functional activity in the absence of nominal antigen to be fully effective. To test this we used an alternative approach where memory cells were parked in secondary Rag-deficient hosts for 30 days before re-immunization. We found that under these conditions antigenic challenge resulted in the production of HEL-specific IgG antibodies and in a 100 fold increase in the number of cells recovered, expansion that largely exceed that observed after immediate challenge (Fig 5G) . The aim of this study was to characterize the fate of activated B cells and the generation of memory B cells. To do this, we adoptively transferred monoclonal B cells into immune deficient hosts followed by immunization in presence of T cell help. This strategy resulted in the development of different B cell memory subsets, namely IgM + and IgG + , as described for in situ generated memory cells [4, 6, 14] . These findings indicate that distinct memory B cell subsets are not the result of the heterogeneity of initially responding naive cells, but originate from the differentiation of a single B cell clone. While studying the respective rate of proliferation of both types of memory B cells, we found the same high rate of proliferation for IgM + and IgG + memory B cells. These results contrast with previous published data. First it was been reported that "in situ" memory B cells persist as resting non-dividing cells [11, 25] . However, we have shown that upon adoptive transfer and in absence of competing cells, B cells increase their division rate to occupy the available empty niche [26] , which may explain the higher division rate observed here using this adoptive cell transfer strategy. Secondly, comparing life spans among heterogeneous memory B cell populations it was previously reported a lower division rate among the IgM + subset compared to the IgG + polyclonal subset [6] . Differences in BCR affinity between IgM + and IgG + memory clones may explain the higher division rate previously observed among the IgG + cells [6] . In contrast we compared memory B cell subsets belonging to the same clone bearing the same high affinity BCR. Overall these observations support the notion that lymphocyte division rates and life spans are not an intrinsic cell property, but rather determined by the environment and the presence of competing populations [27] . They demonstrate that upon the correct conditions memory B cells can persist by cell division. An important question was whether the number of memory B cells depends on the number of initial naïve B cells. We found that, in the presence of an excess of T cell help, that was not the case. However, it was previously reported during polyclonal responses that serum titers of anti-HSA was proportional to the number of cells transferred into irradiated mice [28] . It is possible that limited antigen-specific T-B cell encounters may constraint the number of responding B cells and thus determine linear precursor-progeny between naïve and memory B cells. Our findings indicate that within a single clone the number of precursor naive B cells present in the peripheral B cell pool does determine neither the intensity nor the final number of memory B cells in response to an optimal dose of antigen. They suggest that the size of memory B cell pool may be controlled independently of the number of naïve B cell precursors and that in the absence of clonal competition the memory niche can be filled with a single monoclonal population. Considering diverse polyclonal populations, the limited niche for memory cells will imply strong competition among clones resulting in the selection of best fit (high affinity) cells: rare mutated clones being able to out compete more frequent but less avid clones. In our settings, the transgenic memory B cells are likely to counter select any new mutant clones since they express a very high affinity BCR selected in the course of a secondary immune response [29] . Thus, notwithstanding the expression of AID and proliferation we did not detect any BCR VH and VL Ig-chain nucleotide mutations among the recovered memory B cells (not shown). These findings may have implication for vaccination protocols as they indicate that each new antigenic exposure or unrelated immunization would add extra competing clones supporting the need for repeated antigenic boosts to prevent memory B cell attrition. They also demonstrate that the memory B cell pool can be reconstituted from a relatively small number of antigen-specific cells. It is likely that the relatively poor memory B cell expansion observed after immediate boost after adoptive transfer could be due to the lack in Rag-deficient hosts of the appropriate environment required for memory B cell survival and function. It should be pointed out that B cell transfer into transgenic ML5 Rag-deficient hosts expressing low levels of HEL [29] resulted in rapid cell loss and recovery suggesting that in these hosts, B cells are trapped by antigen in locations were they are unable to survive (not shown). Nevertheless, it has been shown that B cells can drive the maturation of follicular dendritic cells and the organization of lymphoid follicles [30] . Similarly, transferred helper cells may also modify their immediate environment. Thus, by allowing lymphocytes to adapt and modify their immediate environment we improved their response and more important, we recovered the memory B cell pool size present in the original donor mice. In this study we show that it is possible to fully reconstitute a primary response and the establishment of antibody memory in immune deficient mice after adoptive transfer of antigen-specific monoclonal B cells together with a population of monoclonal helper T cells. Indeed, it is generally believed that in immune deficiencies, B cell therapy has restricted application due to intrinsic defects of host's lymphoid organs structure that may prevent development of immune responses, germinal center formation, establishment of antibody memory and limit cell survival. In contrast we showed that after adoptive transfer in immune deficient hosts antigen immunization induced B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production, germinal center formation and the generation of two subsets of AID/YFP + IgM + IgGand AID/YFP + Ig-M -IgG + antigen-experienced B cell subsets able to persist in a lymphopenic environment by cell division mimicking responses obtained in intact non-Tg mice [4] . Upon challenge the AID/YFP + IgM -IgG + cells responded promptly with the production of HEL-specific IgG while the AID/YFP + IgM + B cells secreted only limited amounts of IgM antibodies and fail to produce IgG. In contrast the AID/YFP + IgM + B cells could give rise to new GL7 + B cells, suggesting that full reconstitution of the memory B cell pool may require transfer of the different antigen-experienced B cell subsets. Importantly, we found that the recall responses were more efficient if the transferred memory cells were given the required time to adapt to their new environment, suggesting that a period of accommodation is necessary before the transferred cells are fully capable to respond. Our findings also show that different processes can modify the survival conditions of memory B cells. Finally, we found that the generation of the memory B cell pool in response to an optimal dose of Ag did not rely on the number of the initially responding B cells, suggesting autonomous homeostatic controls for naïve and memory B cells a property that may allow reconstitution of the memory pool in immune-deficient hosts using a limited number of precursor naïve B cells. An autonomous control of the memory B cell pool where each antigenic exposure adds new competing clones supports the notion of vaccination strategies using antigenic boosting to prevent memory B cell attrition. Overall the findings reported demonstrate that it is possible to reconstitute the memory B cell pool of an immune deficient host with an artificially induced population of monoclonal high affinity memory B cells.

What was used to quantify the amount of IgM secreting cells?

Optimization Method for Forecasting Confirmed Cases of COVID-19 in China https://doi.org/10.3390/jcm9030674 SHA: 1d7f8850c5244fdc9b387038e7eeae9bcbbde6d2 Authors: Al-Qaness, Mohammed A. A.; Ewees, Ahmed A.; Fan, Hong; Abd El Aziz, Mohamed Date: 2020 DOI: 10.3390/jcm9030674 License: cc-by Abstract: In December 2019, a novel coronavirus, called COVID-19, was discovered in Wuhan, China, and has spread to different cities in China as well as to 24 other countries. The number of confirmed cases is increasing daily and reached 34,598 on 8 February 2020. In the current study, we present a new forecasting model to estimate and forecast the number of confirmed cases of COVID-19 in the upcoming ten days based on the previously confirmed cases recorded in China. The proposed model is an improved adaptive neuro-fuzzy inference system (ANFIS) using an enhanced flower pollination algorithm (FPA) by using the salp swarm algorithm (SSA). In general, SSA is employed to improve FPA to avoid its drawbacks (i.e., getting trapped at the local optima). The main idea of the proposed model, called FPASSA-ANFIS, is to improve the performance of ANFIS by determining the parameters of ANFIS using FPASSA. The FPASSA-ANFIS model is evaluated using the World Health Organization (WHO) official data of the outbreak of the COVID-19 to forecast the confirmed cases of the upcoming ten days. More so, the FPASSA-ANFIS model is compared to several existing models, and it showed better performance in terms of Mean Absolute Percentage Error (MAPE), Root Mean Squared Relative Error (RMSRE), Root Mean Squared Relative Error (RMSRE), coefficient of determination ( R 2 ), and computing time. Furthermore, we tested the proposed model using two different datasets of weekly influenza confirmed cases in two countries, namely the USA and China. The outcomes also showed good performances. Text: A large family of viruses, called coronaviruses, are severe pathogens for human beings, which infect respiratory, hepatic, gastrointestinal, and neurologic diseases. They are distributed among humans, birds, livestock, mice, bats, and other wild animals [1] [2] [3] . The outbreaks of two previous coronaviruses, SARS-CoV and MERS-CoV in 2003 and 2012, respectively, have approved the transmission from animal to animal, and human to human [4] . In December 2019, the World Health Organization (WHO) received notifications from China for many cases of respiratory illness that were linked to some people who had visited a seafood market in Wuhan [5] . Currently, Wuhan city suffers from the spreading of a novel coronavirus, called COVID-19 (previously, it was called 2019-nCoV). In [6] , the authors concluded that COVID-19 likely originated in bats, because it is more similar to two bat-derived coronavirus strains. However, the source of the COVID-19 is not confirmed yet, and it communities, Hong Kong and Toronto, were 1.2 and 1.32, respectively. Ong et al. [20] proposed a monitoring and forecasting model for influenza A (H1N1-2009). Furthermore, Nah et al. [21] proposed a probability-based model to predict the spread of the MERS. The Adaptive Neuro-Fuzzy Inference System (ANFIS) [22] is widely applied in time series prediction and forecasting problems, and it showed good performance in many existing applications. It offers flexibility in determining nonlinearity in the time series data, as well as combining the properties of both artificial neural networks (ANN) and fuzzy logic systems. It has been applied in various forecasting applications, for example, in [23] , a stock price forecasting model was proposed using ANFIS and empirical mode decomposition. Chen et al. [24] proposed a TAIEX time series forecasting model based on a hybrid of ANFIS and ordered weighted averaging (OWA). In [25] , another time series forecasting method was presented for electricity prices based on ANFIS. Svalina et al. [26] proposed an ANFIS based forecasting model for close price indices for a stock market for five days. Ekici and Aksoy [27] presented an ANFIS based building energy consumption forecasting model. More so, ANFIS is also applied to forecast electricity loads [28] . Kumar et al. [29] proposed an ANFIS based model to forecast return products. Ho and Tsai [30] applied ANFIS to forecast product development performance. However, estimating ANFIS parameters is a challenge that needs to be improved. Therefore, in previous studies, some individual swarm intelligence (SI) methods have been applied to the ANFIS parameters to enhance time series forecasting because these parameters have a significant effect on the performance of ANFIS. The SI methods include the particle swarm optimization (PSO) [31, 32] , social-spider optimization [33] , sine-cosine algorithm (SCA) [34] , and multi-verse optimizer (MVO) [35] . For example, in [34] SCA algorithm was applied to improve the ANFIS model to forecast oil consumption in three countries, namely, Canada, Germany, and Japan. In the same context, in [35] , The MVO algorithm was used to enhance the ANFIS model to forecast oil consumption in two countries. In addition, in [36] the PSO was used with ANFIS to predict biochar yield. However, individual SI algorithms may stock at local optima. Therefore, one solution is to apply hybrid SI algorithms to avoid this problem. In [37] , a hybrid of two SI algorithms, namely GA and SSA, was presented to improve the ANFIS model. The proposed new model called GA-SSA-ANFIS was applied to forecast crude oil prices for long-term time series data. However, the previously mentioned methods suffer from some limitations that can affect the performance of the forecasting output such as slow convergence and the ability to balance between exploration and exploitation phases can influence the quality of the final output. This motivated us to propose an alternative forecasting method dependent on the hybridization concept. This concept avoids the limitations of traditional SI techniques by combining the strengths of different techniques, and this produces new SI techniques that are better than traditional ones. In the current study, we propose an improved ANFIS model based on a modified flower pollination algorithm (FPA) using the salp swarm algorithm (SSA). The FPA is an optimization algorithm proposed by Yang [38] , which was inspired by the flow pollination process of the flowering plants. The FPA was employed in various optimization applications, for example to estimate solar PV parameter [39, 40] , solving sudoku puzzles [41] , feature selection [42] , antenna design [43] , and other applications [44] [45] [46] [47] . Moreover, SSA is also an optimization algorithm proposed by Mirjalili et al. [48] inspired by the behavior of salp chains. In recent years, the SSA was utilized to solve different optimization problems, such as feature selection [49, 50] , data classification [51] , image segmentation [52] , and others [53, 54] . The proposed method called FPASSA is a hybrid of FPA and SSA, in which the SSA is applied as a local search method for FPA. The proposed FPASSA starts by receiving the historical COVID-19 dataset. Then a set of solutions is generated where each of them represents the value for the parameters of the ANFIS model. Then the quality of each solution is calculated using the fitness value, and the solution that has the best fitness value is chosen to represent the best solution. Then the probability of each solution is computed. Then the current solution will be updated, either using global or local strategy in FPA. However, in the case of local strategy, the operators of SSA or FPA will be used according to the probability of the fitness value for each solution. The process of updating the solutions is repeated until reaching the stop condition, and the best parameter configurations are used to forecast the number of confirmed cases of COVID-19. The main contribution points of the current study are as follows: 1. We propose an efficient forecasting model to forecast the confirmed cases of the COVID-19 in China for the upcoming ten days based on previously confirmed cases. An improved ANFIS model is proposed using a modified FPA algorithm, using SSA. We compare the proposed model with the original ANFIS and existing modified ANFIS models, such as PSO, GA, ABC, and FPA. The rest of this study is organized as follows. The preliminaries of ANFIS, FPA, and SSA are described in Section 2. Section 3 presents the proposed FPASSA, and Section 4 presents the experimental setup and results. We conclude this study in Section 5. The principles of the ANFIS are given in this section. The ANFIS model links the fuzzy logic and neural networks [22] . It generates a mapping between the input and output by applying IF-THEN rules (it is also called Takagi-Sugeno inference model). Figure 1 illustrates the ANFIS model where, y and x define the inputs to Layer 1 whereas, O 1i is its output of node i that is computed as follows: where µ denotes the generalized Gaussian membership functions. A i and B i define the membership values of µ. α i and ρ i denote the premise parameters set. The output of Layer 2 (it is also known as the firing strength of a rule) is calculated as follows: Meanwhile, the output of Layer 3 (it is also known as the normalized firing strength) is calculated as follows: The output of Layer 4 (it is also known as an adaptive node) is calculated as follows: where r i , q i , and p i define the consequent parameters of the node i. Layer 5 contains only one node; its output is computed as: Flower Pollination Algorithm is an optimization method proposed by Yang [38] . It simulates the transfer of flowers' pollen by pollinators in nature. This algorithm utilizes the two types of pollination (i.e., self-pollination and cross-pollination). In self-pollination, the pollination occurs with no pollinators, whereas, in cross-pollination, the pollens are moved between different plants. In more detail, the self-pollination can be represented as a local pollination while the cross-pollination can be called global pollination. The global pollination or cross-pollination can be mathematically formed as follows: where x t i defines the pollen i at iteration t. L denotes the pollination's strength or the step size. F * is the target position or best solution. In some cases, insects can fly with different distance steps for a long space; therefore, Levy fly distribution is applied to simulate this movement. where λ = 1.5. Γ(λ) denotes the gamma function. This distribution is available for large steps s > 0. The self-pollination or local pollination can be mathematically formed as follows: where x t i and x k i represent pollens from different flower in the same plant. in the range [0,1] The process of pollination can be done using cross-pollination or self-pollination. Therefore, the random variable p, in the range [0, 1], is used to determine this process. SSA is an optimization technique introduced by [48] . It simulates the Salps' behavior in nature. This behavior is called salp chain. The mathematical model of SSA begins by splinting its population into a leader group and followers group. The leader is the front salp, whereas, the followers are the other salps. The search space is determined in n-dimensions with n variables. Equation (10) works to update the salps' positions. where x 1 j denotes the leader's position in j-th dimension. F j is the target position. ub j and lb j represent the max and min bounds, respectively. c 2 and c 3 denote random numbers in [0, 1]. c 1 is an important parameter; it balances between the exploration and exploitation phases. It is computed as follows: where the current loop number is t and the max loop' number is t max . Then, the followers' position is updated as follows: where x i j defines the i-th position of the follower in j-th dimension. i > 1. This section explains the proposed FPASSA-ANFIS method. It is a time series method for forecasting the confirmed cases of the COVID-19, as given in Figure 2 . The FPASSA-ANFIS utilizes the improved FPA to train the ANFIS model by optimizing its parameters. The FPASSA-ANFIS contains five layers as the classic ANFIS model. Layer 1 contains the input variables (the historical COVID-19 confirmed cases). Whereas Layer 5 produces the forecasted values. In the learning phase, the FPASSA is used to select the best weights between Layer 4 and Layer 5. The FPASSA-ANFIS starts by formatting the input data in a time series form. In our case, the autocorrelation function (ACF) was considered. ACF is one of the methods applied to find patterns in the data; it presents information about the correlation between points separated by various time lags. Therefore, in this paper, the variables with ACF greater than 0.2 are considered i.e., 5-lags. Besides, the training data contains 75% of the dataset, whereas the testing data contains 25% of them. The number of clusters is defined by the fuzzy c-mean (FCM) method to construct the ANFIS model. The parameters of the ANFIS model are prepared by the FPASSA algorithm. In the training phase, the calculation error (as in Equation (13)) between the real data and the predicted data is used to evaluate the parameters' quality. where T is the real data, and P is the predicted data. N s is the sample length. The smaller values of the objective function indicate good ANFIS's parameter. On the other hand, the updating phase of the followers' positions in the SSA algorithm is applied to improve the global pollination phase in the FPA algorithm. In this improvement, there is a random variable (r) used to switch between both phases. If r > 0.5, then the operators of the SSA is used; otherwise, the operators of the FPA are used. In general, The FPASSA starts by constructing the population (X); afterward, the objective function is calculated for each solution. The solution with the lowest error value is saved to the next iteration. This sequence is repeated until meeting the stop condition, which in this paper, is the maximum number of iterations. Then the best solution is passed to train the parameters of the ANFIS model. After finishing the training phase, the testing phase is started with the best solution to compute the final output. The performance of the proposed method is evaluated by comparing the real data with the predicted data using the performance measures. Finally, the FPASSA produces a foretasted value for confirmed cases of COVID-19 in China in the next day. The steps of the proposed FPASSA are presented in Algorithm 1. Input: Historical COVID-19 dataset, size of population N, total number of iterations t max . Divide the data into training and testing sets. Using Fuzzy c-mean method to determine the number of membership functions. Constructing the ANFIS network. Set the initial value for N solutions (X). Return the best solution that represents the best configuration for ANFIS. Apply the testing set to the best ANFIS model. Forecasting the COVID-19 for the next ten days. This section presents the description of the used dataset, the performance measures, the parameter setting for all methods, the experiment results, and discussions. The main dataset of this study is COVID-19 dataset. It was collected from the WHO website (https: //www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/). It contains the daily confirmed cases in China from 21 January 2020 to 18 February 2020, as shown in Table 1 . We used 75% from the dataset to train the model while the rest is used to test it. Moreover, we evaluated the performance of the proposed method using two datasets of weekly influenza confirmed cases. The first one is called DS1; it was collected from the Centers for Disease Control and Prevention (CDC) (https://www.cdc.gov/flu/weekly/). It starts from week number 40 in 2015 and continues until week number 6 in 2020. Whereas, the second one is called DS2. It was collected from the WHO website (https://www.who.int/influenza). It contains the data of weekly influenza confirmed cases in China from week number 1 in 2016 to week number 8 in 2020. The quality of the proposed method is evaluated using a set of performance metrics as follows: • Root Mean Square Error (RMSE): where Yp and Y are the predicted and original values, respectively. • Mean Absolute Error (MAE): • Mean Absolute Percentage Error (MAPE): • Root Mean Squared Relative Error (RMSRE): N s represents the sample size of the data. • Coefficient of Determination (R 2 ): where Y represents the average of Y. The lowest value of RMSE, MAE, MAPE, and RMSRE refers to the best method. The higher value of R 2 indicates better correlation for the method. This paper aims to assess the ability of the FPASSA to forecast the COVID-19 by comparing its performance with other methods, namely the ANFIS and the trained ANFIS models using PSO, GA, ABC, FPA, and FPASSA. The parameters' setting for these models is listed in Table 2 . The common parameters, such as population size, are set to 25 and 100 iterations are applied. Besides, each algorithm is performed for 30 independent runs to fair comparisons. The selected parameters are chosen because they produced good behavior in previous experiments, such as [34, 35, 55, 56] . Table 2 . Parameters' setting. Parameters Setting Max. epochs = 100, Error goal = 0, Initial step = 0.01, Decrease rate = 0.9, Increase rate = 1. In this section, the performance of the proposed FPASSA to predict the DS1 and DS2 is discussed. It can be concluded from Table 3 that the performance of FPASSA outperformed the compared methods in all measures, whereas the FPA is ranked second. The results of DS2 indicate that the FPASSA is ranked first in terms of RMSE, MAPE, R 2 , and the CPU time. Whereas, the PSO is ranked second, followed by the FPA, GA, then ABC. These results denote that the proposed method can optimize the parameters of the ANFIS model effectively and produce good results in terms of the performance measures. Comparison results between the proposed FPASSA and other models to forecast COVID-19 are given in Table 4 . It can be concluded that the FPASSA outperforms other models. For example, by analyzing the results of RMSE, MAE, MAPE, RMSRE, and CPU time(s) it can be observed that the FPASSA achieves the smallest value among the comparison algorithms, and this indicates the high quality of the FPASSA. Meanwhile, the FPA allocates the second rank, which provides better results than the rest of the methods. Moreover, the value of R 2 refers to the high correlation between the prediction obtained by the proposed FPASSA method and the original COVID-19, which has nearly 0.97. This can also be noticed from Figure 3 , which depicts the training of the algorithms using the historical data of the COVID-19 as well as their forecasting values for ten days. Table 5 depicts the forecasting value for the confirmed cases of the COVID-19 in China from 19/2/2020 to 28/2/2020. From these results, it can be noticed that the outbreak will reach its highest level on the day 28/2/2020. The average percentage of the increase over the forecasted period is 10%, the highest percentage is 12% on 28/2/2020, and the lowest percentage is 8.7% on 19/2/2020. From the previous results, it can be concluded that the proposed FPASSA-ANFIS has a high ability to forecast the COVID-19 dataset. These results avoid the limitations of traditional ANFIS because of the combination with the modified FPA method. Moreover, the operators of SSA are combined with the local strategy of FPA to enhance their exploitation ability. However, the time computational of the proposed FPASSA method still requires more improvements. This paper proposed a modified version for the flower pollination algorithm (FPA) using the salp swarm algorithm (SSA). This modified version, called FPASSA, is applied to improve the performance of the ANFIS through determining the optimal value for its parameters. The developed FPASSA-ANFIS model is applied as a forecasting technique for a novel coronavirus, called COVID-19, that was discovered in Wuhan, China at the end of last year and January of the current year. The proposed FPASSA-ANFIS model has a high ability to predict the number of confirmed cases within ten days. Besides, FPASSA-ANFIS outperforms other forecasting models in terms of RMSE, MAE, MAPE, RMSRE, and R 2 . Furthermore, two datasets of weekly influenza confirmed cases in the USA and China were used to evaluate the proposed method, and the evaluation outcomes showed its good performance. According to the promising results obtained by the proposed FPASSA-ANFIS, it can be applied in different forecasting applications.

Where was the coronavirus discovered?