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Influenza surveillance is an important tool to identify emerging/reemerging strains, and defining seasonality. We describe the distinct patterns of circulating strains of the virus in different areas in India from 2009 to 2013. Patients in ten cities presenting with influenza like illness in out-patient departments of dispensaries/hospitals and hospitalized patients with severe acute respiratory infections were enrolled. Nasopharangeal swabs were tested for influenza viruses by real-time RT-PCR, and subtyping; antigenic and genetic analysis were carried out using standard assays. Of the 44,127 ILI/SARI cases, 6,193 (14.0%) were positive for influenza virus. Peaks of influenza were observed during July-September coinciding with monsoon in cities Delhi and Lucknow (north), Pune (west), Allaphuza (southwest), Nagpur (central), Kolkata (east) and Dibrugarh (northeast), whereas Chennai and Vellore (southeast) revealed peaks in October-November, coinciding with the monsoon months in these cities. In Srinagar (Northern most city at 34°N latitude) influenza circulation peaked in January-March in winter months. The patterns of circulating strains varied over the years: whereas A/H1N1pdm09 and type B co-circulated in 2009 and 2010, H3N2 was the predominant circulating strain in 2011, followed by circulation of A/H1N1pdm09 and influenza B in 2012 and return of A/H3N2 in 2013. Antigenic analysis revealed that most circulating viruses were close to vaccine selected viral strains. Our data shows that India, though physically located in northern hemisphere, has distinct seasonality that might be related to latitude and environmental factors. While cities with temperate seasonality will benefit from vaccination in September-October, cities with peaks in the monsoon season in July-September will benefit from vaccination in April-May. Continued surveillance is critical to understand regional differences in influenza seasonality at regional and sub-regional level, especially in countries with large latitude span.

Background The susceptibility of mice and humans to Chandipura virus infection is age-dependent. Upon experimental infection, mice secrete significant amounts of proinflammatory cytokines. Similarly, children who recover from natural infection with the virus show significant amounts of TNF-α production, suggesting that innate immunity plays a major role in the response to Chandipura virus. Toll-like receptors (TLR) are key host molecules involved in innate immune responses in infections. Therefore, the aim of this study was to examine the role of TLR in the response to Chandipura virus infection. Methods The mouse monocyte-macrophage cell line, RAW 264.7, and C3H/HeJ mice were used as models. Micro array techniques were used to identify the type of TLR involved in the response to infection. The results were validated by examining TLR expression using flow cytometry and by measuring the levels of proinflammatory cytokines and nitric oxide (NO) in the culture supernatants using bead assays and the Griess method, respectively. The pathogenic role of Toll-like receptor 4 (TLR4) was studied in a TLR4 mutant strain of mice -C3H/HeJ and the results compared with those from wild-type mice- C3H/CaJ. The pathogenic effects of NO were studied by treating experimentally infected mice with the NO inhibitor, aminoguanidine (AG). Results The micro array results showed that TLR4 was regulated after Chandipura virus infection. At high multiplicities of infection (10 MOI), RAW cells up- regulated cell surface expression of TLR4 and secreted significant amounts of TNF-α, MCP-1, IL-10 and IL-12 and NO. The survival rate of C3H/HeJ mice was higher than those of wild-type C3H/CaJ mice. The survived C3H/HeJ mice secreted significant quantity of MCP-1 and IFN-γ cytokines and cleared virus from brain. Similarly, the survival rate of AG-treated mice was higher than those of the untreated controls. Conclusions Chandipura virus regulates TLR4, which leads to the secretion of proinflammatory cytokines and NO by infected RAW cells. Difference in survival rate in TLR4 mutant mice and nitric oxide inhibitor treated mice, confirmed the role of these molecules in disease pathogenesis. The result is significant in clinical management and designing antiviral intervention for Chandipura virus infection.

Background: Herpes genitalis is an ulcerating sexually transmitted infection, the clinical importance of which lies in its ability to produce painful and recurrent disease in addition to its potential role as a cofactor in acquisition and transmission of human immunodeficiency virus. In recent years, there are increasing reports of genital herpes due to herpes simplex virus (HSV)-1 from various parts of the world. Molecular diagnostic methods such as polymerase chain reaction (PCR) have got both diagnostic and prognostic significance in genital herpes. Aims: The present study was designed to identify the viral serotype in herpes genitalis patients in our locality, using PCR. Materials and Methods: The specimens from forty herpes genitalis patients were subjected to nested PCR and results were evaluated. Results: PCR was positive for HSV in 83% of cases, of which 58% were due to HSV-1. HSV-2 accounted for maximum number of recurrent herpes. Limitation: Higher sample size would have been more representative. Conclusion: A rising trend of type 1 HSV was observed in herpes genitalis in south India probably due to increasing practice of orogenital sex.

Chandipura virus (CHPV) is a tropical pathogen, suggesting its involvement in childhood encephalitis syndrome in India. No reports are available in adult human beings for its pathogenicity. Similarly, in adult mice, the virus does not develop pathogenesis by parenteral route except for intracranial route of infection. The virus is remarkably nonpathogenic to adult immunocompromised nude mice. In vitro in tissue culture, the CHPV infects and kills many types of cells. All of these properties could qualify the CHPV to be a candidate virus for tumor therapy. To prove this, an experimentally induced tumor in a mouse was infected with live CHPV. The results showed that intra-tumoral injection reduced the volume of tumor and increased the longevity of the mice. The study concludes that the CHPV may be a safe tumor therapy virus. More precisely, the discovery of CHPV protein with oncolytic potential may lead to the development of novel drugs/therapeutics.

Back ground Chandipura virus produces acute infection in mice. During infection drastic reduction of CD4+, CD8+ and CD19 + cell was noticed. Depletion of lymphocytes also noticed in spleen. The reduction may be due to the regulatory mechanism of immune system to prevent the bystander host tissue injury. There are several mechanisms like generation of regulatory cells, activation induced cell death (ACID) etc were indicated to control the activation and maintain cellular homeostasis. Role of regulatory cells in homeostasis has been described in several viral diseases. This study was undertaken to characterize CD4+T regulatory cells from the infected mice. Method In this study we purified the CD4+ T cells from Chandipura virus infected susceptible Balb/c mice. CD4+ T regulatory cells were identified by expression of cell surface markers CD25, CD127 and CTLA-4 and intracellular markers Foxp3, IL-10 and TGF-beta. Antigen specificity and ability to suppress the proliferation of other lymphocytes were studied in vitro by purified CD4+CD25+T regulatory cells from infected mice. The proliferation was calculated by proliferation module of Flow Jo software. Expression of death receptors on regulatory cells were studied by flowcytometer. Results The CD4+ T cells isolated from infected mice expressed characteristic markers of regulatory phenotype at all post infective hours tested. The CD4+ T regulatory cells were proliferated when stimulated with Chandipura virus antigen. The regulatory cells did not suppress the proliferation of splenocytes stimulated with anti CD3 antibody when co cultured with them. Interesting observation was, while purification of CD4+ T cells by negative selection, the population of cells negative for CD4 also co purified along with CD4+ T cell. Flow cytometry analysis and light microscopy revealed that CD4 negative cells were of different size and shape (atypical) compared to the normal lymphocytes. Greater percentage of these atypical lymphocytes expressed Fas Ligand and Programmed Death1 (PD-1) receptor. Conclusion From these results we concluded that virus specific CD4+T regulatory cells are generated during Chandipura virus infection in mice and these cells might control the activated lymphocytes during infection by different mechanism.

Introduction: Influenza is an RNA virus that belongs to the Orthomyxoviridae family. It causes a highly contagious acute respiratory illness, has been recognized since ancient times, and is a major health threat throughout the world. An outbreak of influenza-like illness (ILI) was reported from Alappuzha district of Kerala State between late June and July 2011. This investigation was conducted to determine the clinical picture, causative agents, and epidemiological characteristics of the illness. Methodology: The World Health Organization (WHO)’s case definition for ILI was followed throughout the investigation. Nasal or throat swabs were collected from 204 suspected patients. Real-time reverse transcription polymerase chain reaction (RT-PCR)-based diagnosis was performed to detect influenza A and B viruses and their subtypes. Madin-Darby canine kidney (MDCK) cell line was used for virus isolation. One-step RT-PCR was performed to amplify the HA1 gene of influenza A(H3N2). The amplicons for the HA1 gene of influenza A(H3N2) were sequenced, and phylogenetic analysis was done. Results: Analysis of the data revealed that 96 (47.05%) of the 204 respiratory specimens collected were influenza A(H3N2) and only 6 (2.94%) were A(H1N1)pdm09. Phylogenetic analysis revealed that the isolated A(H3N2) was closely related to the 2012–2013 northern hemisphere vaccine strain (A/Victoria/361/2011/H3N2). Conclusions: An influenza A(H3N2) outbreak was confirmed in Alappuzha district of Kerala state with a co-circulation of A(H1N1)pdm09. No substantial difference in the sequence was observed in the etiological agent, and the virus was found to be sensitive to oseltamivir.

Background & objectives: The global pandemic caused by SARS-CoV-2 virus has challenged public health system worldwide due to the unavailability of approved preventive and therapeutic options. Identification of neutralizing antibodies (NAb) and understanding their role is important. However, the data on kinetics of NAb response among COVID-19 patients are unclear. To understand the NAb response in COVID-19 patients, we compared the findings of microneutralization test (MNT) and plaque reduction neutralization test (PRNT) for the SARS-CoV-2. Further, the kinetics of NAb response among COVID-19 patients was assessed. Methods: A total of 343 blood samples (89 positive, 58 negative for SARS-CoV-2 and 17 cross-reactive and 179 serum from healthy individuals) were collected and tested by MNT and PRNT. SARS-CoV-2 virus was prepared by propagating the virus in Vero CCL-81 cells. The intra-class correlation was calculated to assess the correlation between MNT and PRNT. The neutralizing endpoint as the reduction in the number of plaque count by 90 per cent (PRNT90) was also calculated. Results: The analysis of MNT and PRNT quantitative results indicated that the intra-class correlation was 0.520. Of the 89 confirmed COVID-19 patients, 64 (71.9%) showed NAb response. Interpretation & conclusions: The results of MNT and PRNT were specific with no cross-reactivity. In the early stages of infection, the NAb response was observed with variable antibody kinetics. The neutralization assays can be used for titration of NAb in recovered/vaccinated or infected COVID-19 patients.

Several outbreaks of acute encephalitis syndrome (AES) have been reported in Alappuzha district, Kerala State, India, in the past. The aetiology of these outbreaks was either inconclusive or concluded as probable Japanese encephalitis virus (JEV) infection based on clinical presentation. The role of West Nile virus (WNV) in AES outbreaks was also determined. However, the extent of WNV infection has not been studied in this region previously. A population-based cross-sectional serosurvey study was undertaken to determine the seroprevalence of JEV and WNV in Alappuzha district. A total of 30 clusters were identified from 12 blocks and five municipalities as per the probability proportional to size sampling method. A total of 1125 samples were collected from all age groups. A microneutralization assay was performed to estimate the prevalence of JEV and WNV neutralizing antibodies in the sample population. Of 1125 serum samples tested, 235 [21.5%, 95% confidence interval (CI): 15.2-27.8%] and 179 (15.9%, 95% CI: 9.6-22.3%) were positive for neutralizing antibodies against WNV and JEV, respectively. In addition, 411 (34.5%, 95% CI: 26.7-42.2%) were positive for cross-reactive antibodies against flaviviruses. The study showed the seroprevalence of WNV and JEV antibodies in the surveyed area and the WNV seroprevalence was greater than JEV. It is necessary to create awareness in public and adopt suitable policy to control these diseases.

Background & objectives: Public health and diagnostic laboratories are facing huge sample loads for COVID-19 diagnosis by real-time reverse transcription-polymerase chain reaction (RT-PCR). High sensitivity of optimized real-time RT-PCR assays makes pooled testing a potentially efficient strategy for resource utilization when positivity rates for particular regions or groups of individuals are low. We report here a comparative analysis of pooled testing for 5- and 10-sample pools by real-time RT-PCR across 10 COVID-19 testing laboratories in India. Methods: Ten virus research and diagnostic laboratories (VRDLs) testing for COVID-19 by real-time RT-PCR participated in this evaluation. At each laboratory, 100 nasopharyngeal swab samples including 10 positive samples were used to create 5- and 10-sample pools with one positive sample in each pool. RNA extraction and real-time RT-PCR for SARS-CoV-2-specific E gene target were performed for individual positive samples as well as pooled samples. Concordance between individual sample testing and testing in the 5- or 10-sample pools was calculated, and the variation across sites and by sample cycle threshold (Ct) values was analyzed. Results: A total of 110 each of 5- and 10-sample pools were evaluated. Concordance between the 5-sample pool and individual sample testing was 100 per cent in the Ct value ≤30 cycles and 95.5 per cent for Ctvalues ≤33 cycles. Overall concordance between the 5-sample pooled and individual sample testing was 88 per cent while that between 10-sample pool and individual sample testing was 66 per cent. Although the concordance rates for both the 5- and 10-sample pooled testing varied across laboratories, yet for samples with Ct values ≤33 cycles, the concordance was ≥90 per cent across all laboratories for the 5-sample pools. Interpretation & conclusions: Results from this multi-site assessment suggest that pooling five samples for SARS-CoV-2 detection by real-time RT-PCR may be an acceptable strategy without much loss of sensitivity even for low viral loads, while with 10-sample pools, there may be considerably higher numbers of false negatives. However, testing laboratories should perform validations with the specific RNA extraction and RT-PCR kits in use at their centres before initiating pooled testing.