Immunoglobulin G Subclass Response After Chikungunya Virus Infection.

Various vaccines are under development to prevent chikungunya (CHIKV) infection. For the assessment of the CHIKV vaccine-induced antibody response, it is extremely important to understand antibody response after the infection has occurred. Previously, we assessed IgG response in samples from healthy donors using I-CHIKV and found that IgG1 was the predominant subclass induced after CHIKV infection followed by IgG4. However, IgG3 subclass induction is reported in serum samples from patients with acute CHIKV infection. Therefore, in this study, we evaluated serum/plasma from samples of patients with acute CHIKV infection for the presence of IgG and IgG subclasses against I-CHIKV and recombinant E2 protein (rE2). Out of 44 samples that were positive against I-CHIKV, 43 were found reactive against rE2. The positivity of IgG1 either alone or together with other IgG subclasses using I-CHIKV was 89% samples, while 86% samples were positive using rE2. High titers of IgG1 are obtained with I-CHIKV (67%), while raised IgG4 levels are detected using rE2p (72%) in the samples that are positive for both these subclasses. Testing of 22 samples for neutralizing antibodies revealed 100% IgG1 positivity and neutralizing antibodies in 21, 1 sample negative for both. Overall, these data will be useful in assessing IgG subclass-specific CHIKV neutralization and response after CHIKV immunization.

Antibody (IgA, IgG, and IgG Subtype) Responses to SARS-CoV-2 in Severe and Nonsevere COVID-19 Patients.

For the assessment of vaccine-induced immune response and to understand the role of antibodies in neutralization, it is necessary to assess dynamics of various antibodies in patients with different clinical manifestations. This study aims to quantitate circulating levels of IgA/IgG and IgG subtypes induced at different days postonset of symptoms, in severe and nonsevere patients. For this, serum or plasma samples (n = 146) collected from 79 COVID-19 patients were used. Indirect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific IgA, IgG, and IgG subtype specific enzyme-linked immunosorbent assays (ELISAs) were performed. Antibody titers between severe and nonsevere patients were compared at different times postonset of clinical symptoms. Titers in ELISA were compared to neutralizing antibody (Nab) titers determined by plaque reduction neutralization test (PRNT). Over 75% patients were positive for IgA/IgG antibodies in the first week. The ELISA titers did not differ during the first week; however, severe disease exhibited raised titers thereafter. Nab titers correlated with the ELISA titers in mild presentation but not in severe disease. IgA and IgG1 antibodies correlated stronger with Nabs. The findings highlighted that IgA together with IgG play an important in SARS-CoV-2 neutralization. These results will prove useful in assessing efficacy of vaccines and understanding disease pathogenesis.

Age-Dependent Evaluation of Immunoglobulin G Response after Chikungunya Virus Infection.

Current chikungunya antibody prevalence and titers are likely to differ based on the exposure rates before the 2006 reemergence in India. For vaccine usage, such data are of immense importance. This study addresses age-stratified IgG titers and its subtypes in Pune, India, endemic for the disease. 170 age-stratified serum pools from 791 individuals with prior chikungunya exposure, and 15 samples from acute disease phase were analyzed. An indirect ELISA based on inactivated chikungunya virus was used to determine anti-CHIKV-IgG and its subtypes. Neutralizing antibody titers (plaque reduction neutralization test [PRNT]) were compared with binding antibody titers (ELISA). Anti-CHIKV-IgG titers along with IgG1 and IgG4 increased till the age-group of until 11-15 years and remained comparable thereafter till > 65 years. IgG1 was the predominant IgG subtype detected in all the pools, whereas IgG4 was present in 151/170 pools. Strong positive correlation of IgG1 was obtained with CHIKV-PRNT50 titers. None of the sample had anti-CHIKV-IgG2, whereas five pools had IgG3 antibody. In the acute-phase serum sample, IgG1 was present in all the samples, whereas IgG4 was present in 8/15 samples. IgG4 was predominant in four samples. During acute phase and at different times postinfection, IgG1 circulated in high titers followed by IgG4. Higher antibody titers in adults reflect reexposures. The data will prove useful in assessing immune response to CHIKV vaccine in relation to IgG subtype.

Disease-duration based comparison of subsets of immune cells in SARS CoV-2 infected patients presenting with mild or severe symptoms identifies prognostic markers for severity.

INTRODUCTION: Infection with SARS-CoV-2 leads to a spectrum of symptoms. Understanding the basis for severity remains crucial for better management and therapy development. So far, older age, associated-comorbidities, and IL-6 have been associated with severity/mortality. MATERIALS AND METHODOLOGY: As a primary step, we analyzed the frequency and functional profile of innate immune cells (NK cells/dendritic cells/monocytes) and adaptive immunity-driving lymphocytes (B cells/T cells/follicular T helper cells) by flow cytometry. Sixty cases of SARS CoV-2 infection (25 severe, 35 mild) and ten healthy subjects without SARS CoV-2 IgG were included. Disease-duration based analysis of immune profile was explored for early events differentiating the two disease forms. Neutralizing antibody titers were determined by PRNT. RESULTS AND CONCLUSION: Disease severity was found to be associated with impaired maturation of mDCs and hyperactivation of NK, follicular T helper cells, and CD8 T cells. Lower IL-21 receptor expression on memory B cells indicated an imbalance in IL-21/IL-21 R ratio. Lower BCMA positive plasmablast cells in severe cases did suggest a probable absence of long-term humoral immunity. Multivariate analysis revealed a progressive association of PD-1+CD4 T cells with PRNT50 titers. Thus, in addition to identifying probable prognostic markers for severity, our study emphasizes the definite need for in-depth viral antigen-specific functional analyses in a larger patient cohort and with multiple sampling.

Standardization of ELISA for anti-chikungunya-IgG antibodies and age-stratified prevalence of anti-chikungunya-IgG antibodies in Pune, India.

Chikungunya (CHIKV) reemerged in India after a gap of 32 years, in 2005-2006 and has established endemicity in Pune. To assess the degree of CHIKV exposure, we estimated age-stratified prevalence of IgG antibodies to CHIKV in Pune population. This retrospective study utilized age-stratified serum samples collected from 15 wards of Pune in 2017 for dengue (DENV) virus study. Indirect anti-CHIKV-IgG ELISA was developed and used to test 1904 samples. Exposure to CHIKV and DENV was compared in the same population. CHIKV-specific plaque reduction neutralization test (PRNT) was employed to evaluate ELISA positivity and neutralizing potential of anti-CHIKV-IgG antibodies. Indirect ELISA showed 98.5% concordance with commercial ELISA. Seropositivity to CHIKV was 46.4%, one-third children < 15 years being antibody positive. A significant increase (45%, p = 0.026-0.038) was noted at 16-25 years and varied between 48 and 56% until the age 65. In elderly (65 + years), antibody positivity was reduced (41%, p = 0.01). In children, CHIKV-PRNT50 titers increased with age and remained comparable from the age group 11-15 until > 65. Exposure to DENV was higher than CHIKV. Lower exposure of children and elderly could be due to lesser exposure to the vectors. High prevalence of IgG antibodies needs to be addressed while planning vaccine studies for CHIKV.

Comparative assessment of commercial enzyme-linked immunosorbent assay & rapid diagnostic tests used for dengue diagnosis in India.

Background & objectives: Dengue diagnosis is routinely carried out by detection of dengue virus (DENV) antigen NS1 and/or anti-DENV IgM antibodies using enzyme-linked immunosorbent assays (ELISAs) and rapid diagnostic tests (RDTs). This study was aimed at evaluation of quality of diagnostic assays currently in use in India for the identification of DENV infection. Methods: During 2016 dengue season (July-November) in Pune, India, comparative assessment of a few immunoassays was undertaken using (i) WHO-approved Panbio-Dengue-Early-(NS1)-ELISA and Panbio-Dengue-IgM-Capture-ELISA as reference tests, and (ii) Bayesian latent class analysis (BLCA) which assumes that no test is perfect. The assays included J.Mitra-Dengue-NS1-Ag-MICROLISA (JME-NS1), J.Mitra-Dengue-IgM-MICROLISA (JME-IgM), and two RDTs, namely, J.Mitra-Dengue-Day-1-Test (JM-RDT) and SD-BIOLINE-Dengue-Duo (SDB-RDT). Serum samples from patients seeking dengue diagnosis (n=809) were tested using the diagnostic kits. The presence of NS1 and/or IgM was taken as evidence for dengue-positive diagnosis. Results: Panbio-NS1/IgM-ELISAs identified 38.6 per cent patients as dengue positive. With Panbio-ELISA as reference, all the tests were less sensitive for IgM detection, while for NS1, JM-RDT was less sensitive. For combined diagnosis (both markers), sensitivity of all the tests was low (55.7-76.6%). According to BLCA, Panbio-ELISA was 84 per cent sensitive for NS1, 86 per cent specific for IgM and 87 per cent specific for combined diagnosis. Accordingly, performance of the other tests was substantially improved with BLCA; however, sensitivity of both the RDTs for IgM detection remained unacceptable. The NS1 ELISAs and RDTs detected all four DENV serotypes, JME being most efficient. All IgM tests exhibited higher sensitivity in secondary infections. Interpretation & conclusions: These results confirmed superiority of ELISAs, and testing for both NS1 and IgM markers for dengue diagnosis, and emphasized on improvement in sensitivity of RDTs.

Risk of transfusion-associated dengue: screening of blood donors from Pune, western India.

BACKGROUND: Dengue, a mosquito-borne viral disease, is endemic in >125 countries worldwide. The threat of blood-borne transmission of dengue virus (DENV) has been documented. STUDY DESIGN AND METHODS: This study was conducted to assess the potential magnitude of transfusion-associated dengue, by determination of DENV seromarkers in blood donations from Pune, India, during two dengue seasons (2016 and 2017). These included DENV nonstructural protein 1 (NS1), anti-DENV immunoglobulin (Ig) M, anti-DENV IgG (enzyme-linked immunosorbent assay), and DENV RNA (reverse transcription-polymerase chain reaction). RESULTS: NS1 (IgM) reactivity was 1 of 209, 0.48% (11/209, 5.3%) in 2016 and 2 of 311, 0.64% (20/311, 6.4%) in 2017. Of the 34 NS1/IgM reactives, 1 NS1-reactive donor and 10 IgM-reactive donors exhibited evidence of secondary infection. DENV RNA was not detected in any of the 34 NS1/IgM reactives. Among the NS1/IgM negatives, anti-DENV IgG reactivity was high in 2016 (75%) and further increased in 2017 (87%, p = 0.002). CONCLUSION: Although RNA negative, detection of 34 NS1/IgM-reactive donations, of which 11 had evidence of secondary infection, suggests the need for further evaluation on the basis of potential risk to recipients of either dengue transmission or increased risk of secondary infection. These would include multicenter studies followed by cost-benefit analyses.

Stratified sero-prevalence revealed overall high disease burden of dengue but suboptimal immunity in younger age groups in Pune, India.

BACKGROUND: In India, dengue disease is emerging as the most important vector borne public health problem due to rapid and unplanned urbanization, high human density and week management of the disease. Clinical cases are grossly underreported and not much information is available on prevalence and incidence of the disease. METHODOLOGY: A cross sectional, stratified, facility based, multistage cluster sampling was conducted between May 4 and June 27, 2017 in Pune city. A total of 1,434 participants were enrolled. The serum samples were tested for detection of historical dengue IgG antibodies by ELISA using the commercial Panbio Dengue IgG Indirect ELISA kit. Anti-dengue IgG-capture Panbio ELISA was used for detection of high titered antibodies to detect recent secondary infection. We used this data to estimate key transmission parameters like force of infection and basic reproductive number. A subset of 120 indirect ELISA positive samples was also tested for Plaque Reduction Neutralizing Antibodies for determining serotype-specific prevalence. FINDINGS: Overall, 81% participants were infected with dengue virus (DENV) at least once if not more. The positivity was significantly different in different age groups. All the adults above 70 years were positive for DENV antibodies. Over 69% participants were positive for neutralizing antibodies against all 4 serotypes suggesting intense transmission of all DENV serotypes in Pune. Age-specific seroprevalence was consistent with long-term, endemic circulation of DENV. There was an increasing trend with age, from 21.6% among <36 months to 59.4% in age group 10-12 years. We estimate that 8.68% of the susceptible population gets infected by DENV each year resulting into more than 3,00,000 infections and about 47,000 to 59,000 cases per year. This transmission intensity is similar to that reported from other known hyper-endemic settings in Southeast Asia and the Americas but significantly lower than report from Chennai. CONCLUSIONS: Our study suggests that Pune city has high disease burden, all 4 serotypes are circulating, significant spatial heterogeneity in seroprevalence and suboptimal immunity in younger age groups. This would allow informed decisions to be made on management of dengue and introduction of upcoming dengue vaccines in the city.

Comparison of the conventional multiplex RT-PCR, real time RT-PCR and Luminex xTAG® RVP fast assay for the detection of respiratory viruses.

Detection of respiratory viruses using polymerase chain reaction (PCR) is sensitive, specific and cost effective, having huge potential for patient management. In this study, the performance of an in-house developed conventional multiplex RT-PCR (mRT-PCR), real time RT-PCR (rtRT-PCR) and Luminex xTAG(®) RVP fast assay (Luminex Diagnostics, Toronto, Canada) for the detection of respiratory viruses was compared. A total 310 respiratory clinical specimens predominantly from pediatric patients, referred for diagnosis of influenza A/H1N1pdm09 from August 2009 to March 2011 were tested to determine performance characteristic of the three methods. A total 193 (62.2%) samples were detected positive for one or more viruses by mRT-PCR, 175 (56.4%) samples by real time monoplex RT-PCR, and 138 (44.5%) samples by xTAG(®) RVP fast assay. The overall sensitivity of mRT-PCR was 96.9% (95% CI: 93.5, 98.8), rtRT-PCR 87.9% (95% CI: 82.5, 92.1) and xTAG(®) RVP fast was 68.3% (95% CI: 61.4, 74.6). Rhinovirus was detected most commonly followed by respiratory syncytial virus group B and influenza A/H1N1pdm09. The monoplex real time RT-PCR and in-house developed mRT-PCR are more sensitive, specific and cost effective than the xTAG(®) RVP fast assay.

Dynamics of influenza seasonality at sub-regional levels in India and implications for vaccination timing.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Burden of seasonal and pandemic influenza-associated hospitalization during and after 2009 A(H1N1)pdm09 pandemic in a rural community in India.

BACKGROUND: Influenza is vaccine-preventable; however, the burden of severe influenza in India remains unknown. We conducted a population-based study to estimate the incidence of laboratory confirmed influenza-associated hospitalizations in a rural community in western India. METHODS: We conducted active surveillance for hospitalized patients with acute medical illnesses or acute chronic disease exacerbations in Pune during pandemic and post pandemic periods (May 2009-April 2011). Nasal and throat swabs were tested for influenza viruses. A community health utilization survey estimated the proportion of residents hospitalized with respiratory illness at non-study facilities and was used to adjust incidence estimates from facility-based surveillance. RESULTS: Among 9,426 hospitalizations, 3,391 (36%) patients were enrolled; 665 of 3,179 (20.9%) tested positive for influenza. Of 665 influenza positives, 340 (51%) were pandemic A(H1N1)pdm09 and 327 (49%) were seasonal, including A/H3 (16%), A/H1 (3%) and influenza B (30%). The proportion of patients with influenza peaked during August 2009 (39%) and 2010 (42%). The adjusted annual incidence of influenza hospitalizations was 46.8/10,000 during pandemic and 40.5/10,000 during post-pandemic period with comparable incidence of A(H1N1)pdm09 during both periods (18.8 and 20.3, respectively). The incidence of both pH1N1 and seasonal hospitalized influenza disease was highest in the 5-29 year olds. CONCLUSIONS: We document the previously unrecognized burden of influenza hospitalization in a rural community following the emergence of influenza A(H1N1)pdm09 viruses in India. During peak periods of influenza activity circulation i.e during the monsoon period, 20% of all hospital admissions in the community had influenza positivity. These findings can inform development of influenza prevention and control strategies in India.

Kyasanur Forest disease, India, 2011-2012.

To determine the cause of the recent upsurge in Kyasanur Forest disease, we investigated the outbreak that occurred during December 2011-March 2012 in India. Male patients >14 years of age were most commonly affected. Although vaccination is the key strategy for preventing disease, vaccine for boosters was unavailable during 2011, which might be a reason for the increased cases.

Development of a multiplex one step RT-PCR that detects eighteen respiratory viruses in clinical specimens and comparison with real time RT-PCR.

Rapid and accurate diagnosis of viral respiratory infections is crucial for patient management. Multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) is used increasingly to diagnose respiratory infections and has shown to be more sensitive than viral culture and antigen detection. Objective of the present study was to develop a one-step mRT-PCR that could detect 18 respiratory viruses in three sets. The method was compared with real time RT-PCR (rRT-PCR) for its sensitivity and specificity. Clinical specimens from 843 pediatric patients with respiratory symptoms were used in the study. 503 (59.7%) samples were detected positive by mRT-PCR. Of these 462 (54.8%) exhibited presence of a single pathogen and 41 (4.9%) had multiple pathogens. rRT-PCR detected 439 (52.1%) positive samples, where 419 (49.7%) exhibited one virus and 20 (2.4%) showed co-infections. Concordance between mRT-PCR and rRT-PCR was 91.9% and kappa correlation 0.837. Sensitivity and specificity of mRT-PCR were 99.5% and 83.7% while that of rRT-PCR was 86.9% and 99.4% respectively. Rhinovirus (17.2%) was the most frequently detected virus followed by respiratory syncytial virus B (15.4%), H1N1pdm09 (8.54%), parainfluenza virus-3 (5.8%) and metapneumovirus (5.2%). In conclusion, mRT-PCR is a rapid, cost effective, specific and highly sensitive method for detection of respiratory viruses.

Antibody persistence after Pandemic H1N1 2009 influenza vaccination among healthcare workers in Pune, India.

The healthcare workers having seroprotection at 3 weeks (n = 127) following Pandemic H1N1 2009 influenza vaccination were followed up for antibody persistence. Seroprotection at 12 mo (60.2%) was significantly lower as compared with 3 weeks (74.7%), 3 mo (77.8%) and 6 mo (75.4%). The vaccine provided seroprotection up to one year.

Validity of clinical case definitions for influenza surveillance among hospitalized patients: results from a rural community in North India.

OBJECTIVE: Clinical case definitions used for influenza surveillance among hospitalized patients vary and need systematic evaluation. DESIGN, SETTING AND SAMPLE: During July 2009-August 2011, we collected clinical data and specimens (nasal and throat swabs) from rural patients hospitalized for acute medical illnesses. Specimens were tested by rRT-PCR for influenza viruses. MAIN OUTCOME MEASURES: Case definitions evaluated the following: influenza-like illness (ILI: measured fever plus cough or sore throat); severe acute respiratory illness (SARI: ILI with difficulty breathing in ≥5 years, Integrated Management of Childhood Illness-defined pneumonia or severe pneumonia, or physician diagnosed lower respiratory infection in <5 years); acute respiratory infection (ARI: ≥1 of cough, nasal discharge, difficulty breathing or sore throat); febrile acute respiratory illness (FARI: fever plus either cough, sore throat, runny nose, difficulty breathing, or earache). Variants that included "reported fever" and additional sign-symptom combinations were also evaluated. RESULTS: We enrolled 1043 hospitalized patients, including 257 children <5 years of age (range 1 day-86 years). Seventy-four patients tested influenza virus positive (including 28 A(H1N1)pdm09). Sensitivity(95% CI) and specificity (95% CI) for influenza infection were 78% (67-87) and 60% (57-63) for ILI (measured/reported fever); 37% (26-49) and 78% (75-80) for SARI (measured/reported fever); 82% (72-90) and 57% (54-60) for FARI (measured/reported fever); 88% (78-94) and 45% (42-49) for ARI; and 74% (63-84) and 61% (58-64) for measured/reported fever plus cough. Case definitions including only measured fever had lower sensitivity. CONCLUSION: ILI and FARI with measured/reported fever provided good balance between sensitivity and specificity among hospitalized patients. The simpler case definition of measured/reported fever plus cough is suited for field surveillance.

Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the influenza A virus subtypes responsible for the 20th-century pandemics.

BACKGROUND: PB1F2 is the 11th protein of influenza A virus translated from +1 alternate reading frame of PB1 gene. Since the discovery, varying sizes and functions of the PB1F2 protein of influenza A viruses have been reported. Selection of PB1 gene segment in the pandemics, variable size and pleiotropic effect of PB1F2 intrigued us to analyze amino acid sequences of this protein in various influenza A viruses. METHODS: Amino acid sequences for PB1F2 protein of influenza A H5N1, H1N1, H2N2, and H3N2 subtypes were obtained from Influenza Research Database. Multiple sequence alignments of the PB1F2 protein sequences of the aforementioned subtypes were used to determine the size, variable and conserved domains and to perform mutational analysis. RESULTS: Analysis showed that 96·4% of the H5N1 influenza viruses harbored full-length PB1F2 protein. Except for the 2009 pandemic H1N1 virus, all the subtypes of the 20th-century pandemic influenza viruses contained full-length PB1F2 protein. Through the years, PB1F2 protein of the H1N1 and H3N2 viruses has undergone much variation. PB1F2 protein sequences of H5N1 viruses showed both human- and avian host-specific conserved domains. Global database of PB1F2 protein revealed that N66S mutation was present only in 3·8% of the H5N1 strains. We found a novel mutation, N84S in the PB1F2 protein of 9·35% of the highly pathogenic avian influenza H5N1 influenza viruses. CONCLUSIONS: Varying sizes and mutations of the PB1F2 protein in different influenza A virus subtypes with pandemic potential were obtained. There was genetic divergence of the protein in various hosts which highlighted the host-specific evolution of the virus. However, studies are required to correlate this sequence variability with the virulence and pathogenicity.

Genetic characterization and molecular clock analyses of the Crimean-Congo hemorrhagic fever virus from human and ticks in India, 2010-2011.

A nosocomial outbreak of Crimean Congo hemorrhagic fever (CCHF) was reported among humans in Ahmadabad district, Gujarat, India during January, 2011. In the present study we provide the complete genomic sequences of four CCHFV isolates derived from two human patients and two pools of Hyalomma anatolicum ticks during the period of this outbreak and the complete S segment sequence of two retrospective human serum samples, positive for CCHFV in 2010. Sequence-based molecular characterization of the Indian CCHFV showed that they possessed the functional motifs known to occur in the S, M and L gene segment products as in other CCHF viruses. The S segment of the six Indian CCHF viruses showed 99.8% nucleotide identity. Notably both tick isolates shared 100% nucleotide identity with one of the Indian human isolates of 2011. Phylogenetic analysis based on the S segment demonstrated that the Indian CCHFV isolates formed a distinct cluster in the Asian-Middle East group IV of CCHF viruses. The S segment was closest to a Tajikistan strain TADJ/HU8966 of 1990 (98.5% nucleotide identity) and was of South-Asia 2 type while the M segment was of type M2. Both M and L segments were closest to an Afghanistan strain Afg09-2990 of 2009 (93% and 98% nucleotide identity, respectively). The Indian isolates were thus identified as a South-Asia 2/M2 far-east virus combination and the differing parental origin in the S and L/M segments is suggestive that it may be an intra-genotypic reassortant. Molecular clock studies further revealed that the ancestry of the viruses was not very recent and dated back to about 33years on the basis of the S segment while it was about 15years based on the M segment. Thus though the 2011 outbreak may not have resulted from a very recent introduction, considering that so far there is no evidence of multiple circulating strains in the country, the possibility of a recent re-introduction of the virus from any of the neighboring countries cannot be ruled out. The study thus warrants the need for continued surveillance and increased sampling of CCHFV in different parts of the country.

Suitability of specimen types for isolation of avian influenza viruses from poultry.

In view of the outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus in poultry in India, its impact on global public health and growing concerns of avian influenza (AI) viruses, surveys in wet poultry markets were conducted in the states of Maharashtra, West Bengal and Jharkhand in India during the period 2009-2012. During these surveys various types of samples from poultry were collected. During outbreaks and surveys in poultry, tracheal swabs (TS), cloacal swabs (CS), poultry drinking water (PDW) samples and fecal samples (FS) are preferred samples for AI diagnosis. The suitability of various types of poultry samples for AI virus isolation was analyzed. The parameters such as availability of specimen, ease of collection, quality of the specimen for the presence of contaminants such as organic debris or solid matter were considered for the analysis. A total of 2,405 samples were collected, which included 1,297 TS, 1,012 CS, 79 PDW, and 17 FS. Out of 2,309 TS and CS samples 1,752 samples were paired samples, collected from 876 birds. All samples were processed for virus isolation and identification. Of the 2,405 samples AI H9N2 was isolated from 199 samples (8.27 %). The virus isolation rate was significantly higher in PDW samples (21.5 %) (P < 0.05) and TS samples (12.1 %), in comparison with CS (2.3 %) (P < 0.001). Other viruses isolated were AI H4N6 and HPAI H5N1viruses; however the number of isolates of AI H4N6 and H5N1 were not sufficient for comparison. In conclusion, the PDW and TS samples were suitable for AI H9N2 virus isolation from poultry.