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The emergence of novel variants of SARS-CoV-2 in several countries has been associated with increased transmissibility or reduced neutralization potential of antibodies against the Wuhan virus (wild type). From August 2021 onwards, India experienced a progressive decline in the number of active SARS-CoV-2 infections, indicative of a downward trend in the explosive second wave. This prospective study was conducted quarterly for one year (May 2020 to June 2021) at a tertiary care hospital in the city of Pune in western India. Receptor-binding domain (RBD, n = 319) and full genome (n = 20) sequences from viral-RNA-positive nasopharyngeal swabs of COVID-19 patients representing the first and second waves were used for analysis. No Brazilian, South African, or California variants were detected in this study. Until December 2020, only the wild-type strain was prevalent. Concurrent with the upsurge of the second wave in March 2021, 73% (33/45) of RBD sequences harboured L452R/E484Q mutations characteristic of the Kappa variant. In April 2021, co-circulation of Kappa (37%) and Delta (L452R/T478K, 59%) variants was recorded. During May and June 2021, the Delta variant became the predominant circulating variant, and this coincided with a significant decline in the number of COVID-19 cases. Of the 20 full genome sequences, six isolates each exhibited signature mutations of the Kappa and Delta variant. With several states witnessing a reduction in the number of COVID-19 cases, continuous monitoring of newer mutations and assessment of their effect on virus transmissibility and their impact on vaccinated or previously exposed individuals is necessary. Supplementary Information The online version contains supplementary material available at 10.1007/s00705-021-05320-7.

Purpose COVID-19 pandemic remains a serious public health threat worldwide. In view of the limited data on the risk of perinatal transmission of SARS-CoV-2 and transfer of maternal anti-SARS-CoV-2 antibodies, the present study was undertaken. Methods A prospective study including 57 pregnant women with a positive SARS-CoV-2 RNA test (SARS-CoV-2-RNA+) and 59 neonates born to them was conducted at Pune, India. 39 viral RNA negative (SARS-CoV-2-RNA-negative) pregnant women and their 39 neonates were included as controls. Neonatal nasal swab/cord blood samples were subjected to SARS-CoV-2 RNA detection by RT-PCR for investigation of perinatal transmission. Transfer of maternal antibodies was studied using ELISA and PRNT. Results 10/57 SARS-CoV-2-RNA+ mothers were symptomatic. The duration between COVID-19 diagnosis and delivery was ≤ 7 days for 82.4%. Perinatal transmission as evidenced by viral RNA in the neonatal nasal swab/cord blood (CB) was 3.6%. IgG-anti-SARS-CoV-2 positivity was 21.6%. Of the 39 neonates born to SARS-CoV-2-RNA-negative mothers, 20 (51%) and none, respectively, were positive for IgG-anti-SARS-CoV-2 and viral RNA. Preterm deliveries were higher in SARS-CoV-2-RNA+ (18.6%) than SARS-CoV-2 RNA-negative (0/39) mothers (p

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.

Patients with SARS-CoV-2 infection have a wide spectrum of clinical presentations, from asymptomatic infection, to mild illness, to severe disease with recovery or fatal outcome. Immune correlates of protection are not yet clear. To understand the association between presence and titers of neutralizing antibodies (NAb) with recovery, we screened 82 COVID-19 patients classified in mild (n = 56) and severe (n = 26) disease groups on different days post onset of disease and 27 viral RNA–positive asymptomatic contacts examined within 1 week of the identification of index cases. Of 26 patients with severe disease, six died and 20 recovered. Anti-SARS-CoV-2 NAb levels in plasma and serum were measured using a plaque reduction neutralization test with live virus. The proportion of asymptomatic and symptomatic infections was 1:7.8 in males and 1:1 in females, with males predominating the severe disease group (21/26, 80.7%). At the time of presentation, NAb positivity and titers were comparable among groups with asymptomatic and mild infections. Notably, patients with severe disease exhibited higher NAb seropositivity and titers (25 of 26, 96.2%; 866 ± 188) than those in the mild category (39 of 56, 69.6%; 199 ± 50, P < 0.0001) and asymptomatic individuals (21 of 27, 77.8%; 124 ± 28, P = 0.0002). Within first 2 weeks of onset, NAb titers were significantly higher among patients with severe disease than those with mild presentation. Our data suggest that irrespective of fatal outcome, progression to disease severity was associated with induction of early and high levels of NAb. In our patient series, clinical disease, severity and fatality were predominantly seen in males. The role of NAbs in immunopathogenesis or protection needs to be defined.

In view of the rapidly progressing COVID-19 pandemic, our aim was to isolate and characterize SARS-CoV-2 from Indian patients. SARS-CoV-2 was isolated from nasopharyngeal swabs collected from the two members of a family without any history of (H/O) travel abroad. Both the virus isolates (8003 and 8004) showed CPE on day 3 post-inoculation, viral antigens by immunofluorescence assay and produced distinct, clear and uniform plaques. Infectious virus titers were 5 × 106 and 4 × 106 Pfu/ml by plaque assay and 107.5 and 107 by CPE-based TCID50/ml, respectively. Phylogenetic analysis grouped our isolates with the Italian strains. On comparison with Wuhan strain, 3 unique mutations were identified in nsp3 (A1812D), exonuclease (P1821S) of Orf1ab and spike protein (Q677H) regions, respectively. Both the viruses grouped with Italian strains of SARS-CoV-2 suggesting possible source being the virus imported from Italy. These fully characterized virus isolates will be useful in developing neutralization/virological assays for the evaluation of vaccines/antivirals.

Background & objectives: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) continues to be a devastating pandemic. This study was aimed at performance assessment of SARS-CoV-2 IgM and IgG ELISAs, and investigation of their utility for patient diagnosis and sero-epidemiologic investigations. Methods: Serum/plasma samples from COVID-19 patients or asymptomatic contacts (n=180) and healthy donors (n=90) were tested in parallel using two commercial IgM ELISAs (Erbalisa and Inbios), and four IgG ELISAs (Kavach, Euroimmun, Erbalisa and Inbios) along with an indigenous β-propiolactone inactivated virus-based ELISA (IRSHA-IgG-ELISA). Plaque reduction neutralization test (PRNT) was used as reference test. Results: Among 180 COVID-19 patients, 125 tested positive by PRNT. Inbios-IgM-ELISA showed sensitivity (Se)/specificity (Sp)/positive predictive value (PPV)/negative predictive value (NPV) of 93.6/97.8/98.4/94.4 per cent in relation to PRNT, and performed better than Erbalisa-IgM-ELISA (Se: 48%, Sp: 95.6%, PPV: 95.2%, NPV: 65.2%). During the first week of disease, only 47.4 per cent of the COVID-19 patients tested IgM positive by Inbios-IgM-ELISA, detection improving at two weeks and beyond (~86-100%). Among IgG tests, Inbios-IgG-ELISA ranked first in terms of sensitivity (83.2%), followed by IRSHA (64.8%), Euroimmun (64%), Erbalisa (57.6%) and Kavach (56%) tests. For all IgG tests, sensitivity improved during the third (73.9-95.7%) and fourth week (100%) of illness. The specificity (96.7-100%) and PPV (96.2-100%) of all IgG tests were high; NPV ranged between 71.9 and 87.1 per cent with Inbios-IgG-ELISA scoring highest. Interpretation & conclusions: Our results show that IgM detection by the current, most sensitive ELISAs cannot replace molecular diagnosis, but may aid as a supplement test. The available IgG tests are suitable for serosurveys for the assessment of previous virus exposure.

BackgroundDengue is a public health problem worldwide. Therapeutic monoclonal antibodies (MAbs) against dengue virus (DENV) are likely to be available soon. In view of the feasibility issues pertaining to pretreatment viraemia quantitation for therapy decisions, we conducted this study for investigation of a correlation between patient serostatus (NS1/immunoglobulin M [IgM]/IgG) and viraemia levels among Indian dengue patients at the time of first diagnosis.MethodsThe study included 297 serum samples from dengue patients in Pune, India. The samples were tested for NS1, IgM and IgG (capture enzyme-linked immunosorbent assay [ELISA] for identifying secondary dengue) using Panbio ELISAs. Quantitation of viraemia was conducted using an NS1 ELISA-based 50% tissue culture infectious dose (TCID50) test in Vero cells.ResultsViraemia was detectable only among NS1-positive patients (n = 229, range 0.5–8.3 logTCID50/ml) with a mean titre of 1.9 logTCID50/ml. Among the NS1-positive patients, DENV titres were higher in IgM-negative than IgM-positive patients (p < 0.0001) and in primary (IgG < 18 Panbio units) versus secondary (IgG > 22 Panbio units) dengue patients (p = 0.002). Virus titres were higher during the first 3 days of illness and decreased later (p = 0.005).ConclusionsThe study provides a range of infectious DENV titres in relation to serologic status among dengue patients in India. The data suggest the possibility of using serological markers (NS1/IgM) as a basis for treatment decisions.

Dengue is the fastest spreading mosquito-borne viral infection across the globe affecting over 40% of world populations. Co-existence of 4 distinct serotypes, disease severity due to secondary infection, and continuous evolution of dengue viruses over the years intensify the need for a better and effective virus surveillance program. In this study, we evaluated NS1 antigen detection method to screen large number of clinical samples for dengue virus isolation. Patient’s serum samples were added onto in vitro tissue culture grown C6/36 or Vero cell lines. Seven days post-infections, culture supernatants were harvested for testing of NS1 antigen by ELISA to confirm the presence of dengue viruses. Significantly higher rate of virus isolation was observed in Vero cells at 7 days post-infection in comparison to C6/36 cells. NS1 antigen could be detected earliest at day 3 post-infection in culture supernatants of both C6/36 and Vero cells. Use of Vero cells in a single 35-mm culture dishes resulted in 69.7% DENV isolations from NS1 alone positive samples. The method is economical, easy to perform, and useful in handling large number of clinical samples and will be of value in virus surveillance, especially post-vaccination.

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.

Understanding of the basis for severity and fatal outcome of SARS-CoV-2 infection is of paramount importance for developing therapeutic options and identification of prognostic markers. So far, accumulation of neutrophils and increased levels of pro-inflammatory cytokines are associated with disease severity in COVID-19 patients. In this study, we aimed to compare circulatory levels of neutrophil secretory proteins, alpha-defensins (DEFA1), calprotectin (S100A8/A9), and myeloperoxidase (MPO) in COVID-19 patients with different clinical presentations. We studied 19 healthy subjects, 63 COVID-19 patients with mild (n=32) and severe (n=31) disease, 23 asymptomatic individuals identified through contact tracing programme and 23 recovering patients (1-4 months post-disease). At the time of disease presentation, serum levels of DEFA1 were significantly higher in patients with mild (mean230 ± 17, p

The emergence of novel variants of SARS-CoV-2 in several countries has been associated with the increased transmission/ reduced neutralization potential of antibodies against the Wuhan virus (wild type). Currently, India is recovering from an explosive second wave of COVID-19. This prospective study was conducted quarterly for one year (May 2020 to June 2021) at a tertiary care hospital, Pune city, western India. Receptor binding domain (RBD, n = 319) and full genome (n = 20) sequences from viral RNA positive nasopharyngeal swabs of COVID-19 patients representing first and second waves were used for analysis. We did not detect Brazil, South Africa and California variants. Till December, only wild type strain was prevalent. Concurrent with the upsurge of second wave in March-2021, 73% (33/45) of RBD sequences harboured L452R/E484Q mutations characteristic of Kappa variant. In April, co-circulation of Kappa (37%) and Delta (L452R/T478K, 59%) variants was recorded. During May and June, delta variant became the predominant circulating variant and coincided with a significant decline in the number of COVID-19 cases. Of the 20 full genome sequences, six isolates each exhibited signature mutations of Kappa and Delta variants respectively. With several states witnessing reduction in the number of COVID-19 cases, continuous monitoring of newer mutations and assessment of these mutations in influencing virus transmissibility and impact on vaccinated / previously exposed individuals would be necessary.

Hepatitis A, an acute inflammatory liver disease caused by hepatitis A virus (HAV) infection from close contact with infected people, is highly endemic in the Indian subcontinent. Due to poor sanitary conditions, most of the population is exposed to the virus in childhood. At this age, the disease is asymptomatic and provides life-long protection against the disease. Due to rapid socioeconomic development in some areas, however, pockets of the population are reaching adolescence/adulthood without prior exposure to the virus and are thus susceptible to infection. At these ages, infection carries a higher risk of symptomatic disease and complications including mortality. This review of epidemiology and burden of disease studies in the Indian subcontinent, published since 2005, shows increasing evidence of a shift from high to intermediate endemicity in high-income—typically urban—populations. The prevalence of anti-HAV antibodies (previously reported at > 90%) is lower now in adolescents and young adults (e.g., around 80% in Bangladesh and 55% in 5–15 years in India). As a result, HAV is responsible for more acute viral hepatitis predominantly in this age group (e.g., > 15 years: 3.4% in 1999 to 12.3% in 2003 or high socioeconomic status 13–20 years: 27% in 1999 to 62% in 2003), with a greater clinical and economic burden. Numerous outbreaks due to HAV have been reported [e.g., Sri Lanka (2009–2010): > 13,000 affected; Kashmir (2015–2017): 12 outbreaks; Kerala (2012–2016): 84 outbreaks] from water or food contamination. Due to current shifts in endemicity, a growing proportion of the population is no longer exposed in childhood. As the disease remains highly endemic, it also provides a source for more severe disease in susceptible people at an older age and for outbreaks. Well-tolerated and effective vaccines are available and help prevent disease burden and provide long-term protection. These should now be used more widely to protect more patients from the growing disease burden of hepatitis A. Funding GlaxoSmithKline Biologicals SA. Plain Language Summary Plain language summary available for this article—please see Fig. 1 and the following link: 10.6084/m9.figshare.9963044.Fig. 1Plain Language Summary. Highlights the context of the article, the endemicity shift and the burden of hepatitis A in adolescents and adults and steps to be taken to address the impact of this disease Electronic supplementary material The online version of this article (10.1007/s40121-019-00270-9) contains supplementary material, which is available to authorized users.

High mortality in pregnant women is a characteristic of hepatitis E virus (HEV) infection. Role of monocytes/T cells in HEV infection during pregnancy is still unclear. We compared CD14+monocytes and CD4+T cells by flow-cytometry in hepatitis-E patients including 13 pregnant (Antenatal care, ANC), 25 non-ANC patients and respective controls (12 and 20). Non-ANC-patients showed significantly higher frequency of monocytes with increased expression of CD80, CD86 and HLA-DR than control individuals (p Healthy pregnancy was associated with increased frequency of monocytes with higher CD80 expression and lower levels of HLA-DR (p 0.05). Compared to respective controls, CD137+ and CD152+CD4+T cells were higher (p 0.05). We found higher and reduced levels of circulating inflammatory cytokines (IL12, TNFα, IL6 and IL8; miliplex-assay) in non-ANC and ANC-patients respectively. In conclusion, on contrary to the classical activation of CD14+monocytes in the non-ANC-patients, impaired response was evident in the ANC-patients while the CD4+T cell populations were similar in the patient groups.

Respiratory syncytial virus (RSV) causes significant infant mortality worldwide and a vaccine may be available soon. This study determined age-stratified anti-RSV antibody positivity (enzyme-linked immunosorbent assay [ELISA]) at Pune, India (cord blood-85 years). Antibody positivity declined from 100% at birth to 71.3% (3 months), and 0.7% (6 months). A significant rise was noted at 15 months (16%), 16 to 24 months (64.5%) and 4 years (95.2%) with concomitant IgM-anti-RSV positivity indicative of recent infection. Antibody decline was higher in infants born preterm than full-term. Across subsequent age groups including the elderly, antibody positivity was similar and comparable, suggestive of repeated exposure to the virus. Early protection/vaccination is essential for the infant population.

Background Immunization of children with vaccines against Measles, Mumps, Rubella, and Varicella (MMRV) is practiced globally with varied recommendations. In India, measles vaccine is administered alone or as MMR at 9 months age. Varicella vaccine is not routinely used. Immunization age is a function of disappearance of maternal antibodies and natural exposure of the children to the pathogens. In view of the measles-WHO-initiative, we aimed to assess if the current immunization age for measles is still valid. In addition, the kinetics of IgG and IgM antibodies against rubella, mumps and varicella viruses was also examined. Methods This cross-sectional study was conducted at a tertiary care hospital in Pune, India. A total of 600 children, 150 each in 6-month/9-month (no vaccination) and 12-month/15-month (minimum 4 weeks post-measles-vaccine) cohorts were included. History of these infections and birth status (term/preterm) was recorded. All serum samples were screened for IgG-anti-MMRV-antibodies while IgG-positives were tested for specific IgM antibodies (ELISA). Results At 6-months, the prevalence of MMRV antibodies was 4.7%, 2.7%, 10.7%, 5.3% respectively depicting disappearance of maternal antibodies in majority of the children. Birth status did not influence antibody positivity. Despite vaccination at ∼9-months, >25% children were still susceptible to measles virus at the age of 12/15-months. The ratio of clinical:subclinical infections was 4:10 (measles) and 12:1 (varicella). All the mumps/rubella IgM positives (1 and 2 respectively) represented subclinical infections. Conclusion Demonstration of early disappearance of maternal antibodies against MMRV viruses leading to the risk of these infections at an early age emphasize need for early immunization of Indian children. Suboptimal response to measles vaccine needs to be seriously addressed especially in view of the WHO’s initiative for measles eradication.

Seasonal outbreaks of acute encephalitis syndrome (AES) at Gorakhpur, India have been recognized since 2006. So far, the causative agent has not been identified. Use of next generation sequencing identified human parvovirus 4 (HPARV4) sequences in a CSF/plasma pool. These sequences showed highest identity with sequences earlier identified in similar patients from south India. Real-time PCR detected HPARV4 DNA in 20/78 (25.6%) CSF and 6/31 (19.3%) plasma of AES patients. Phylogenetic analysis classified three almost complete genomes and 24 partial NS1 sequences as genotype 2A. The observed association of HPARV4 with AES needs further evaluation. ELISAs for the detection of IgM and IgG antibodies against scrub typhus (Orientia tsutsugamushi, OT) showed ∼70% IgM/IgG positivity suggestive of etiologic association. Prospective, comprehensive studies are needed to confirm association of these agents, singly or in combination with AES in Gorakhpur region.

Childhood vaccination plays critical role in protecting infants from several dreaded diseases. Of the global 15 million preterm (PT) infants with compromised immune system born annually, India contributes to >3.5 million. Generation of adequate vaccine-induced immune response needs to be ensured of their protection. Immune response of Indian PT (n = 113) and full-term (FT, n = 80) infants to pentavalent vaccine administered as per the national recommendation was studied. Antibody titers against component antigens of pentavalent vaccine, immune cells profiling (T and B cells, monocytes and dendritic cells) and plasma cytokines were determined pre- and post-vaccination. Additionally, cell-mediated recall immune responses to pentavalent antigens were evaluated after short time antigenic exposure to infant PBMCs. Irrespective of gestational age (GA), all the infants developed adequate antibody response against tetanus, diphtheria, and protective but lower antibody levels for Haemophilus influenzae type-b and hepatitis B in preterm infants. Lower (~74%) protective antibody response to pertussis was independent of gestational age. PT-infants exhibited lower frequencies of CD4 T cells/dendritic cells/monocytes, increased plasma IL-10 levels and lower proliferation of central and effector memory T cells than in term-infants. Proliferative central memory response of FT-infants without anti-pertussis antibodies suggests protection from subsequent infection. Responder/non-responder PT-infants lacked immunological memory and could be infected with Bordetella. For hepatitis B, the recall response was gestational age-dependent and antibody status-independent. Humoral/cellular immune responses of PT-infants were dependent on the type of the immunogen. Preterm infants born before 32 weeks of gestation may need an extra dose of pentavalent vaccine for long lived robust immune response.

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.

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., Understanding pathogenesis of severity in current SARS‐CoV2 pandemic is of prime importance. Another concern is the recent observations (including ours) of early, higher neutralizing antibody titers in severe disease. India is reporting >80,000/day for several weeks now. Identification of prognostic markers for severity may help identifying “extra‐care‐needed” patients. As a first step, this flow‐cytometry based study was planned to understand (1) differences in immunologic modulations in patients with mild and severe disease (2) if we can identify early events associated with severity (3) prognostic markers for severity (4) association of functionality of immune cells with higher antibody titers in severe disease.

Background: Being a long-term preventive measure, COVID-19 vaccines are used in global populations. In India, country-wide immunization drive was initiated in January 2021. Methods: To assess immune response of health-care-workers to COVISHIELD(n=187) and COVAXIN(n=21), blood samples collected pre-vaccination and 1month-post-1/post-2 dose, administered 28days apart, were tested for IgG-anti-SARS-CoV-2 (ELISA) and neutralizing (Nab, PRNT50) antibodies. Spike protein-specific T cells were quantitated by IFN-γ ELISPOT and Flow-cytometry-based Intra-cellular-secretion (ICS) for IFN-γ/IL2. Findings: Among pre-vaccination-antibody negative (pre-negatives, n=120) and positive (pre-positives, n=67) COVISHIELD recipients, %Nab seroconversion and median (IQR) Nab titers were 55.1%/95.6% and 16(IQR 2.5-36.3)/(64.5, IQR 34.5-154.2, p

To assess modulation of neutralizing antibody titers in COVID-19 patients and understand association of variables such as age, presence of comorbidity, BMI and gender with antibody titers.Patients (n = 100) diagnosed from 20th March 2020 to 17th August 2020 and treated at two large hospitals from Pune, India were included and followed up (clinical and serologic) for varied periods. IgG-anti-SARS-CoV-2 (Spike protein-based ELISA) and neutralizing antibody titers (NAb, PRNT) were determined in all the samples.Of the 100 patients enrolled initially (median 60 days of diagnosis), follow up samples were collected from 70 patients (median 106 days of diagnosis). Overall, NAb titers reduced significantly (p 0.001) and as early as 3-4 months. During two visits, 20% and 7.1% patients reported some symptoms. At the first visit, NAb titers were higher in patients with severe disease (p 0.001), comorbidities (p 0.005), age50 years (p 0.05) and male gender (p 0.05). Multivariate analysis identified older age (p 0.001), duration post-diagnosis and female gender as independent variables influencing NAb titers (negative correlation, p 0.05). During the follow-up, reduction in NAb titers was recorded in patients with comorbidity (p 0.05), mild disease (p 0.05), age50 years (p 0.05), higher BMI (p 0.05) and male gender (p 0.001). Serology identified six cases of asymptomatic reinfections.Decline of NAb titers was associated with age50 years, mild disease, comorbidities, higher BMI and male gender. At the time of follow up, 8/70 (11.4%) patients lacked neutralizing antibodies. Evidence of 6 probable asymptomatic reinfections suggests waning of immunity, but, probable protection from clinical disease needing hospitalization.

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.

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 children15 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-PRNT

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.

Dengue is the most common mosquito-borne viral infection in tropical and sub-tropical countries. In the recent years, frequent dengue outbreaks are being reported in many parts of India. DENV circulates as four independent serotypes posing a major public health threat around the globe. Phylogenetic and full genome sequence analyses of 19 complete DENV genome sequences presenting all the four serotypes in Pune, India (2016-2017) revealed no change in the circulating genotypes i.e., genotype V clade C (D1), genotype IVB (D2), genotype III lineage III (D3) and genotype I clade D (D4). Additionally, unique amino acid substitutions that may potentially influence viral fitness and virulence in host cells were identified. Mapping of the unique amino acid substitutions onto the T cell epitopes of the reference strains revealed that 8/10 (D1), 14/15 (D2), 3/4 (D3) and 21/74 (D4), amino acids were involved in T-cell epitope presentation for a maximum number of HLA alleles associated with disease outcome. Selection pressure analysis documented a positive selection pressure to be acting on few amino acid sites indicating continuous evolutionary changes in the viral RNA. Overall, the evolutionary and selection pressure data generated during this study may help in better understanding of DENV evolution and epidemiology.

Background Hepatitis E (HE) is prevalent in developing countries in both epidemic and sporadic forms and is characterized by high mortality during pregnancy. miR-122, the major hepatic microRNA has been shown to be modulated during liver diseases. Lack of data in HE led to investigations in non-pregnant (NPR) and pregnant (PR) patients. Results Self-recovering NPR patients and pregnant women presenting with clinical (PR-acute) or subclinical (PR-SC) HE and respective healthy controls were studied. Serum samples were tested for miR-122 levels using qRT-PCR. Acute-phase, NPR patients circulated lower miR-122 levels, reducing further during convalescence. In contrast to previous reports, circulating miR-122 levels did not correlate with serum aminotransferase (ALT). In PR-acute patients, miR-122 levels were significantly lower that reflected pregnancy status and not HEV effect. In PR-SC patients, miR-122 levels were lower than the pregnant controls and reduced further when examined one month apart. A pregnant, fulminant HE patient circulated very high miR-122 levels that increased further during convalescence. Correlation analysis of miR-122 and circulating cytokines showed moderate correlation with CCL2 (subclinical, pregnant) and IL-6 (NPR). This first report revealed downregulation of circulating miR-122 levels in self-recovering NPR-acute patients despite liver damage (raised serum ALT) suggestive of alternate mechanism of secretion in blood. Conclusion HEV infection during pregnancy led to differential modulation of serum miR-122 levels that correlated with clinical presentation. Utility of miR-122 as a prognostic marker for severe disease during pregnancy needs to be evaluated.

Highlights • Development of indirect IgG ELISA using β-propiolactone-inactivated SARS-CoV-2. • Detection of anti-SARS-CoV-2 IgG during early disease phase. • Higher IgG seropositivity and OD values in COVID-19 patients with severe disease., Background Coronavirus disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome – coronavirus-2 (SARS-CoV-2) continues to affect many countries and large populations. Serologic assays for antibody detection aid patient diagnosis and seroepidemiologic investigations. Methods An indirect IgG ELISA was developed indigenously using β-propiolactone (BPL) inactivated SARS-CoV-2. This assay was used for screening 200 healthy donor sera collected prior to COVID-19 emergence (2017–2019), 185 serum/plasma samples of confirmed COVID-19 patients (n = 137) and 57 samples of viral RNA positive asymptomatic contacts (n = 51). The IgG response was studied in relation to duration and severity of illness. Results The ELISA demonstrated 97 % specificity and IgG detection in >50 %, 80 %, 93.8 % and 100 % of the patients respectively during the first, second, third and fourth week of illness. IgG detection rate was higher in patients with severe disease (SD, 90.9 %) than those with mild disease (MD, 68.8 %) during the second week of illness (P = 0.027). IgG seropositivity among asymptomatic contacts was 64.7 %. IgG ELISA absorbance values were higher in SD than MD patients during the first 2 weeks of illness (P

Background Liposome encapsulated neutralizing epitope protein of Hepatitis E virus (HEV), rNEp, our Hepatitis E vaccine candidate, was shown to be immunogenic and safe in pregnant and non-pregnant mice and yielded sterilizing immunity in rhesus monkeys. Methods The current study in Balb/c mice assessed the levels and persistence of anti-HEV IgG antibodies by ELISA, frequencies of B, memory B, T and memory T cells by flow cytometry and HEV-specific IgG secreting memory B cells by ELISPOT till 420 days post immunization (PI) with 5 μg rNEp encapsulated in liposome based adjuvant (2 doses, 4 weeks apart). Mice immunized with a lower dose (1 μg) were assessed only for anamnestic response post booster dose. Results Vaccine candidate immunized mice (5 μg dose) elicited strong anti-HEV IgG response that was estimated to persist for lifetime. At day 120 PI, frequency of memory B cells was higher in immunized mice than those receiving adjuvant alone. Anti-HEV IgG titers were lower in mice immunized with 1 μg dose. A booster dose yielded a heightened antibody response in mice with both high (>800 GMT, 5 μg) and low (⩽100 GMT, 1 μg) anti-HEV IgG titers. At day 6th post booster dose, HEV-specific antibody secreting plasma cells (ASCs) were detected in 100% and 50% of mice with high and low anti-HEV IgG titers, respectively, whereas the frequencies of CD4+ central and effector memory T cells were high in mice with high anti-HEV IgG titers only. Conclusions Taken together, the vaccine candidate effectively generates persistent and anamnestic antibody response, elicits participation of CD4+ memory T cells and triggers memory B cells to differentiate into ASCs upon boosting. This approach of assessing the immunogenicity of vaccine candidate could be useful to explore the longevity of HEV-specific memory response in future HEV vaccine trials in human.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis and a major public health problem in India. There are four mammalian HEV genotypes worldwide. In India, genotype 1 (HEV-1) is restricted to humans whereas genotype 4 (HEV-4) circulates in pigs. Studies from our laboratory have shown that HEV-4 (swine) virus can establish experimental infection in rhesus monkeys; however, HEV-1 (human) virus cannot infect pigs. Viral and/or cellular factors responsible for this host specificity are not yet known. We developed 12 different genotype 1-4 chimeric full genome clones with pSK-HEV2 as the backbone and by replacing structural (ORF2 and ORF3), non-structural (ORF1) and non-coding regions (NCR) with corresponding segments from the HEV-4 clone. S10-3 (human hepatoma) and PK-15 (pig kidney) cells were transfected with transcripts generated from the above clones to test their replication competence. Transfected cells were monitored for successful virus replication by detecting replicative intermediate RNA and capsid protein (immunofluorescence assay). All the chimeric constructs were able to replicate in S10-3 cells. However, only two chimeric clones, HEV-1 (HEV-4 5'NCR-ORF1) and HEV-1 (HEV-4 ORF1), containing 5'NCR-ORF1 and ORF1 regions from the HEV-4 clone, respectively, were able to replicate in PK-15 cells. We demonstrate for the first time the crucial role of ORF1 polyprotein in crossing the species barrier at the cellular level. These results indicate the importance of interactions between ORF1 protein domains and host cell specific factors during HEV replication and the critical role of cellular factors as post-entry barrier/s in virus establishment.

The degree of transmission of hepatitis A virus (HAV) is inversely proportional to the socioeconomic status of a community. Serosurveys conducted at Pune, India during 1982-98 documented significant reduction in HAV exposure of paediatric, higher socioeconomic status (HSS) population. Anti-HAV positivity (ELISA) in age-stratified Pune population representing HSS and lower middle socioeconomic status (LMSS) (n = 1065) and infants till the age of 15 months (n = 690) was determined in 2017. Anti-HAV positivity in the LMSS population decreased significantly in 2017 while an increase was seen in the HSS category. The surprising rise in anti-HAV positivity in the HSS population reflected vaccine- and infection-induced antibodies while only infection-induced antibodies were present in the LMSS category. Lowest antibody prevalence in infants was at 12 months, the recommended age for hepatitis A vaccination. Improved hygiene and selective immunization practices impacted HAV exposure of the LMSS population. The data emphasize the need for hepatitis A vaccination irrespective of socioeconomic status.

The molecular mechanisms of liver pathology and clinical disease in hepatitis E virus (HEV) infection remain unclear. MicroRNAs (miRNAs) are known to modulate viral pathogenesis either by directly altering viral gene expression or by enhancing cellular antiviral responses. Given the importance of microRNA-122 (miR-122) in liver pathobiology, we investigated possible role of miR-122 in HEV infection. In silico predictions using HEV genotype 1 (HEV-1), HEV-2, HEV-3, and HEV-4 sequences showed that the majority of genomes (203/222) harbor at least one miR-122/microRNA-122-3p (miR-122*) target site. Interestingly, HEV-1 genomes showed a highly (97%) conserved miR-122 target site in the RNA-dependent RNA polymerase (RdRp) region (RdRp c ). We analyzed the significance of miR-122 target sites in HEV-1/HEV-3 (HEV-1/3) genomes by using a replicon-based cell culture system. HEV infection did not change the basal levels of miR-122 in hepatoma cells. However, transfection of these cells with miR-122 mimics enhanced HEV-1/3 replication and depletion of miR-122 with inhibitors led to suppression of HEV-1/3 replication. Mutant HEV-1 replicons with an altered target RdRp c sequence (CACTCC) showed a drastic decrease in virus replication, whereas introduction of alternative miR-122 target sites in mutant replicons rescued viral replication. There was enrichment of HEV-1 RNA and miR-122 molecules in RNA-induced silencing complexes in HEV-infected cells. Furthermore, pulldown of miR-122 molecules from HEV-infected cells resulted in pulldown of HEV genomic RNA along with miR-122 molecules. These observations indicate that miR-122 facilitates HEV-1 replication, probably via direct interaction with a target site in the viral genome. The positive role of miR-122 in viral replication presents novel opportunities for antiviral therapy and management of hepatitis E. IMPORTANCE Hepatitis E is a problem in both developing and developed countries. HEV infection in most patients follows a self-limited course; however, 20% to 30% mortality is seen in infected pregnant women. HEV superinfections in patients with chronic hepatitis B or hepatitis C virus infections are associated with adverse clinical outcomes, and both conditions warrant therapy. Chronic HEV infections in immunocompromised transplant recipients are known to rapidly progress into cirrhosis. Currently, off-label use of ribavirin (RBV) and polyethylene glycol-interferon (PEG-IFN) as antiviral therapy has shown promising results in both acute and chronic hepatitis E patients; however, the teratogenicity of RBV limits its use during pregnancy, while alpha IFN (IFN-α) increases the risk of transplant rejections. Experimental data determined with genotype 1 virus in the current study show that miR-122 facilitates HEV replication. These observations present novel opportunities for antiviral therapy and management of hepatitis E.

Hepatitis E infection caused by hepatitis E virus (HEV), a major public health concern in developing countries, is responsible for sporadic and epidemic acute viral hepatitis in adults. Pathogenesis of hepatitis E infection is poorly understood. Toll-like receptors (TLRs) are the key players of innate immunity recognize pathogen-associated molecular patterns (PAMPs). Previously, we found higher TLR4 expression (at protein and gene level) with impaired cytokine response upon stimulus of PBMCs with LPS in HEV-infected patients. In view of the earlier observations of the association of polymorphisms in TLR4 genes (A299G, C399T) with liver diseases, we investigated TLR4 polymorphisms in HEV-infected patients. We observed the significant association of TLR4-399CC and CT alleles with hepatitis E (both subclinical and acute patients). Carrier frequency of TLR4-399 CT was lower in patients' categories in comparison with the controls. Higher frequency of allele TLR4-399C significantly correlated with disease progression. Acute hepatitis E patients showed the higher frequency of CG and TA haplotypes, while the rare haplotype (TG) was more frequent in controls. The other single nucleotide polymorphism (SNP) at TLR4-299 (A>G) did not show any difference. We report here for the first time the association of TLR4 polymorphism with hepatitis E and suggest that TLR 4 hyporesponsiveness during HEV infection might be related to its polymorphism.

Background & objectives : Since the 2006 massive outbreaks, chikungunya (CHIK) is a major public health concern in India. The aim of this study was to assess envelope specific immune responses in patients with chikungunya infection. Methods: This study included 46 hospitalized patients with chikungunya virus infection (encephalitis, n=22, other systemic involvement, OSI, n=12, classical, n=12) and six controls from Ahmedabad city, Gujarat, India. T cell responses and the levels of Th1, pro/ anti-inflammatory cytokines against the CHIK virus envelope antigens were assessed by lymphocyte proliferation assay and by cytometric bead array in flow cytometry, respectively. Results: Lymphoproliferative response was uniform among the patients. Comparisons of cytokines revealed significantly higher levels of interleukin (IL)-4 and IL-5 in encephalitis, OSI and classical patients versus controls. The levels of tumour necrosis factor (TNF)-α were higher in classical patients categories compared to the controls. Interferon (IFN)-γ levels were lower in encephalitis patients versus control. Interpretation & conclusions : Our findings showed recognition of T cell epitopes on the envelope region of chikungunya virus by all patient categories. Lower level of IFN-γ may be associated with the severity of disease in these patients.

Dengue is the most common mosquito-borne viral infection in tropical and sub-tropical countries. In recent years, India has reported increased incidences of concurrent infection with multiple serotypes of dengue viruses (DENV). In the present study, we have characterized DENV circulating during a single season of 2016 in Pune, India. A total of 64 serum samples from NS1 ELISA positive dengue patients were used for PCR amplification of CprM region of the viral genome and sequencing. Phylogenetic analysis documented circulation of all the four DENV serotypes with predominance of DENV-2 (40.6%). DENV genotyping classified DENV-1 to Genotype V, DENV-2 to Genotype IV, DENV-3 to Genotype III and DENV-4 to Genotype I. Further analysis revealed emergence of a novel clade (D) of genotype I of DENV-4. Subsequent isolation of three DENV-4 viruses in cell culture followed by complete genome sequence analysis confirmed this observation. Additionally, a new genotype within serotype-4 with >6.7% sequence variation from other genotypes was identified. This first report of significant co-circulation of all the four serotypes in a single outbreak in Pune reconfirms need for molecular monitoring of DENV.

We documented earlier that Mw (heat-killed suspension of Mycobacterium indicus pranii) adjuvant when used with conserved antigens, nucleoprotein (NP), and ectodomain of matrix (M2) protein (M2e) provided complete protection against homologous (clade 2.2) virus challenge in mice. The present study extends these observations to inter-clade challenge (clade 2.3.2.1) H5N1 virus and attempts to understand preliminary immunologic basis for the observed protection. Female BALB/c mice immunized with a single or two doses of vaccine formulations (clade 2.2 antigens) were challenged with 100LD50 homologous or heterologous (clade 2.3.2.1) virus. To understand the preliminary immunologic mechanism, we studied proportions of selected immune cell types, immune response gene expression, and Th1/Th2 cytokines induced by antigen-stimulated splenocytes from immunized mice, at different time points. Complete protection was conferred by Mw-HA, Mw-HA + NP, and Mw-HA + NP + M2e against homologous challenge. The protection correlated with IgG2a antibody titers indicating important role of Th1 response. Despite high inter-cladal antigenic differences, complete protection against the heterologous strain was achieved with Mw-HA + NP + M2e. Of note, a single dose with higher antigen concentrations (50 µg HA + 50 μg NP + 50 μg M2e) led to 80% protection against clade 2.3.2.1 strain. The protection conferred by Mw-HNM correlated with induction of IFN-γ, CD8+ T cytotoxic cells, and CD4+ T helper cells. Mw-adjuvanted HA + NP + M2e combination represents a promising vaccine candidate deserving further evaluation.

Hepatitis E virus (HEV) causes high mortality among pregnant women. Pathogenesis of HEV, especially during pregnancy, is poorly understood. Our aim was to assess the role of Toll-like-receptors (TLRs) in hepatitis E patients with pregnancy (Antenatal care, ANC) or without pregnancy (non-ANC). The patient categories included acute-phase, non-ANC (n=46) and ANC patients (2nd/3rd trimesters, n=13) and non-ANC patients (n=31) during convalescence. Controls included apparently healthy non-ANC (n=30) and ANC subjects in the first (n=10) and later (2nd/3rd, n=20) trimesters. TLR2/TLR3/TLR4/TLR7/TLR8 levels were determined by flow-cytometry. Cytokine responses induced by TLR-specific-ligands-stimulated-PBMCs from ANC/non-ANC-patients and TLR-signaling-molecules (non-ANC-patients) were measured. PBMCs were used to assess gene expression levels by TaqMan-Low-Density-Array. Compared to the temporal activation of TLR4/TLR7/TLR8 at protein and mRNA levels, the ANC-patients and controls exhibited reduced TLRs indicative of impaired TLR response. Stimulation of PBMCs with TLR-specific ligands led to the induction of type-I interferons, IFNβ by the non-ANC group and IFNα by the ANC category. Involvement of MyD88-independent (TLR3/TLR4) and MyD88-dependent (TLR4/TLR7/TLR8) pathways and association of TLR4/TLR7/TLR8 with recovery was documented in the non-ANC-patients. Except for robust type-I-interferon response, HEV infection could not modulate pregnancy-related diminished immune response. The results have implications in the understanding of HEV pathogenesis.

Retrospective serologic screening of 1077 serum samples collected from the primitive tribe from north-eastern India in 1963 revealed high prevalence of HBV (15% HBsAg carrier rate) and HCV (7% anti-HCV positivity) and co-circulation of multiple HBV genotypes-A, C, D and G. Full genome sequencing classified all the G-genotype samples as genotype-I. Comparison of genotype-I-HBV full-genome sequences representing 1963 (n=5, this study) and 2005 (reported earlier) showed identical recombination break-points of genotypes-A/G/C. Genotype-C and genotype-C-fragment of I-genotype circulating in 1963 were distinctly different. The data demonstrates that the recombination events were not recent. Molecular clock analysis predicted existence of genotype-I in this tribe during 1920s.

Identification and characterization of virus host interactions is an essential step for the development of novel antiviral strategies. Very few studies have been targeted towards identification of chikungunya virus (CHIKV) interacting host proteins. In current study, virus overlay protein binding assay (VOPBA) and matrix-assisted laser desorption/ ionization time of flight analysis (MALDI TOF/TOF) were employed for the identification of CHIKV binding proteins in mammalian cells. HSP70 and actin were identified as virus binding proteins in HEK-293T and Vero-E6 cells, whereas STAT-2 was identified as an additional protein in Vero-E6 cells. Pre-incubation with anti-HSP70 antibody and miRNA silencing of HSP70 significantly reduced the CHIKV production in HEK-293T and Vero-E6 cells at early time points. These results suggest that CHIKV exploits the housekeeping molecules such as actin, HSP70 and STAT-2 to establish infection in the mammalian cells.