Varsha A. Potdar

Genetic Characterization of the Influenza A Pandemic (H1N1) 2009 Virus Isolates from India

Background The Influenza A pandemic H1N1 2009 (H1N1pdm) virus appeared in India in May 2009 and thereafter outbreaks with considerable morbidity and mortality have been reported from many parts of the country. Continuous monitoring of the genetic makeup of the virus is essential to understand its evolution within the country in relation to global diversification and to track the mutations that may affect the behavior of the virus. Methods H1N1pdm viruses were isolated from both recovered and fatal cases representing major cities and sequenced. Phylogenetic analyses of six concatenated whole genomes and the hemagglutinin (HA) gene of seven more isolates from May-September 2009 was performed with reference to 685 whole genomes of global isolates available as of November 24, 2009. Molecular characterization of all the 8 segments was carried out for known pathogenic markers. Results The first isolate of May 2009 belonged to clade 5. Although clade 7 was the dominant H1N1pdm lineage in India, both clades 6 and 7 were found to be co-circulating. The neuraminidase of all the Indian isolates possessed H275, the marker for sensitivity to the neuraminidase inhibitor Oseltamivir. Some of the mutations in HA are at or in the vicinity of antigenic sites and may therefore be of possible antigenic significance. Among these a D222G mutation in the HA receptor binding domain was found in two of the eight Indian isolates obtained from fatal cases. Conclusions The majority of the 13 Indian isolates grouped in the globally most widely circulating H1N1pdm clade 7. Further, correlations of the mutations specific to clade 7 Indian isolates to viral fitness and adaptability in the country remains to be understood. The D222G mutation in HA from isolates of fatal cases needs to be studied for pathogenicity.

Pandemic Influenza (H1N1) 2009 Is Associated with Severe Disease in India

Background Pandemic influenza A (H1N1) 2009 has posed a serious public health challenge world-wide. In absence of reliable information on severity of the disease, the nations are unable to decide on the appropriate response against this disease. Methods Based on the results of laboratory investigations, attendance in outpatient department, hospital admissions and mortality from the cases of influenza like illness from 1 August to 31 October 2009 in Pune urban agglomeration, risk of hospitalization and case fatality ratio were assessed to determine the severity of pandemic H1N1 and seasonal influenza-A infections. Results Prevalence of pandemic H1N1 as well as seasonal-A cases were high in Pune urban agglomeration during the study period. The cases positive for pandemic H1N1 virus had significantly higher risk of hospitalization than those positive for seasonal influenza-A viruses (OR: 1.7). Of 93 influenza related deaths, 57 and 8 deaths from Pune (urban) and 27 and 1 death from Pune (rural) were from pandemic H1N1 positive and seasonal-A positive cases respectively. The case fatality ratio 0.86% for pandemic H1N1 was significantly higher than that of seasonal-A (0.13%) and it was in category 3 of the pandemic severity index of CDC, USA. The data on the cumulative fatality of rural and urban Pune revealed that with time the epidemic is spreading to rural areas. Conclusions The severity of the H1N1 influenza pandemic is less than that reported for ‘Spanish flu 1918’ but higher than other pandemics of the 20th century. Thus, pandemic influenza should be considered as serious health threat and unprecedented global response seems justified.

Multisite virological influenza surveillance in India: 2004-2008.

Please cite this paper as: Chadha et al. (2011) Multi site Virological Influenza Surveillance in India: 2004–2008. Influenza and Other Respiratory Viruses 6(3), 196–203.

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.

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

Abstract 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.

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. J. Med. Virol. 88:51–57, 2016.

Natural infection by simian retrovirus-6 (SRV-6) in Hanuman langurs (Semnopithecus entellus) from two different geographical regions of India.

We have previously reported natural infection of Hanuman langurs (Semnopithecus entellus) from Lucknow, India by a novel simian retrovirus, SRV-6, a beta-retrovirus (type D retrovirus). Here we describe infection by a closely related SRV-6 in an isolated feral population of Hanuman langurs from Jodhpur in the Northwestern desert region of India. Serological analyses, using in-house ELISA and WB, genomic amplification, and sequencing of env region (gp70 and gp20) of the viral genome were carried out. SRV-6-infected langurs from the two regions were serologically cross-reactive. The env gene was used for phylogenetic analyses, being the most variable part of a retroviral genome. The surface glycoproteins (gp70) were almost identical between the two SRV-6 isolates and related to but distinct from equivalent regions from other exogenous SRVs. We could sequence the transmembrane glycoprotein gp20 from SRV-6 infecting the Jodhpur langurs, which was again shown to be related to but unique compared to the other known SRVs. The study suggests that natural infection by related strains of SRV-6 occurs in wild langurs from different parts of India.

Whole-Genome Sequence Analysis of Postpandemic Influenza A(H1N1)pdm09 Virus Isolates from India

ABSTRACT The pandemic influenza A(H1N1)pdm09 virus was first detected in India in May 2009 and continued to circulate in the postpandemic period. Whole-genome sequence analysis of postpandemic A(H1N1)pdm09 viruses showed the circulation of clade 6 and clade 7 viruses. The hemagglutinin (HA) gene showed increased diversity compared with that in the pandemic phase.

Respiratory viruses in acute exacerbations of chronic obstructive pulmonary disease.

Objective: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) cause significant morbidity, mortality, and an inexorable decline of lung function. Data from developed countries have shown viruses to be important causes of AECOPD, but data from developing countries like India are scant. We set out to determine the contribution of viruses in the causation of hospitalized patients with AECOPD. Methods: Twin nasopharyngeal/oropharyngeal swabs collected from 233 patients admitted with an acute AECOPD and tested for respiratory viruses including respiratory syncytial virus A and B, parainfluenza were (PIV) 1, 2, 3, and 4, human metapneumovirus (hMPV) A and B, influenza A and B, enterovirus, corona NL65, OC43, and 229E viruses, adenovirus 2 and 4, rhinovirus, and bocavirus, by duplex real time reverse-transcription polymerase chain reaction (qRT-PCR) using CDC approved primers and probes. Samples positive for influenza A were subtyped for A/H1N1pdm09 and A/H3N2 whereas influenza B samples were subtyped into B/Yamagata and B/Victoria subtypes, using primers and probes recommended by CDC, USA. Results: Respiratory viruses were detected in 46 (19.7%) cases, influenza A/H3N2 and rhinoviruses being the most common viruses detected. More than one virus was isolated in four cases consisting of hMPV-B + adeno-2 + Inf-B; rhino + H3N2, PIV-1 + rhino; and PIV-1+ hMPV-B in one case each. Ancillary supportive therapeutic measures included bronchodilators, antibiotics, steroids, and ventilation (noninvasive in 42 and invasive in 4). Antiviral therapy was instituted in influenza-positive patients. Three patients with A/H3N2 infection died during hospitalization. Conclusions: We conclude that respiratory viruses are important contributors to AECOPD in India. Our data calls for prompt investigation during an exacerbation for viruses to obviate inappropriate antibiotic use and institute antiviral therapy in viral disease amenable to antiviral therapy. Appropriate preventive strategies like influenza vaccination also need to be employed routinely.

An outbreak of influenza A(H3N2) in Alappuzha district, Kerala, India, in 2011

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.