Surveillance in Eastern India (2007-2009) revealed reassortment event involving NS and PB1-F2 gene segments among co-circulating influenza A subtypes.

BACKGROUND: Influenza A virus encodes for eleven proteins, of which HA, NA, NS1 and PB1-F2 have been implicated in viral pathogenicity and virulence. Thus, in addition to the HA and NA gene segments, monitoring diversity of NS1 and PB1-F2 is also important. METHODS: 55 out of 166 circulating influenza A strains (31 H1N1 and 24 H3N2) were randomly picked during 2007-2009 and NS and PB1-F2 genes were sequenced. Phylogenetic analysis was carried out with reference to the prototype strains, concurrent vaccine strains and other reference strains isolated world wide. RESULTS: Comparative analysis of both nucleotide and deduced amino acid sequences, revealed presence of NS gene with A/PR/8/34(H1N1)-like mutations (H4N, Q21R, A22V, K44R, N53D, C59R, V60A, F103S and M106I) in both RNA-binding and effector domain of NS1 protein, and G63E, the HPAI-H5N1-like mutation in NEP/NS2 of five A/H1N1 strains of 2007 and 2009. NS1 of other A/H1N1 strains clustered with concurrent A/H1N1 vaccine strains. Of 31 A/H1N1 strains, five had PB1-F2 similar to the H3N2 strains; six had non-functional PB1-F2 protein (11 amino acids) similar to the 2009 pandemic H1N1 strains and rest 20 strains had 57 amino acids PB1-F2 protein, similar to concurrent A/H1N1 vaccine strain. Interestingly, three A/H1N1 strains with H3N2-like PB1-F2 protein carried primitive PR8-like NS gene. Full gene sequencing of PB1 gene confirmed presence of H3N2-like PB1 gene in these A/H1N1 strains. CONCLUSION: Overall the study highlights reassortment event involving gene segments other than HA and NA in the co-circulating A/H1N1 and A/H3N2 strains and their importance in complexity of influenza virus genetics. In contrast, NS and PB1-F2 genes of all A/H3N2 eastern India strains were highly conserved and homologous to the concurrent A/H3N2 vaccine strains suggesting that these gene segments of H3N2 viruses are evolutionarily more stable compared to H1N1 viruses.

Pandemic and seasonal influenza viruses among patients with acute respiratory illness in Kashmir (India).

BACKGROUND: With the emergence of pandemic influenza A (2009A/H1N1) virus in India, we sought to determine the prevalence and clinical presentations of seasonal and pandemic influenza viruses among acute respiratory illness (ARI) patients from Srinagar, a temperate climate area in northern India, during the peak winter season. METHODS: Combined throat and nasal swabs, obtained from 194 (108 male) presenting with ARI from January to March 2010 (Week 53-week 10), were tested by RT-PCR for influenza A and B, including 2009A/H1N1 viruses. HA1 gene of selected 2009A/H1N1-positive samples was sequenced, and phylogenetic analysis was carried out. RESULTS: Twenty-one (10·8%, age 15-80 years, median age 40 years) patients tested positive for influenza viruses: 13 (62%) for 2009A/H1N1 virus, 6 (28·5%) for seasonal influenza A (H3N2), and 2 (9·5%) for influenza B. Twelve of the 13 patients with 2009A/H1N1 presented with febrile ARI, and eight had associated comorbidities. All of the patients recovered. Phylogenetic analysis of HA gene (n = 8) revealed that all strains from Srinagar clustered in 2009A/H1N1 clade seven along with the other 2009A/H1N1 strains from India. Amino acid substitutions in the HA protein defining clade seven (P83S, S203T, and I321V) were found in almost all isolates from Srinagar. CONCLUSIONS: Both seasonal and 2009A/H1N1 viruses appear to be associated with ARI in Srinagar. The 2009A/H1N1 in Srinagar is genetically similar to globally circulating clade 7 strains, with unique signature sequences in the HA gene. Further investigations into ascertain the role of these mutations in possible alteration of the virulence and transmissibility of the virus are needed.

Molecular characterization and comparative analysis of pandemic H1N1/2009 strains with co-circulating seasonal H1N1/2009 strains from eastern India.

During the peak outbreak (July-September 2009), a total 1886 patients were screened in eastern India, of which 139 (7.37%) and 52 (2.76%) were positive for pH1N1 and seasonal H1N1, respectively. Full-length HA1, NA, NS1 and PB1-F2 genes of representative strains were sequenced. Phylogenetic analysis of deduced amino acid sequences of pH1N1 strains revealed HA1 and NS1 to be of North American swine lineage, and the NA gene of Eurasian swine lineage. Consistent with previous reports, the PB1-F2 gene of pH1N1 strains was unique due to a mutation resulting in a truncated protein of 11 aa. The HA, NA and NS1 genes of H1N1/2009 strains clustered with H1N1 strains of 2000-2009, whereas a subset of strains contained a pH1N1-like truncated PB1-F2. The truncated PB1-F2 may confer the advantage of lower pathogenicity but higher replication and infectivity to the human H1N1 strains. This is the first report of seasonal H1N1/2009 strains with a pH1N1/2009-like gene segment.

Genetic characterization of circulating seasonal Influenza A viruses (2005-2009) revealed introduction of oseltamivir resistant H1N1 strains during 2009 in eastern India.

Influenza surveillance was implemented in Kolkata, eastern India in 2005 to identify the circulating subtypes and characterize their genetic diversity. Throat and nasal swabs were collected from outpatients with influenza-like illness (ILI). Of 2844 ILI cases identified at two referral hospitals during October 2005-September 2009, 309 (10.86%) were positive for Influenza A by real time RT-PCR, of which 110 (35.60%) were subtyped as H1N1 and 199 (64.40%) as H3N2. Comparison of the nucleotide (nt) and amino acid (aa) sequences of the HA1 gene for H1N1 and H3N2 strains showed that a subset of strains precede WHO recommended contemporary strains by 1-2 years. The Kolkata H1N1 strains clustered in Clade II, subgroup 2B with A/Brisbane/59/2007 but were distant from the corresponding vaccine strains (New Caledonia/20/99 and A/Solomon Island/3/06). The 2005-06 and 2007 H3N2 strains (15/17) clustered either A/Brisbane/10/2007-like (n=8) or A/Nepal/921/2006 like (n=7) strains, whereas 2008 strains (8/12) and 2009 strains (4/4) were similar to the 2010-11 vaccine strain A/Perth/16/2009. More aa substitutions were found in HA or NA genes of H3N2 than in H1N1 strains. No mutation conferring neuraminidase resistance was observed in any of the strain during 2005-08, however in 2009, drug resistant marker (H275Y) was present in seasonal H1N1, but not in co-circulating H3N2 strains. This is the first report of genetic characterization of circulating Influenza A strains from India. The results also highlight the importance of continuing Influenza surveillance in developing countries of Asia for monitoring unusual strains with pandemic potential and mutations conferring antiviral resistance.

Comparative evaluation of real-time PCR and conventional RT-PCR during a 2 year surveillance for influenza and respiratory syncytial virus among children with acute respiratory infections in Kolkata, India, reveals a distinct seasonality of infection.

Acute respiratory tract infections (ARTIs) are one of the most common causes of morbidity and mortality in young children worldwide. Influenza virus and respiratory syncytial virus (RSV) are the predominant aetiological agents during seasonal epidemics, and thus rapid and sensitive molecular tests for screening for such agents and timely identification of epidemics are required. This study compared real-time quantitative PCR (qPCR) with conventional RT-PCR for parallel identification of influenza A virus (IAV) or influenza B virus (IBV) and RSV. A total of 1091 respiratory samples was examined from children with suspected ARTIs between January 2007 and December 2008. Of these, 275 (25.21 %) were positive for either influenza or RSV by qPCR compared with 262 (24 .01%) positive by RT-PCR. Overall, IAV, IBV and RSV were detected in 121 (11.09 %), 59 (5.41 %) and 95 (8.71 %) samples, respectively. In spite of overlapping clinical symptoms, RSV and influenza virus showed distinct seasonal peaks. IAV correlated positively and RSV negatively with rainfall and temperature. No distinct seasonality was observed in IBV infections. This is, to the best of our knowledge, the first report of a systemic surveillance of respiratory viruses with seasonal correlation and prevalence rates from eastern India. This 2 year comparative analysis also confirmed the feasibility of using qPCR in developing countries, which will not only improve the scope for prevention of epidemics, but will also provide crucial epidemiological data from tropical regions.

Prevalence of respiratory syncytial virus group B genotype BA-IV strains among children with acute respiratory tract infection in Kolkata, Eastern India.

BACKGROUND: Although recognized as a significant respiratory pathogen, there is a dearth of information on genetic diversity of human respiratory syncytial virus (HRSV) strains among children in India. OBJECTIVES: To study prevalence and genomic diversity of HRSV strains among children (<5 years of age) with acute respiratory tract infection (ARTI) at Dr. B.C. Roy Memorial Hospital for Children, Kolkata, Eastern India. STUDY DESIGN: During September 2005 to August 2008, nasal and/or throat swabs from children with ARTI were screened for presence of HRSVs by RT-PCR of nucleocapsid (N) and glycoprotein (G) genes. Classification of G gene of HRSV strains were achieved with different primer pairs designed for amplification of N'- and/or C'-terminal hypervariable regions (HVR1 and HVR2) of HRSV A and B strains. The HVR1 and HVR2 of G gene of 27 and 22 HRSV B strains were sequenced. RESULTS: One hundred seventy seven of 1720 clinical samples were positive for HRSVs. Group B strains were detected at higher rates (95% against 5%, n=80) than A in 2005-2006, whereas in consecutive years, the rate of detection of group A were higher (94.84% against 5.16%, n=97). The group B strains were genetically related to globally spreading BA genotype, exhibiting conserved nature of stop codon, six nucleotide deletions in HVR1 and formed single phylogenetic clusters for both HVR1 and HVR2. CONCLUSIONS: The detection of significant high rates of group B strains in 2005-2006 followed by increase in prevalence of A strains in subsequent years highlight the dynamic nature of prevalence of HRSV subtypes among children with ARTI in Eastern India.