Molecular epidemiology and complete genome characterization of H1N1pdm virus from India.

BACKGROUND: Influenza A virus is one of world's major uncontrolled pathogen, causing seasonal epidemic as well as global pandemic. This was evidenced by recent emergence and continued prevalent 2009 swine origin pandemic H1N1 Influenza A virus, provoking first true pandemic in the past 40 years. In the course of its evolution, the virus acquired many mutations and multiple unidentified molecular determinants are likely responsible for the ability of the 2009 H1N1 virus to cause increased disease severity in humans. Availability of limited data on complete genome hampers the continuous monitoring of this type of events. Outbreaks with considerable morbidity and mortality have been reported from all parts of the country. METHODS/RESULTS: Considering a large number of clinical cases of infection complete genome based sequence characterization of Indian H1N1pdm virus and their phylogenetic analysis with respect to circulating global viruses was undertaken, to reveal the phylodynamic pattern of H1N1pdm virus in India from 2009-2011. The Clade VII was observed as a major circulating clade in phylogenetic analysis. Selection pressure analysis revealed 18 positively selected sites in major surface proteins of H1N1pdm virus. CONCLUSIONS: This study clearly revealed that clade VII has been identified as recent circulating clade in India as well globally. Few clade VII specific well identified markers undergone positive selection during virus evolution. Continuous monitoring of the H1N1pdm virus is warranted to track of the virus evolution and further transmission. This study will serve as a baseline data for future surveillance and also for development of suitable therapeutics

(A) Monthly trend of pandemic influenza A H1N1 and seasonal flu reported from September 2010 to December 2011.

<p>(B) Age and sex wise distribution of the influenza A H1N1 2010–2011 suspected ILI cases.</p

Details of the genome sequences of the H1N1pdm virus isolates retrieved and investigated in the whole genome and complete HA gene based phylogenetic analysis in this study.

<p>Note: The isolates in bold font are sequenced in this study.</p

Phylogenetic tree of concatenated whole genome of representative global H1N1pdm viruses including four Indian viruses sequenced in this study generated by Bayesian method.

<p>Each strain is abbreviated with virus subtype, country of origin, strain name and year of isolation in parenthesis. Scale bar indicates number of nucleotide substitutions per site. The Indian isolates sequenced in this study are highlighted in different font in clade VII. Other Indian isolates are highlighted by solid diamond in respective clades. Each clade is defined by long branch and nodes supported by high Bayesian posterior probability (BPP) values (100%).</p

Details of positive cases for H1N1pdm virus during investigation of suspected samples from 2010–2011.

<p>Details of positive cases for H1N1pdm virus during investigation of suspected samples from 2010–2011.</p

Confirmation of H1N1pdm virus.

<p>(<b>A</b>) Microscopic photograph of healthy and Influenza A (H1N1pdm) virus infected Madin Darby Canine Kidney Cells. (<b>B</b>) Immunofluorescence assay. (<b>C</b>) Haemagglutination assay. (<b>D</b>) WHO CDC Real-Time PCR amplification. Real time amplification curve of positive clinical samples showing amplification of all four probes.</p

Description of major/important non-conservative and clade specific amino acid substitutions among the four Indian H1N1pdm virus (sequenced in this study) compared to prototype H1N1pdm strain (California/04/2009) and other Indian (A/Pune/NIV6447/2009) virus strain (sequenced previously).

<p>Note: Major non conservative changes involving basic to acidic amino acid are written in bold font and also underlined; The hydrophobic to hydrophilic amino acid substitutions and vice-versa are written in bold font. The substitutions involving charged residues to uncharged residues; cyclic to acyclic and vice versa are written in italics. The clade specific substitutions (NP:V100I; NA:V106I; NS1:I123V; HA:S220T, I338V) are written in normal font.</p>*<p>The residue position for the HA is the numbering considered inclusive of signal peptide.</p

Selection pressure analysis of HA protein (566 codons); NA protein (469 codons), M1 Protein (252 codons) and M2 Protein (97 codons) of H1N1pdm virus using SLAC, FEL,REL,MEME and FUBAR methods. (www.datamonkey.org).

<p>Note: The sites found under positive selection by atleast two methods are shown*. Site present in B-cell epitope region are highlighted in bold font.</p>*<p> <b>Significance value.</b></p><p>SLAC P value–0.5.</p><p>FEL P value- 0.25.</p><p>REL Bayes factor- 50.</p><p>MEME P value- 0.1.</p><p>FUBAR Posterior probability- 0.9.</p

Phylogenetic tree among H1N1pdm viruses generated by Bayesian method based on Full HA gene (1701 nucleotides).

<p>Each strain is highlighted with virus subtype, country of origin, strain name, year of isolation and accession number in parenthesis. Each clade is defined by long branch and nodes supported by high Bayesian posterior probability (BPP) values (90%). Scale bar indicates number of nucleotide substitutions per site.</p