Genetic sequencing of influenza A (H1N1) pdm09 isolates from South India, collected between 2011 and 2015 to detect mutations affecting virulence and resistance to oseltamivir.

BACKGROUND

Influenza A viruses evolve continuously and the two surface antigens, hemagglutinin (HA) and neuraminidase (NA) have been the target proteins for research as they are vital components in determining the virulence, immune effectiveness, pathogenicity, transmission and resistance.

METHODS

Both HA and NA (partial genes) of 45 pandemic influenza A(H1N1)pdm09 isolates were sequenced. Phylogenetic analysis was performed with reference to representative global isolates retrieved from Influenza Virus Resource (IVR), GISAID EpiFluTM and GenBank and evolutionary analyses. Nucleotide and amino acid sequences were aligned using ClustalW/ Clustal Omega/MEGA version 6 with reference to vaccine strain (A/California/07/2009).

RESULTS

All the isolates clustered along with the clade 7 virus, irrespective of the year of isolation. The study isolates exhibited 98.5% and 98.8% nucleotide homology to the reference strain A/California/07/2009(H1N1) for HA and NA, respectively. Overall, there was limited genetic diversity observed over a period of 3 years (2012-2015). Two samples collected from expired patients had D239N (D222G or D225G) mutation in HA. This mutation which is associated with dual-binding specificity of the virus has been well-correlated with severe disease outcomes. All the study isolates possessed H274 residue and 7 strains had N295S, the next most common mutation found in oseltamivir-resistant variants.

CONCLUSION

In this study, although H274Y mutation associated with oseltamivir resistance has not been noted, significant mutations have been noted in both HA and NA genes including D239N, N295S, V106I, Q136K, N248D, V267A. In both HA and NA gene analysis, multiple mutations were found more in 2015 strains when compared to 2012 strains. Hence such accumulation of mutations has to be monitored continuously to determine the efficacy of annual flu vaccines and anti-influenza drugs.