The complete nucleotide sequence of the NS gene and partial sequence of PB1 gene segment encoding full-length PB1-F2 of representative influenza A (H1N1/H3N2) positive samples collected from the out-patient departments (OPDs) of local hospitals were compared with the concurrent influenza A (H1N1/H3N2) strains, circulating worldwide. Cumulative point mutations and reassortment events due to segmented RNA genome contribute to continuous genetic and antigenic variation in circulating influenza viruses resulting in seasonal epidemics. Unlike HA and NA surface glycoproteins, mutations in the NS genes appeared to be sequential, suggesting that reassortment has probably not contributed significantly to the evolution of the NS gene of these human viruses which is in agreement with previous studies [28, 29]. In addition, due to relatively conserved nature of NS gene, reassortment events may have precluded detection. In 1978 recombinant H1N1 viruses with P1, P2, P3 and NP genes from H3N2 still carried HA, NA, M, and NS gene from parent H1N1 subtype . However, significant differences in the NS genes of the influenza A H1N1 and H3N2 subtypes during this study were identified, which allowed detection of an NS gene reassortment .
With respect to PB1-F2 protein coding region, five out of thirty-one H1N1 strains (2007-2009) with functional PB1-F2 were evolutionarily close to co-circulating A/H3N2 strains, whereas, corresponding NS gene showed H1N1 origin (Figure 1 and 5). Presumably these five 2007 H1N1 strains arose by reassortment between co-circulating H1N1 and H3N2 viruses in the region. For confirmation HA and NA genes (partial) and M1 (full length) were sequenced, which on analysis confirmed nucleotide identity with A/H1N1 strains. The PB1 gene segment of these strains, however, clustered with A/H3N2 strains suggesting that although these viruses were of H1N1 origin, they probably had derived PB1 segment from an H3N2 virus. The significance of selectively lateral transmission of PB1-F2 gene among co-circulating strains is not clear, but since PB1-F2 protein is associated with pathogenesis, it may confer improved infectivity or replication efficiency. As reported earlier by our group , six A/H1N1 strains had truncated 11 aa PB1-F2 similar to 2009 pH1N1 viruses. Rest twenty A/H1N1 strains with 57 aa PB1-F2 peptide were similar to the concurrent A/H1N1 vaccine strains (Figure 6).
Surprisingly, NS nucleotide sequences of five A/H1N1 strains was highly homologous (>97%) with the 1934 prototype strain [A/Puerto Rico/8/34(H1N1)] (Figure 2). In addition, these five strains contained G63E substitution in NEP, similar to the highly pathogenic avian influenza H5N1 viruses, which may confer higher pathogenicity . To verify possible cross contamination, BLAST search of HA, NA, M1 and NS1 gene sequences showed only NS1 having sole identity with A/PR/8/34(H1N1). Thus, a chance of cross contamination with laboratory PR8 strain was ruled out. Though the frequency of vaccination in India is very low but since the WHO approved vaccines with PR8 backbone are used, possibility of reassortment with the vaccine strain can not be ruled out. Moreover, 3 out of five PR8-like NS1 carrying A/H1N1 2007 strains, had H3N2-like PB1-F2 gene, whereas, 2/5 had non-functional PB1-F2 similar to pandemic A/H1N1 strains of 2009 (Figure 1 and 5). Thus the circulation of prototype NS gene carrying A/H1N1 strains in 2007 and 2009, with PB1-F2 gene from diverse origin underlines the complexity of influenza virus genetics and evolution. In contrast to A/H1N1 strains, all A/H3N2 (n = 24) strains analyzed in this study revealed highly conserved NS and PB1-F2 gene, with >98.5% homology to concurrent A/H3N2 strains circulating worldwide.