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

Abstract

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.

Figure 1

Phylogenetic analysis of the NS gene. Phylogenetic analysis of the NS gene (nucleotides) of seasonal influenza A (H1N1/H3N2) strains circulating in eastern India with respect to allele A and allele B gene pool [A/mallard/Gloucestershire/PD374/1985(H10N4) and A/Mallard Duck/ALB/302/1977(H10N7)], concurrent vaccine and other worldwide strains during 2007-2009 was carried out taking B/Lee/40 as an out-group. The tree was created by using neighbor-joining method and bootstrapped values of ≥70% were given for each node. The 2007 and 2009 A/H1N1 eastern India strains with A/Puerto Rico/8/1934(H1N1)-like NS gene are highlighted (♦).

Figure 2

Alignment of deduced amino acid sequences of NS1 protein. Alignment of deduced amino acid sequences of NS1 protein of 2009 seasonal Eastern India A/H1N1 strains with respect to the prototype [A/Puerto Rico/8/34(H1N1)] and concurrent vaccine strain [A/Brisbane/59/2007(H1N1)] was carried out. The five 2007 and 2009 seasonal H1N1 Eastern India strains having PR8-like NS1 protein are indicated (*).

Figure 3

Result of ConSurf server. ConSurf server identified the conserved regions among the deduced amino acid sequences of NS1 and NS2 protein of eastern India strains, with respect to concurrent vaccine strains [A/Brisbane/59/2007(H1N1) and A/Brisbane/10/2007(H3N2)]. The degree of conservation was subdivided into nine grades, with grade 1 being the least and grade 9 being most conserved. Figure 3A, 3B and 3C, 3D represents the NS1 and NS2 protein of A/H1N1 and A/H3N2, respectively. Although amino acid residues especially at positions 4, 21, 22, 44, 53, 59, 60, 103 and 106 were conserved among the vaccine strains as well as all other eastern India strains, ConSurf server predicted the lowest score for these amino acid residues suggesting them as highly variable residues of grade 1.

Figure 4

Alignment of deduced amino acid sequences of NEP/NS2 protein. Alignment of deduced amino acid sequences of NEP/NS2 protein of 2009 A/H1N1/2009 strains with respect to the prototype [A/Puerto Rico/8/34(H1N1)] and concurrent vaccine strain [A/Brisbane/59/2007(H1N1)] was carried out. The five 2007 and 2009 seasonal H1N1 eastern India strains having PR8-like NEP/NS2 protein are indicated (*).

Figure 5

Phylogenetic analysis of PB1-F2 gene. Phylogenetic analysis of PB1-F2 gene (nucleotide) of seasonal inf A (H1N1/H3N2) strains circulating in eastern India with respect to representative vaccine, prototype, pH1N1/2009 and concurrent A/H1N1 strains reported worldwide was carried out taking B/Lee/40 as an out-group. The tree was created by using neighbor-joining method and bootstrapped values of ≥70% were given for each node. The 2007 A/H1N1 eastern India strains which showed A/H3N2-like PB1-F2 are highlighted (♦).

Figure 6

Alignment of deduced amino acid sequences of PB1-F2 protein. Alignment of deduced amino acid sequences of PB1-F2 protein of 2007 seasonal Eastern India A/H1N1 strains with respect to the prototype [A/Puerto Rico/8/34(H1N1)] and concurrent vaccine strains [A/Brisbane/59/2007(H1N1); A/Wisconsin/67/2005(H3N2)] was carried out. The five 2007 seasonal H1N1 eastern India strains having A/H3N2-like PB1-F2 protein are indicated (*).

Figure 7

Phylogenetic analysis of PB1 gene. Phylogenetic analysis of PB1 gene (nucleotide) of seasonal inf A (H1N1/H3N2) strains circulating in eastern India with respect to representative vaccine, prototype, pH1N1/2009 and concurrent A/H1N1 strains reported worldwide was carried out taking B/Lee/40 as an out-group. The tree was created by using neighbor-joining method and bootstrapped values of ≥70% were given for each node. The 2007 A/H1N1 eastern India strains which showed A/H3N2-like PB1are highlighted (♦).