Study On The Characteristic Within The Whole Genome Of Influenza Virus Subtype A/H3N2 Strains Circulated In Zhejiang Province During 1998 To 2009

ObjectiveTo analyze the relationship between the influenza epidemic and the genetic characteristic, including mutations of amino acid, alteration of glycosylation sites, evolution condition and the potential antigenic region, on the whole genome of influenza virus subtype A/H3N2 strains isolated in Zhejiang province during 1998 to 2009. To study the consistency of mutations condition between the whole genome and HA gene of influenza virus subtype A/H3N2 strains.MethodsThe sequences of whole genome of 19 Zhejiang Influenza virus isolates circulated during 1998 to 2009, which conserved by influenza laboratory of Zhejiang provincial Centre for Disease Prevention and Control, were amplified using RT-PCR assays. After sequenced, sequence of whole genome were spliced, compared and analyzed by using bioinformatics software including MEGA4.0, DNAStar5.0.221.0, BioEdit5.0.6 and PAML3.15. The obtained sequences were used to conduct phylogenetic analysis with 10 contemporaneous vaccine strains. Three methods, including comparison of the amino acid substitutions, calculation of the entropy value and the filtering of positive selection sites, were used to confirm the mutable sites on each gene.ResultsThe highest mutation happened within HA and NA genes and the amino acid divergent ratios were 13.98% and 10.00%. Amongst the six internal proteins, the amino acid divergent ratios of NP,M2 and NS1 were 6.43%,6.19% and 3.48% respectively, and the others were lower than 3%. There still were 29% and 77% mutations of HA and NA genes occurred on the non-epitope regions respectively. Within the internal genes, parts of the amino acid sites were also prone to mutation. Based on almost all of genes, the homology divergence between Zhejiang Influenza virus strains and contemporaneous vaccine strains in 1998 and 2002 is bigger than that between Zhejiang epidemic strains in 2007-2009. Analysis on the complete sequence was more comprehensive than the HA gene to reflect the genetic relationship and law of evolution among the influenza virus subtype A/H3N2 strains. the phenomenon of genetic recombination is showed on the phylogenetic tree of NP gene and other internal genes.Conclusion 1 A new round influenza epidemic will be caused by a new strain whose key sites of HA gene and internal genes were mutated together.2 A growing trend is showed on glycosylation sites of HA and NA genes. For the different genes or the different regions on the same gene, alteration of glycosylation sites may play different roles to the genetic characteristic of influenza strains.3 There is no transition of lineages had been observed based on Zhejiang epidemic strains. Influenza virus subtype A/H3N2 strains may not follow the laws of the periodic variation.4 All of the eight genes are presented in different degrees of variation when the influenza prevailed widely. The time series and tendency of regional aggregation can be reflected more comprehensively by phylogenetic tree which constructed based on the complete sequence, besides, the law of evolution among the influenza virus subtype A/H3N2 strains can be reflected by the phylogenetic trees which constructed based on both HA sequence and complete sequence.5 Except NS gene, there was an obvious genetic lag between vaccine strains recommended by the WHO and the contemporary Zhejiang epidemic strains within other genes. Vaccine strains can't protect our people very well.6 The potential antigenic sites on Influenza virus subtype A/H3N2 mainly including 3, 225, 361 aa of HA gene; 93, 143, 147, 150, 372 aa of NA gene; 113, 576, 586 aa of PB1 gene; 101, 256, 382, 421, 437 aa of PA; 377, 450 aa of NP gene; 218 aa of M1 gene and 31 aa of M2 gene.7 Based on the whole genome, evolutionary rate of influenza virus subtype A/H3N2 strains was high in the years when the influenza subtype A/H3N2 prevailed widely.