Study On Molecular Epidemiological Characteristics Of2009 Pandemic Influenza A(H1N1) Virus In Fujian Province, China During 2009~2014

Objective: In order to elucidate the genetic variation and molecular evolution of 2009 pandemic influenza A(H1N1)(influenza A(H1N1)pdm09) virus, to understand its future epidemic trend, to evaluate the effect of the vaccine and to provide scientific evidence for prevention of the new outbreak, this study was carried out to analyze the molecular epidemiological characteristics of influenza A(H1N1)pdm09 virus from 2009 to 2014 in Fujian Province combined with its regular surveillance data.Methods: A total of 106 influenza A(H1N1)pdm09 virus strains isolated from patients with influenza-like illness(ILI) in 17 sentinel hospitals in Fujian Province were collected. The entire sequences of hemagglutinin(HA) and neuraminidase(NA) were obtained by using PCR and sequencing technique. The bioinformatical and statistical softwares were applied to align and analyze all the sequences. Comapred with the international vaccine strain A/California/07/2009(H1N1), the molecular epidemiology of influenza A(H1N1)pdm09 in Fujian Province was analyzed from several aspects.Results: 1、 The influenza A(H1N1)pdm09 has been presenting high or low prevalent repeatedly in Fujian during the period of 2009~2014. The magnitude of epidemic became larger in 2013 and 2014, however, it was much smaller in the second half of 2014 which will probably trigger a new epidemic outbreak. 2、 Homology analysises revealed that the nucleotide and amimo acid homologies of HA in the influenza A(H1N1)pdm09 virus from Fujian were 97.6-99.6% and97.2-99.1%, and 98.7-99.6% and 97.4-99.6% for NA compared with the standard vaccine strain respectively. Both of HA-based and NA-based phylogenetic trees were gradually clustered into six distinct clades as times went on. The substitutions of S143 G, H138 R, V249 L, V234 I, K163 Q, S185 T and S451 N in HA together with K432 E and V241 I in NA were speculated to be the significant reasons for all the virus isolate’s clustering into different clades, among them, S143 G and H138 R were located on and near Ca antigenic site, and K163 Q and S185 T were located on Sa and Sb antigenic sites, respectively. 3、 Antigenicity analysis demonstrated that 97 amino acid substitutions were found in the HA protein, among which there were 17 amino acid changes locating in total of 4 antigenic epitopes. Moreover, some mutations were presented in the RBD of HA, with the 190 helix and the 220-loop included. More than 10 substitutions of amino acid have been found since 2012, which resulted in larger variation of antigenicity, and antigenic drift have been found as well. There were significant statistic differences in the substitutions at position 34, 44, 200, 241, 321, 369, 386 and 432 between antigenic drift isolates and non-antigenic drift isolates. 4、 All the strains investigated showed low pathogenicity, however, the mutations of D222 G and Q293 H might probably enhance viral virulence. 5、 The oseltamivir resistance mutation H275 Y, together with simultaneous mutations of V241 I and N369 K, which might enhance the surface expression and enzymatic activity of NA protein in H275 Y were detected. 6、 It was found that NKS162-164 or NQS162-164 on Sa antigenic site were emerged as new glycosylation sites of HA in three isolates, of which the latter two isolates were derived from the outbreak epidemic. Moreover, some strains had acquired the glycosylation site NQS42-44, which was considered to be associated with the oseltamivir-resistance. The lack of glycosylation site NGT146-148 of NA, which might have an effect on viral tissue invasion especially on neural invasion, was also detected in the isolate of A/Fujianfuzhou/SWL11724/2009(H1N1).Conclusions: The genetic variation and molecular evolution have been occurred in influenza A(H1N1)pdm09 virus in Fujian Province. Compared with vaccine strine, the genetic homologies of HA and NA genes of influenza A(H1N1)pdm09 virus in Fujian were gradually reduced as time went on. Antigenic drift was found in 51 isolates among all the 106 isolates, of which 8 positions of mutations in NA, probably associated with the antigenic drift were discovered, and the oseltamivir resistance mutation H275 Y was found as well. It is forecasted that the new outbreak of influenza A(H1N1)pdm09 may happen in Fujian Province in future.