福州市甲型H1N1流感病毒血凝素基因遗传特性研究

Genetic characterization of the hemagglutinin gene of 2009 H1N1 influenza viruses isolated in Fuzhou,Fujian

目的探究福州市甲型H1N1流感病毒血凝素(hemagglutinin,HA)基因的遗传变异特点及种系进化分布,为研究甲型H1N1流感病毒进化、致病性和流行规律提供科学依据。方法采用MDCK细胞培养法和real-time PCR法进行流感病毒分离、鉴定。采用逆转录-聚合酶链反应扩增流感病毒血凝素基因,扩增产物经纯化后,进行血凝素基因HA区核苷酸序列测定并推导出其氨基酸序列,然后用DNASTAR MegAlign序列分析软件进行基因种系进化特性分析。结果福州市甲型H1N1流感病毒与参考株A/California/04/2009相比具有高度同源性,HA蛋白有4个位点氨基酸发生了替换,与猪流感代表株A/swine/Iowa/15/1930(H1N1)存在6个HA抗原决定簇位点变异。福州市甲型H1N1流感病毒株都有8个糖基化位点,其中6个位于HA1区,分离株HA蛋白不具有多碱基裂解位点,具有低致病性流感病毒特点。与美国流感病毒A/California/04/2009(H1N1)株共聚类为一个独立的小分支,进化关系最近,甲型H1N1流感病毒的HA源于古典型猪H1N1流感病毒,与A/swine/Iowa/1/1976(H1N1)的种系关系密切。结论福州市首例甲型H1N1流感病毒株与北美流行株高度同源,相对于以往古典型猪流感代表株出现HA蛋白抗原性漂移,应进一步加强流感病原学变异监测。

Objective To understand the variation characteristics and evolutionary distribution of hemagglutinin gene among H1N1 influenza viruses isolated in 2009.Methods The H1N1 influenza virus was isolated from patients by MDCK cell culture and identified by real-time PCR assay.The hemagglutinin（HA） gene of H1N1 influenza viruses was sequenced for the representative strains in Fuzhou.Finally,the phylogenetic tree was generated and analyzed with DNASTAR MegAlign soft ware.Results The H1N1 influenza virus isolated in Fuzhou had four amino acid substitutions in HA region compared with that of the reference viral strain of A/California/04/2009.The isolated viral strains had 6 antigenic determinant cluster variations and 8 glycosylation sites,6 of which were located in HA1 region and compared with the representative strain of swine influenza,A/swine/Iowa/15/193.The isolated viral strains did not contain the multiple basic amino acids at cleavage sites,indicating the low pathogenicity of influenza virus.Conclusions The influenza HlN1 viral strains isolated in Fuzhou were highly homologous with the epidemic strains in North America.The antigenic drift was confirmed in the HA region of the isolated viruses.Continuous monitoring of gene variation is important for the prevention of flu epidemic and viral pathogenic modification.