2009～2014年度浙江南部呼吸道合胞病毒分子流行病学研究

A molecular epidemiological study of respiratory syncytial virus circulating in southern Zhejiang Province,China, from 2009 to 2014

目的了解浙江南部呼吸道合胞病毒(RSV)基因型流行趋势,并研究A亚型G蛋白基因特征。方法采集2009年7月至2014年6月浙江南部3家医院因肺炎和毛细支气管炎住院的5岁以下儿童下呼吸道分泌物,应用直接免疫荧光法检测RSV抗原。在每个流行年度(7月至次年6月)RSV阳性标本中随机抽取200份,应用RT-PCR方法检测RSV亚型,扩增A亚型G蛋白近全基因序列并测序,鉴定其基因型。结果 2009～20145个年度共采集25 449份下呼吸道分泌物标本,其中6 416份(25.21%)RSV阳性。随机抽样的1 000份RSV阳性标本中检出A亚型462株(46.2%),B亚型538株(53.8%),A亚型依次占各年度的22.5%、74.5%、84.5%、19.0%、30.5%。每年度随机抽样25株RSV A亚型病毒株外送双向测序,结果获得A亚型阳性52株,其中得到4种A亚型基因型,包括NA1(39株)、NA4(1株)、ON1(10株)、GA2(2株)基因型,其中NA1是2009～2012年度的优势基因型,ON1是2013～2014年度A亚型的唯一基因型。A亚型G蛋白基因与原型株A2的核苷酸同源性为80.7%～89.3%,氨基酸同源性为74.4%～82.6%;分离株之间的核苷酸及氨基酸同源性分别为81.5%～100%和80.2%～100%。结论 2009～2014年度浙江南部地区出现RSV A、B亚型共循环,A亚型有多个不同的基因型共循环,其G蛋白基因变异明显。

Objective To find out the prevalence of respiratory syncytial virus（RSV） genotypes in southern Zhejiang Province, China, and to study the genetic characteristics of G protein from subtype A of RSV. Methods The lower respiratory tract secretions of children under 5 years of age who were hospitalized for pneumonia and bronchiolitis in three hospitals in southern Zhejiang Province from July 2009 to June 2014 were collected. Direct immunofluorescence assay was used to detect RSV antigens from the collected secretions. A total of 200 samples were randomly selected from RSV-positive specimens in each prevailing year（from July of a specific year to June of the next year）. RT-PCR was used to determine RSV subtypes, and the near-full length gene sequence of G protein from subtype A was amplified and sequenced to identify the genotype. Results A total of 25 449 samples of lower respiratory tract secretions were collected from 2009 to 2014, among which 6 416（25.21%） samples were RSV-positive. Among the 1 000 RSV-positive specimens randomly sampled, 462 strains（46.2%） were subtype A, and 538 strains（53.8%） were subtype B. Subtype A accounted for 22.5%, 74.5%, 84.5%, 19.0%, and 30.5% of the total strains in each year from 2009 to 2014. A total of 25 RSV subtype A strains were randomly sampled and sent out for bidirectional sequencing in each year, which confirmed 52 positive subtype A strains. Four genotypes of subtype A strains were obtained from the above strains, including NA1（39 strains）, NA4（1 strain）, ON1（10 strains）, and GA2（2 strains）. NA1 was the dominant genotype between 2009 and 2012,and ON1 was the only genotype of subtype A during 2013-2014. The nucleotide homology and amino acid homology between the G protein of subtype A and the prototype strain A2 were 80.7%-89.3% and 74.4%-82.6%, respectively. The nucleotide homology and amino acid homology between the isolates of subtype A were 81.5%-100% and 80.2%-100%, respectively. Conclusions In southern Zhejiang Province from 2009 to 2014, there was a co-circulation of RSV subtypes A and B, as well as a co-circulation of several different genotypes of RSV subtype A, which had highly variableG protein genes.