长沙A(H1N1)亚型季节性流感暴发疫情的实验室诊断及其HA基因特性分析

An Outbreak of Seasonal Influenza Viruses A(H1N1) in Changsha Was Diagnosed by Laboratory and the HA Gene Characteristic Was Analyzed

对2009年长沙麓山国际学校流感暴发疫情进行实验室诊断，并探索新分离的A（H1N1）亚型流感病毒血凝素（HA）的基因特性。对流感暴发疫情的25份鼻／咽拭子标本进行RT—PCR检测和流感病毒分离，然后利用CEQ8000Genetic Analysis System对病毒分离株（A／Yuelu／314／2009）进行测序，测序结果提交至GenBank（登录号：FJ912843）并用ClustalX和Mega4．1软件进行序列分析。结果显示，分离出A（H1N1）亚型流感毒株18株，检出21份A（H1N1）亚型流感病毒核酸阳性：A／Yuelu／314／2009（H1N1）HA基因序列与2008～2009年疫苗株（A／Brisbane／59／2007）比较显示：核苷酸和氨基酸同源性均为99％，有6个位点的氨基酸发生了变异（V148A、S158N、G202A、1203D、A206T、W435R），其中一个S158N氨基酸变异位于B抗原表位，HA基因序列上共有潜在糖基化位点9个（27、28、40、71、151、176、303、497、536），与A／Brisbane／59／2007相同且氨基酸序列保守。本实验诊断出此次流感暴发疫情的病原体为A（H1N1）型季节性流感病毒，研究还发现A／Yuelu／314／2009（H1N1）长沙分离株与A／Brisbane／59／2007疫苗株基因序列比较显示并未形成一个新的变种，推测是由于分离株与疫苗株之间基因特性的改变和人群对A（H1N1）亚型流感病毒免疫力降低导致了此次长沙麓山国际学校A（H1N1）,亚型流感的暴发。

To determine the etiologic agent of an outbreak of influenza viruses from Changsha Foothill Mountain International School in 2009, and to analyze the HA Gene Characteristic of the H1N1 influenza viruses. Twenty-five nasopharyngeal swab specimens from the outbreak of influenza viruses were tested by conventional RT-PCR and influenza viruses isolated simultaneously. Virus isolated （A/Yuelu/314/2009） from the outbreak was sequenced by CEQTM 8000 Genetic Analysis System and the sequencing results submitted to GenBank （Accession No： FJ912843）, then the sequencing data was analyzed by ClustalX and Mega4.1 softwares. Results showed the influenza viruses A（H1N1） of positive were 18 cases by influenza viruses isolated tests and 21 cases by conventional RT-PCR, respectively. The nucleotide and amino acid sequence homology of the HA gene of A/Yuelu/314/2009 are 99% compare with the vaccine strain （AfBrisbane/59/2007） in 2008-2009 years. The HA sequence data also showed that had 6 amino acid mutations （V148A, S158N, G202A, I203D, A206T, W435R）, and the S158N located at antigenic site B of HA protein. Nine potential glycosylation sites （27, 28, 40, 71, 151, 176, 303, 497, 536） in the HA sequence of A/Yuelu/314/2009 is the same with A/Brisbane/59/2007, and the sequences of potential glycosylation sites were conserved. In this study, laboratory evidence diagnosed seasonal influenza A virus （H1N1） as the etiologic agent of the outbreak. The virus isolated （A/Yuelu/314/2009） strain of H1N1 subtype is not a new variant in Changsha in 2009 compare with the vaccine strain （A/Brisbane/59/2007）, the outbreak of influenza A virus （H1N1） from Changsha Foothill Mountain International School maybe are caused by the change in genetic characteristics between vaccine strains and the decreased of immunity to influenza A virus （H1N1） in the crowd.