浙江省2008-2012年肠道病毒相关病毒性脑炎病原谱及分子流行病学特征分析

摘要

目的 了解2008-2012年浙江省肠道病毒相关病毒性脑炎病原谱及分子流行病学特征.方法 从监测点采集疑似病毒性脑炎患者脑脊液和粪便样本利用RD和Hep-2细胞分离病毒,采用肠道病毒标准血清定型,并对分离株VP1基因测序,进行同源性与进化分析.结果 从610例患者616份样本中分离到人类肠道病毒(HEV) 127株(20.6％),其中柯萨奇病毒(CV)60株,埃可病毒(ECHO:E) 67株,病毒血清型分别为CVA9、CVBl 、CVB3 ～5、E3、E4、E6、E9、E14、E25、E30.2008-2012年优势株分别为CVB3、CVB5、E6、E30和E30.各分离株VP1基因序列全长834 ～918个核苷酸,与相应原型株核苷酸(nt)和氨基酸(aa)的同源性分别为76.7％～85.0％和91.1％～ 97.9％;浙江分离株型内差异最大为E6,nt和aa差异分别为20.4％和4.8％.基于VP1基因的进化分析结果表明,浙江分离株均位于HEV-B分支上,并显示出一定地域和时间效应;E6血清型内部又分成2小分支.结论 2008-2012年浙江省病毒性脑炎的主要病原为HEV-B,涉及12个血清型;不同年份优势流行株不断变化,E30为绝对优势株;浙江E6分离株存在2个亚类流行株.%Objective In order to investigate etiology and molecular-epidemiological characteristics of enterovirus associated encephalitis (EAE) in Zhejiang,2008-2012.Method Cerebrospinal fluid and stool specimens were collected from suspected EAE patients,who were admitted to our hospitals.RD and Hep-2 cell lines were used to isolate enterovirus (EV).Serotypes of these EV isolates were identified through neutralization test by using serotype specific anti-sera.VP 1 genes of these isolates were sequenced,compared and used for the construction of phylogenetic tree.Results 127 (20.6％) human enterovirus (HEV) strains were isolated from 616 samples,which were collected from 610 patients.Serotypes of these EV isolates,including 60 coxsackievirus (CV),and 67 Echovirus (E) appeared to be CVA9,CVB1,CVB3-5,E3,E4,E6,E9,El4,E25 and E30,respectively.Predominant EV serotypes on EAE from 2008 to 2012 were seen as CVB3,CVB5,E6,E30 and E30,respectively.The full length of VP1 genes from different EV isolates was between 834 and 918 nucleotides.The VP1 gene similarities between these isolates and the reference strains were from 76.7％ to 85.0％ (nucleotides level) and 91.1％ to 97.9％ (amino acids level).The VP1 genes from E6 serotype isolates appeared most diverged,reaching 20.4％ (nucleotides level) attd 4.8％ (amino acids level).Based on the generated phylogenetic tree,all the EV isolates were fallen on the same branch of HEV-B,and the isolates in the same serotype formed one sub-branch,suggesting there existed geographical and temporal effects.E6 isolates diverged into two branchlets.Conclusion EVs from HEV-B were the etiologic agents for EAE in Zhejiang province from 2008 to 2012.All these EV isolates showed 12 serotypes,with predominant isolates varied every year.E30 was determined as the most dominant serotype while serotype E6 diverged into two sub-genetypes.