无菌性脑膜炎暴发中柯萨奇B3病毒的基因特征分析

目的 明确2008年山东省临沂市郯城县无菌性脑膜炎暴发的病原,对分离到的柯萨奇B3病毒的基因特征、遗传变异规律及进化来源进行分析.方法 采集暴发病例的粪便、脑脊液标本,应用RD、Hep-2细胞进行病毒分离;阳性分离物分别采用中和试验、逆转录-聚合酶链式反应(RT-PCR)和核苷酸序列测定方法进行定型,最后与GenBank中检索到的其他CVB3毒株进行同源性分析,并构建VP1完整编码区亲缘进化树研究其遗传进化规律.结果 共采集22份粪便和120份脑脊液标本,分离到病毒35株,经鉴定34株为CVB3,1株为ECH030.34株CVB3分离株VP1区部分序列(381 bp)核苷酸同源性为90.5%～100%,氨基酸同源性为98.4%～100%;与Nancy原型株的核苷酸同源性为79.5%～81.6%,氨基酸同源性为98.4%～99.2%.012/2008TC/SD/CHN和177/2008TC/SD/CHN与安徽2008年分离的Fuyang19株同源性最高,核苷酸同源性分别为98.2%和91.0%,氨基酸同源性分别为99.2%和98.9%;进化树分析显示,中国大陆分离株独处一支,且呈单源性发生关系.结论 此次脑膜炎暴发的病原是CVB3.它与其他的中国分离株同源性高,与国外分离到的CVB3具有不同的基因特征.

Molecular characterization of coxsackievirus B3 isolated from an outbreak of aseptic meningitis in Shandong province,China

Objective To identify the pathogen caused an outbreak of aseptic meningitis in Tancheng county of Shandong province in 2008,and to analyze the molecular characterization of VP1 gene of the Coxsackievirus B3(CVB3)isolates.Methods Stool and cerebrospinal fluid(CSF)specimens were collected from this outbreak for virus isolation with RD and Hep-2 cell.After typing by neutralization test.the VP1 gene of the isolates were amplified by RT-PCR and sequenced.Homologous comparison and phylogenetic analysis were performed.Results 35 strains of enteviruse were isolated from 22 stools and 120 CSFs(7 from stools and 28 from CSFs),34 strains identified as CVB3 and 1 as Echovirus 30(ECHO30)by neutralization test.The nucleotide homologies were 90.5%-100.0% in the partial VP1 gene(381 bp)mnong 34 CVB3 isolates.Homology comparisons indicated that Shandong strains have the identity of 79.5%-81.6% with the CVB3 prototype strain Nancy.012/2008TC/SD/CHN and 177/2008TC/SD,CHN showed the highest nucleotides homologies(98.2% and 91.0% respectively)with Fuyang19 strain of Anhui province in 2008 in complere VP1 gene.The phylogenetic tree based on complete VP1 genes showed that all the CVB3 correlated with aseptic meningitis in China recently came from the same evolution linkage and formed a monophyletic cluster.Conclusion The causative agent of this outhreak of aseptic meningitis wag CVB3.CVB3 circulated in China wag genetically different from other countries.