柯萨奇B组1型病毒株MSH-KM9-09的分离鉴定及其VP1基因序列特征分析

Isolation,identification and character of VP1 gene sequence of MSH-KM9-09 strain of Coxsackievirus B1

目的对2009年昆明市无菌性脑膜炎的病原柯萨奇B组1型病毒(Coxsackievirus B1,CVB1)分离株MSH-KM9-09进行鉴定,并分析其VP1基因的序列特征。方法采用RD细胞和Hep-2细胞对22份疑似无菌性脑膜炎患者脑脊液标本进行病毒分离,采用微量中和试验,经WHO肠道病毒组合血清及单价血清对分离株进行鉴定,RT-PCR法扩增病毒全长VP1基因,进行序列测定,并采用Mega 4.1等软件进行分析。结果所分离的毒株经微量中和试验定型为CVB1亚型,编号为MSH-KM9-09。经RT-PCR扩增获得834 bp的VP1基因,与国内CVB1分离株核苷酸和氨基酸序列的同源性分别在86.1%和97.8%以上,与国外CVB1分离株的核苷酸和氨基酸序列同源性分别为80.2%～83.2%和94.3%～96.0%;在系统进化树上,MSH-KM9-09株与国内分离株属于同一个分支,而国外分离株分属其他两个不同的分支。结论 MSH-KM9-09分离株为肠道病毒CVB1。

Objective To identify the MSH-KM9-09 strain of Coxsackievirus B1（CVB1）causing aseptic meningitis in Kunming City,China in 2009,and analyze the character of its VP1 gene sequence.Methods Virus strains were isolated from 22 specimens of cerebrospinal fluids（CSFs）of suspected patients with aseptic meningitis by using RD and Hep-2 cells,and identified by micro-neutralization test with compound and monovaluent enterovirus specific sera recommended by WHO.Full-length VP1 gene was amplified from the strains by RT-PCR and sequenced,of which the result was analyzed by Mega 4.1 software.Results The isolated strain was defined as CVB1 by micro-neutralization test,and named as MSH-KM9-09,from which VP1 gene at a length of 834 bp was amplified by RT-PCR.The homologies of nucleotide and amino acid sequences of VP1 gene of the strain were more than 86.1% and more than 97.8% to those isolated in China,while were 80.2% ~ 83.2% and 94.3% ~ 96.0% to those isolated abroad,respectively.Phylogenetic analysis revealed that the sequences of strains isolated in China,including MSH-KM9-09,were segregated into one distinct cluster,while those isolated abroad into other two clusters.Conclusion The isolated MSH-KM9-09 strain was defined as CVB1.