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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 sequence of MSH-KM9-09 of Coxsackievirus B1 (CVB1) isolates from aseptic meningitis in Kunming City in 2009 and analyze the sequence characteristics of VP1 gene. Methods Twenty-two samples of cerebrospinal fluid (CSF) from patients with suspected aseptic meningitis were isolated by RD cells and Hep-2 cells. The micro-neutralization assay was used to identify the isolates from the serum and monovalent sera of WHO enteroviruses. RT-PCR The full-length virus VP1 gene was amplified by PCR, sequenced and analyzed by software such as Mega 4.1. Results The isolated strains were identified as CVB1 subtype by micro-neutralization test and numbered as MSH-KM9-09. The VP1 gene of 834 bp was obtained by RT-PCR amplification. The nucleotide and amino acid sequences of the CVB1 isolates were 86.1% and 97.8% homologous to those of the domestic CVB1 isolates respectively. The nucleotide and amino acid sequences of CV1 The phylogenetic tree showed that MSH-KM9-09 belonged to the same branch as the domestic isolate, while the foreign isolates belonged to two other branches. Conclusion The MSH-KM9-09 isolate is enterovirus CVB1.