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目的:为了查明引起2008-2009年浙江省疑似病毒性脑炎的病原,分析病原的分子特征。方法:采用Hep-2和RD细胞从疑似患者脑脊液样本中分离肠道病毒,对分离株扩增VP1基因,测序并进行同源性与进化分析。结果:从患者11份脑脊液样本中分离到4株柯萨奇B1病毒(CVB1);测得VP1基因全长为834核苷酸(nt),无插入和缺失,推导编码278个氨基酸(aa)。4株浙江CVB1分离株(ZJ/CVB1)之间在VP1区nt和aa的同源性分别为96.3%~99.9%和100%。浙江CVB1分离株BLAST比对nt同源性最高的毒株为2006年山东株(SD/168/06),同源性为98.7%;与CVB1原型株(Conn-5/86)的nt同源性为78.9%。VP1区氨基酸进化树显示浙江CVB1分离株在CVB1 Clade3进化支上,与浙江株亲缘关系最近的毒株为山东CVB1株,均在同一进化支上。结论:从病毒分离结果证实浙江省疑似病毒性脑炎是由肠道病毒CVB1引起,2008-2009年在浙江循环的CVB1病毒之间没有明显差异,浙江CVB1流行株与山东株亲缘关系最近。
OBJECTIVE: To identify the etiological agents responsible for the suspected viral encephalitis in Zhejiang province from 2008 to 2009 and to analyze the molecular characteristics of pathogens. Methods: Enterovirus was isolated from samples of cerebrospinal fluid of suspected patients by using Hep-2 and RD cells. The VP1 gene was amplified from the isolate and sequenced and analyzed for homology and evolution. Results: Four strains of Coxsackievirus B1 (CVB1) were isolated from 11 cerebrospinal fluid samples of patients. The total length of VP1 gene was 834 nucleotides (nt), without insertions and deletions. The deduced cDNA encoding 278 amino acids (aa) . The isolates nt and aa of 4 strains of CVB1 isolates (ZJ / CVB1) in Zhejiang province were 96.3% -99.9% and 100%, respectively. The isolates with the highest homology of BLAST from Zhejiang CVB1 isolates were Shandong strain (SD / 168/06) in 2006 with a homology of 98.7%. Compared with the nt homology of the CVB1 prototype strain (Conn-5/86) Sex is 78.9%. The phylogenetic tree of VP1 showed that the CVB1 isolate of Zhejiang was on the clade3 of CVB1 clade. The closest strain of Zhejiang isolates was Shandong CVB1 strain, both of which were on the same clade. Conclusion: The results of virus isolation confirmed that Zhejiang province was suspected of viral encephalitis caused by enterovirus CVB1. There was no significant difference between Zhejiang CVB1 virus circulating in Zhejiang province in 2008-2009, and the most recent genetic relationship between Zhejiang CVB1 strain and Shandong strain.