浙江省临海市2004年病毒性脑膜炎暴发疫情的病原学及其分子特征研究

目的 为确证2004年浙江省临海市病毒性脑膜炎暴发疫情的病因,分析病原的遗传变异及进化关系.方法 采集患者脑脊液样本60份,采用RD和Hep-2细胞同时分离病毒,中和试验法鉴定病毒型别;对分离株VP1和VP4/VP2基因测序,进行同源性与进化分析.结果 从60份脑脊液样本中分离到埃柯病毒30型(E30)19株,分离率为31.7％;对4株E30分离株VP1区核苷酸序列测定,其长度均为876个核苷酸(nt),编码292个氨基酸(aa).临海分离株与E30原型株Bastianni在VP1区的nt和aa同源性分别为82.4％～84.1％和93.5％ ～ 94.2％;4株临海E30株之间nt和aa的同源性分别为87.1％～99.9％和97.9％ ～ 100.0％.临海分离株分成两类,同类病毒株间的差异很小,而两类病毒株间的差异很大,nt和aa的最大差异率分别为12.9％和2.1％.与临海E30株同源性最高的为2002-2003年浙江E30株.在VP1基因进化树上,临海E30株分别位于G和H基因亚型分支上,其中临海G基因株与2003年浙江、江苏和山东E30株位于同一进化分支,临海H基因株与2002年浙江诸暨株位于同一进化分支.VP4/VP2区进化分析结果与VP1区相似.结论 2004年临海市病毒性脑膜炎暴发疫情由E30G和H不同基因亚型的E30流行株引起;临海E30株与2002-2003年浙江、江苏和山东E30株具有密切的亲缘关系;H基因亚型株推测为新的E30变异株,首先分离于2002年浙江省.

Study on the pathological and molecular characteristics of viral meningitis outbreaks in Linhai county, Zhejiang province, 2004

Objective In order to confirm the causes of viral meningitis outbreaks in Linhai county,Zhejiang province in 2004,and to analyze the relationship between hereditary variation and evolution of the pathogen.Methods 60 cerebrospinal fluid(CSF)specimens were collected from the suspected patients.Virus strains from the specimens were isolated with RD and Hep-2 cell lines,and identified through neutralization test.VP1 and VP4/VP2 genes of the isolated viruses were sequenced.Both phylogcnetic and homological trees were also constructed.Results 19 Echovirus type 30(E30)strains were isolated from 60 CSFs,in which E30 accounted for 31.7％.All of the complete VP1 genes in 4 sequenced virus isolates of E30 were composed of 876 nt,encoding 292 amino acids(aa).The identity of nucleotide and amino acid in VP1 gene were 82.4％-84.1％ and 93.5％-94.2％ between the 4 Linhai strains and the prototype strain Bastianni of E30,were 87.1％-99.9％ and 97.9％-100.0％ among the 4 virus strains of E30 from Linhai,respectively.The 4 Linhai strains could be classified into two classes.The diversity of nt and aa was minimal in the same class but obvious between the two classes,with the range of diversities as 12.9％ and 2.1％,respectively.The Linhai E30 strains had maximum similarity with the Zhejiang E30 strains in 2002-2003.The 4 Linhai strains of E30 in the phylogenetic tree of the VP1 gene were attributed into two branches of the G and H genotype,respectively.The G branch also included the E30 strains from Zhejiang,Jiangsu and Shangdong in 2003,while the H branch including E30 strains from Zhuji,Zhejiang in 2002.The phylogenetic tree of VP4/VP2 genes was similar to that of VP1 gene.Conclusion The outbreak of viral meningitis in Linhai county in 2004 was caused by the two classes of E30 strains with G and H genotype existed simultaneously.The Linhai E30 strains had maximum genetic relations to the Zhejiang,Jiangsu and Shangdong strains of E30.The H genotype was inferred to be a new variant strain,which was first isolated in Zhejiang province in 2002.