四川省分离的基因1型乙型脑炎病毒分子特征分析

目的 从四川省巴中市采集的蚊虫标本中分离乙型脑炎(简称乙脑)病毒(JEV),确定其基因型别,并分析相关的基因1型乙脑病毒PrM和E基因区段氨基酸序列特征.方法 对2004年采集蚊虫标本进行病毒分离,对新分离的乙脑病毒进行生物学、血清学及分子生物学鉴定.逆转录聚合酶链反应(RT-PCR)扩增新分离JEV的PrM、E区段核苷酸序列,测序后应用Clustal X软件做碱基配对分析,MEGA4软件完成病毒进化分析,GENEDOC(3.2)软件完成氨基酸位点分析,根据蜱传脑炎病毒可溶性蛋白晶体结构为模板进行乙脑病毒E蛋白三维结构模拟预测分析.结果 共采集4668只蚊虫标本,主要是骚扰阿蚊和库蚊,分离到6株病毒,经鉴定均属于基因1型的乙脑病毒.将四川省分离的6个毒株结合我国新分离的基因1型乙脑病毒与减毒活疫苗株SA14-14-2株的PrM区段和E区段氨基酸比较,发现PrM区段在PrM2、64和65位存在基因1型乙脑病毒独有的氨基酸位点差异,E区段存在14处共同的氨基酸位点差异,其中在E129、222、327和366位点为中国目前分离到的基因1型乙脑病毒所特有的位点特征.结论 从四川省巴中市首次分离到基因1型的乙脑病毒,并发现基因1型乙脑病毒与减毒活疫苗株之间PrM、E基因区段存在氨基酸差异,但现行疫苗株理论上可以保护新分离的基因1型乙脑病毒.

Molecular character analysis of Japanese encephalitis virus isolated from Sichuan province, China

Objective To analyze the genotype of Japaneso encephalitis virus (JEV) strains isola-ted in 2004 from mosquitoes collected in Bazhong city, Sichuan province of China, and the characters of amino acid in the PrM and E gene. Methods The isolated virus strains from mosquitoes were identified by biological, serological and molecular biology. PrM and E segments of the isolated JEV were amplified by RT-PCR, the PCR products were purified and sequenced. Multiple alignment, phylogenetic and amino acid (AA) analysis were carried out by Clustal X (1.8) , MEGA4 and GENEDOC (3.2) . Results The total of 4688 mosquitoes were collected including Armigeres and Culex. Six isolates were identified be-longing to genotype 1 JEV. The comparison between new genotype 1 JEV strains and live attenuated vaccine strain SA14-14-2 in PrM and E gene showed that total 3 sites amino acid differences in PrM gene and 14 sites in E gene, respectively. Three sites (PrM2, 64 and 65 ) in PrM protein and four sites (E129, 222,327 and 366) in E protein were only belonging to genotype 1 JEV. Conclusion The new isolated JEV strains in Sichuan province belong to genotype 1. It suggests that the vaccine strain SA14-14-2 currently used for preventing Japanese encephalitis is able to protect people against JEV, although in the segments of it had some amino acid differences between vaccine strain and the epidemic genotype 1 JEV strains in PrM and E gene.