武汉市新分离2株乙型脑炎病毒E基因分型及序列分析

Analysis of sequence and genotype of E gene of the Newly Isolated Japanese encephalitis virus strains in Wuhan, Hubei Province

目的 对2009年武汉市新分离的2株乙型脑炎（简称乙脑）病毒进行基因分型和序列分析，了解本地乙脑病毒株的分子生物学特性。方法 将2009年从三带喙库蚊中分离的两株乙型脑炎病毒用RT-PCR法扩增E基因，将其进行测序，并用DNAstar and MegAlign软件与其他基因型代表株进行比对。结果 16组样品检出两株阳性（WHJX9-09、WHJX10-09），这两株阳性均属于GI型。两株新分离JEV之间的核苷酸和氨基酸同源性分别为98.9％和100％。同目前在武汉市使用的疫苗株SA-14-14-2相比，核苷酸同源性分别为87.4％、87.9％，氨基酸同源性为96.9％。共有15个氨基酸发生变异分布在3个不同结构域，中和位点没有变异但是神经毒力位点仍然存在。结论 武汉市本地新分离乙脑病毒基因型为GI型，不同于1988年在武汉检出的GⅢ型的基因型，和疫苗株SA-14-14-2相比，其神经毒力并没有减弱，但疫苗产生的抗体对新出现的GI型乙脑病毒仍有中和作用。因此提高乙脑疫苗的接种率并配合防蚊灭蚊措施对控制乙脑疫情依然至关重要。同时有必要对本市蚊虫及乙脑患者进行长期的病原学监测工作，为乙脑预测预警体系的建立提供科学依据。

Objective To analyze the molecular characteristics of the newly isolated two Japanese encephalitis virus strains（JEV） in Wuhan. Methods The mosquitoes were collected in Wuhan from April to October in 2009. The envelope （E） protein gene of JEV was detected using RT-PCR and sequenced. Sequence comparisons and phylogenetic analysis were conducted using DNAstar and MegAlign. Results Two Japanese encephalitis virus （ JEV ） strains （ WHJX09-9, WHJX09-10 ） were isolated from Culex tritaeniorhynchus among 16 mosquito pools and identified as genotype I. The result showed that the homology of the two strains was 98. 9% in nucleotides and 100% in deduced amines. The comparison between the new genotype 1 JEV strains and live attenuated vaccine strain SA14-14-2 in E gene showed that the homology of nucleotide sequence was 87.4% and 87.9% ,the homology of amino acid was 96.9% （ totol 15 amino acid were different） in E gene. The mutation sites of amino acid distributed among three different coding domain, but no antigen binding site and neurotoxin-involved site of amino acid were changed. Conclusion Wuhan had appeared a new genotype of JEV which was different from the former strain isolated in Wuhan, the new JEV strains still had neurotoxicity but had high homology with the vaccine strains adopted in Wuhan. The vaccine could still be adopted to prevent Japanese encephalitis if steps were take to eradicate mosquitos at the same time. laboratory surveillance were also an important task to build an early-warning mechanism against JEV.