四川省虫媒病毒调查研究

目的通过对四川省虫媒病毒调查,了解乙脑主要流行区蚊虫及其虫媒病毒的分布状况,为防制提供依据。方法 2006-2008年在调查点蚊虫孳生高峰期采集蚊虫标本,进行分类鉴定,并用BHK细胞分离病毒,连续3代观察细胞病变情况;收获细胞病变阳性分离物进行血清学和分子生物学鉴定;采用MEGA3.1生物学软件完成氨基酸序列和病毒进化树分析。结果三带喙库蚊和骚扰阿蚊为乙脑流行区优势种群,分离11株虫媒病毒,其中8株经IFA和RT-PCR鉴定为乙脑病毒(JEV),均属于基因Ⅰ型JEV。E基因区段核苷酸和氨基酸比较发现,8株病毒之间核苷酸和氨基酸同源性分别为98.1%～100.0%和99.7%～100.0%;与2004年四川分离株的核苷酸同源性在97.8%～99.6%,氨基酸同源性在99.3%～100.0%。另外3株病毒在BHK细胞上出现病变时间较JEV快,用20种不同种属的虫媒病毒免疫腹水IFA鉴定和相关的引物进行PCR扩增,结果均为阴性。结论在四川省乙脑流行区有5种蚊虫,其中三带喙库蚊和骚扰阿蚊为优势种群,乙脑病毒由三带喙库蚊携带,其E区核苷酸和氨基酸相对保守,有利于采取针对性防控措施。同时,分离出3株不能鉴定的虫媒病毒,提示为我国不常见种型或尚未发现的新毒种,其生物学特性和对人类的致病性有待进一步研究。

Survey on Arbovirus in Sichuan Province

Objective To understand the distribution of mosquitoes and relevant arboviruses in the Japanese encephalitis （JE） epidemic area in Sichuan and provide evidence for the disease prevention and control.Methods Mosquitoes collected in local mosquito breeding places of survey sites from 2006 to 2008 were classified.Meanwhile,virus was isolated by inoculating homogenates of the mosquitoes in BHK cells and cytopathogenic effect （CPE） were observed for successive three passages.Positive isolates were then identified by serological and molecular biological methods.Sequence alignment and phylogenetic analysis were carried out by MEGA3.1 software.Results Culex tritaeniorhynchus and Armigeres Subalbatus were predominant species in the JE epidemic area.Of 11 isolates that caused obvious CPE in BHK cells,8 were confirmed as JEV by IFA and RT-PCR.Phylogenetic analysis on E gene segment showed that the 8 isolates all belonged to genotype Ⅰ,which shared homology of 98.1%-100.0%and of 99.7%-100.0% in nucleotide and amino acid sequences respectively;Compared with isolates in 2004 in Sichuan,homology were from 97.8% to 99.6% and 99.3% to 100.0% in nucleotide and amino acid sequences respectively.Another three strains showed more rapid CPE in BHK cells than JEV,and were non-reactive to antibodies of 20 different species of arboviruses by IFA.RT-PCR also failed to amplify corresponding segments.Conclusion Five species of mosquitoes were found in JE epidemic area in Sichuan,and Culex tritaeniorhynchus and Armigeres Subalbatus were predominant.JEV were carried by Culex tritaeniorhynchus and their E segment gene was relatively conserved,which contributed to the effective control of disease.However,the other three strains could not be identified by 20 species serum as well as RT-PCR,which suggested that they were unusual or undiscovered strains.It is necessary to conduct further study on their biological characteristics and possible pathogenicity to human.