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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。
Objective To understand the distribution of mosquitoes and their arboviruses in the major epidemic areas of JE through the investigation of arboviruses in Sichuan Province and provide basis for prevention and control. Methods From 2006 to 2008, mosquitoes were collected at the peak of mosquito breeding at the investigation point for classification and identification. BHK cells were used to isolate the virus for three consecutive generations to observe the cytopathic effect. Cytoplasm and molecular biology were used to identify the positive cells. MEGA 3.1 biology software completes amino acid sequence and virus phylogenetic tree analysis. Results Eleven strains of Arbovirus were isolated from Culex tritaeniorhynchus and A. haemolyticus, and eight were identified as JEV by IFA and RT-PCR. All of them belonged to genotype Ⅰ JEV. The nucleotide and amino acid homology between the eight strains of viruses was 98.1% -100.0% and 99.7% -100.0%, respectively. Compared with the homology of nucleotides and amino acids of Sichuan isolate The source was 97.8% ~ 99.6%, the amino acid homology was 99.3% ~ 100.0%. The other 3 viruses showed faster pathological changes in BHK cells than JEV. IFA of ascites was immunized with 20 different species of arboviruses and the related primers were used for PCR amplification. The results were negative. Conclusions There are five kinds of mosquitoes in the endemic area of JE in Sichuan Province, among which Culex tritaeniorhynchus and Anemone mosquitoes are the dominant species, and JEV is carried by Culex tritacis, and the nucleotide and amino acid in E region are relatively conservative Conducive to take targeted control measures. At the same time, three strains of unidentified arboviruses were isolated, suggesting that they are rare species not yet found in our country. Their biological characteristics and pathogenicity to human beings need to be further studied.