目的对新分离乙型脑炎病毒鉴定,了解病毒E基因区段的氨基酸序列特征及基因分型. 方法 2001年在上海市采集蚊虫标本,用组织培养的方法分离到7株病毒.进行病毒一般生物学鉴定,对病毒PrM和E基因区段约2000nt进行了扩增、克隆、测序.应用Clustal X软件做碱基配对和比较分析,种系发生采用PHYLIP软件包分析. 结果 7株病毒可以引起BHK-21细胞规律病变,病变时间为60 h.乳鼠脑内接种病毒后72 h死亡.所有新分离病毒与标准乙型脑炎病毒抗体阳性反应.用病毒PrM区段(456～695位核苷酸)进行的基因分型分析,新分离的7株病毒属于基因Ⅰ型乙型脑炎病毒.以减毒活疫苗株SA14-14-2为标准,对7株病毒的E基因区段进行分析,7株病毒之间核苷酸同源性在97.7%以上,氨基酸同源性在99.2%以上;7株病毒与疫苗株SA14-14-2核苷酸差异在12.2%左右,氨基酸差异在2.8%～3.2%之间,共有14个共同的氨基酸位点差异. 结论 2001年在上海采集的蚊虫中新分离的7株病毒属于基因Ⅰ型乙型脑炎病毒,为我国的首次报道.
Objective To analysis the genotype and the character of amino acid(AA) sequences of the E protein of the newly isolated Japanese encephalitis viruses(JEV) from mosquitoes collected in Shanghai 2001. Methods Virus isolation was performed inoculating mosquito homogenates onto BHK-21. The isolated strains were identified by biological and molecular methods. The PrM and E gene of the newly isolated JEV strains were cloned and sequenced. Phylogenetic analysis was achieved by Phylip program. Two hundred and forty nucleotides sequences of PrM gene(map position 456-695) region in the new isolated JEV strains were selected and compared with the reference strains from GenBank, isolated from different countries, to do phylogenic analysis. Nucleotide sequence and AA sequence of the E gene were studied by bioinformatics software. Results Seven virus strains were isolated, with all causing cytopathogenic effect(CPE) in BHK-21 cells and fatal to suckling mice. Serological tests showed that all of these strains were positive to the antibody of JEV. The phylogenetic results showed that the newly isolates allbelonged to genotype Ⅰ, JEV. Sequence analysis showed that the homology of nucleotide sequence in these 7 strains was over 97.7% and that of AA sequence 99.2%. Compared with differences of the nucleotide sequence between these seven strains and the standard JE vaccine strain SA-14-14-2, were up to 12.2%, and differences of AA sequence ranged from 2.8% to 3.2%. Compared with the vaccine strain, there were 14 common AA variations in all the 7 strains. Conclusion Newly isolated JEV strains belonged to genotype Ⅰ, which was reported for the first time in China. [
Chinese Journal of Microbiology and Immunology