湖北省新分离的二株乙型脑炎病毒全基因组序列特征分析

Molecular characteristics of the full-length genome of two new Japanese encephalitis virus isolated from mosquitoes in Hubei province

目的对2008年湖北省新分离的2株乙型脑炎（简称乙脑）病毒进行全基因组序列测定和分析，了解当地乙脑病毒株的分子生物学特性。方法病毒复苏鉴定后，使用覆盖乙脑病毒全基因组的16对特异性引物，RT-PCR法扩增乙脑病毒HBZG08-09株和HBZG08-55株的全基因组片段，直接测序拼接获得全基因组序列。使用ClustalX 1．83、MegAlign、Mega4和Genedoc3．2等生物学软件进行序列比对、核苷酸和氨基酸同源性分析、系统进化分析及氨基酸位点差异分析。结果新分离的2株乙脑病毒基因组全长均为10965个核苷酸，从97位到10392位为开放阅读框，编码3432个氨基酸，2株间的全基因组核苷酸和氨基酸同源性分别是98．2％和99．7％。进一步的基因型研究显示HBZG08-09株和HBZG08-55株均属于基因Ⅰ型乙脑病毒。2株病毒与我国近年来在河南及浙江的乙脑病毒分离株进化关系最近。与目前使用的减毒活疫苗株sA—14—14-2相比较，HBZG08-09株全基因组共存在82个氨基酸差异，HBZG08-55株存在84个氨基酸差异。但影响毒力或抗原的关键氨基酸位点未发生改变。结论新分离的2株乙脑病毒均属于基因Ⅰ型，与疫苗株相比关键氨基酸位点未见变异。

Objective To sequence and analyze the complete genome of two new Japanese encephalitis virus （ JEV ） strains isolated from mosquitoes collected in Hubei province in 2008, and to understand the molecular biological characteristics of JEV in this area. Methods RT-PCR was used to amplify the fragments of HBZG08-09 strain and HBZG08-55 strain with 16 pairs overlapping primers after they had been recovered and identified, then the full-length genome was obtained by sequencing and splicing. Biological sequence alignment, nucleotide and amino acid sequence analysis, phylogenetic analysis and analysis of amino acid differences were performed by the software of ClustalX （ 1.83 ）, MegMign, Mega （4.0） and Genedoc （3.2）. Results The genome of two new strains were both 10 965 nucleotides in length with a single open reading frame from 96 to 10 392 coding for a 3432 amino acid poly-protein,the homology of nucleotide and amino acid sequence between two isolates were 98. 2% and 99.7% respectively. Further study showed that the new strains were both belonging to genotype Ⅰ Two new strains were most closelyrelated to isolates obtained from Henan and Zhejiang province in recent years. Compared with the live attenuated vaccine strain SA-14-14-2 in China, HBZG08-09 strain had 82 amino acid divergence; HBZG08- 55 had 84 amino acid divergences. But the amino acid difference occurred in sites were not the key ones affecting the toxicity or antigenic of JEV. Conclusion Two new JEV isolates were both belonging to genotype Ⅰ , and the key sites of amino acid were not changed.