河南农业科学2011，Vol.40Issue(5)：152-157,6.
猪流行性乙型脑炎病毒种猪精液分离株的鉴定及进化分析
Characterization and Phylogenetic Analysis of the Field Japanese Encephalitis Virus Strains Isolated from Seminal Fluid of Diseased Stock Boars
摘要
Abstract
For the isolation of field prevalent Japanese encephalitis virus (JEV),the seminal fluids of diseased stock boars were collected from Henan province. Using two specific designed primer pairs,both of JEV M and E genes were amplified from boar seminal fluid (BSF) samples by reverse transcription polymerase chain reaction (RT- PCR), which was further verified by gene cloning and DNA sequencing. Animal experiments also showed that two of the supernatant of seminal fluids caused typical clinical nervous symptoms or death of suckling mouse at about 10 days post intracellular inoculation and the JEV M and E genes can be similarly amplified from the brains of diseased suckling mouse. The virus infections of the 2 new isolates on BHK - 21 cells were detected by indirect immunofluorescence assay (IFA) using JEV-specific monoclonal antibody at 60 h post-infection. This indicated that these two isolates, namely as BSF. ZZ - 1 and BSF. ZZ- 3 respectively,were virulent JEV strains. Virus titration showed that the highest virus titers of BSF. ZZ- 1 and BSF. ZZ- 3 propagated in BHK- 21 cells were 104.5±0.1TCID50/mL or 104. 2±0. 2 TCID50/mL, respectively. Using bio-software MEGA 4. 0, two phylogenetic trees based on the M or E gene sequences of the new isolates together with other reference JEV strains were constructed and showed that BSF. ZZ - 1 and BSF. ZZ - 3 were adjacent to each other and both belonged to the genotypeⅢ of JEV.关键词
流行性乙型脑炎病毒/分离鉴定/M基因/E基因/系统进化树/基因Ⅲ型Key words
Japanese encephalitis virus/ Isolation and identification/ M gene/ E gene/ Phylogenetic tree/ Genotylpe Ⅲ分类
农业科技引用本文复制引用
王兴涛,罗俊,滕蔓,樊剑鸣,鄂巍,禹斌,刘力,张改平..猪流行性乙型脑炎病毒种猪精液分离株的鉴定及进化分析[J].河南农业科学,2011,40(5):152-157,6.基金项目
国家公益性行业(农业)科研专项(200803015) （农业）
