摘要
利用RT-PCR技术对河南省某规模化猪场乙脑疑似病例的种猪精液进行分析,从部分样本中扩增出与流行性乙型脑炎病毒(JEV)M基因和E基因具有高度同源性的预期产物。在3日龄乳鼠脑内接种种猪精液上清,10 d左右即出现典型神经症状,并且病死鼠脑组织RT-PCR结果均为阳性。用病死鼠脑组织研磨上清接种BHK-21细胞,感染60 h后用JEV单抗进行间接免疫荧光鉴定均为阳性,这表明分离毒株均为JEV流行毒株,分别命名为BSF.ZZ-1和BSF.ZZ-3。对新分离毒株进行体外细胞培养,BSF.ZZ-1和BSF.ZZ-3连续传代3次后病毒量均获得明显增加,最高分别可达104.5±0.1TCID50/mL和104.2±0.2TCID50/mL。用生物学软件MEGA 4.0分别构建JEV基因的系统进化树,发现BSF.ZZ-1和BSF.ZZ-3处于同一进化分支,均属于基因Ⅲ型。
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±0.2TCID50/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.
出处
《河南农业科学》
CSCD
北大核心
2011年第5期152-157,共6页
Journal of Henan Agricultural Sciences
基金
国家公益性行业(农业)科研专项(200803015)
