一株猪源乙型脑炎病毒的分离及鉴定

Isolation and identification of Japanese encephalitis virus from swine

从江苏某发病猪场采集的母猪流产胎儿脑组织样本中成功分离到一株猪源乙型脑炎病毒(JEV)毒株,并对该毒株的相关生物学特性进行了鉴定。利用BHK-21细胞传代和脑内接种小鼠方法,对疑似阳性病料做病毒分离。通过RT-PCR,CPE和IFA等方法对JEV分离株进行鉴定,并测定其TCID50。结果成功分离获得一株JEV毒株(JEV JS01株),该毒株能够在BHK-21细胞上增殖并产生细胞病变,TCID50为每1 m L107.0,免疫荧光检测可观察到JEV特异性荧光。RT-PCR扩增E基因并测序分析表明,与Gen Bank中登录的JEV毒株序列具有较高的同源性,属基因Ⅲ型。小鼠致病力试验表明该分离株对易感动物具有较高致病性。本研究为进一步开展乙脑病毒流行病学与疫苗免疫研究奠定了基础。

A strain of Japanese encephalitis virus（ JEV） was isolated from the brain of aborted fetus of a JEV infected pregnant sow in Jiangsu Province. The biological characteristics of this isolate were identified. The JEV positive sample was inoculated and passaged on BHK-21 cells,and inoculated intracranial on mice. The isolate was identified by RT-PCR,CPE,and confirmed by immunofluorescence assay（ IFA）. The TCID50 of this isolate was measured using BHK-21 cells. The results indicated that the JEV strain was successfully isolated. It could induce cytopathogenic effect（ CPE） on BHK-21 cells,and specific fluorescence could be observed. The TCID50 of this strain was 107. 0.The E protein＇s amino acid sequence was compared with those of different genotype of JEV strains available in GenBank. A phylogenetic tree was constructed based on E gene sequences of the isolate and some other JEV strains. The isolate belonged to JEV genotype Ⅲ. The pathogenicity to the mice demonstrated that the isolate was highly neuroinvasiveness. This study provided a basis for further research on epidemiology and vaccine of the virus.