伪狂犬病毒GDSH株的分离鉴定及gE基因的序列分析

Isolation and Identification of GDSH Strain of Pseudorabies Virus and Sequence Analysis of gE Gene

从广东四会某猪场分离到一株疑为猪伪狂犬病病毒(PRV)的病毒,病毒在猪肾细胞上出现细胞变圆、拉网、融合等典型病变,并具有细胞泛嗜性特点。在MDCK细胞上测得其TCID50值为10-8/0.1mL,能被伪狂犬病病毒标准阳性血清中和。将0.1mL病毒液接种小鼠后发生奇痒并麻痹致死,接种猪3天后发病,7天死亡,从攻毒病死猪的脑组织病理切片上观察到典型的病毒性脑膜脑炎及血管套现象。通过PCR扩增到PRVgD基因,由此进一步证明所分离病毒为猪伪狂犬病毒,并命名为GDSH株。根据GenBank中发表的序列,设计一对扩增PRVgE基因的特异性引物,建立可以区分PRV野毒株与疫苗株的PCR诊断方法。以此方法对病毒的细胞培养液进行检测,结果证实所分毒株为PRV野毒株,经克隆测序后与GenBank收录的其它PRVgE基因序列进行比较,发现所测毒株的核苷酸序列与其它PRV毒株的同源性介于98.3%～99.9%之间,其中与PRVEa株的亲缘关系最近为99.9%。

Isolates were got from brains and visceral organ samplesf of piglets. The typical cytopathic effect（CPE） could be seen when the isolates were cultured on the PK-15 cells and its TCID50 was 10^-8/0.1mL. Typical clinical signs could be observed when the pigs and mice were injected with the isolate. The isolate was named as GDSH strain of Pseudorabies virus. According to the genomic sequence of PRV in GenBank, PCR primer was designed. PCR method was established to differentiate wild PRV and vaccine strains. And then the PCR product was cloned and sequenced.The sequence data of PRV isolate was analysed using the DNAStar software. The results demonstrated that the necleotide identity of PRV gE-gene was 98.3%-99.9% comparing with other PRV isolates published in GenBank, and with 99.9% necleotide identity compared with PRV strain Ea.