致病性猪伪狂犬病病毒PRV-JF株的分离与鉴定

Isolation and identification of pathogenic porcine pseudorabies virus PRV-JF strain

从临床疑似猪伪狂犬病发病仔猪的脑组织病料中,经聚合酶链式反应(PCR)证实为猪伪狂犬病病毒(PRV)野毒感染,采用无PCV1污染的猪肾细胞系(PK-15)分离培养,经蚀斑克隆纯化,培育1株细胞培养适应毒,命名为PRV-JF株。该分离株经细胞培养传代,能够产生典型的细胞病变,病毒滴度随代次显著增加,第24代毒价达108.5 TCID50/mL。免疫过氧化物酶单层细胞试验(IPMA)检测病毒抗原分布在细胞核及细胞质内。病毒感染细胞可被已知PRV阳性血清中和。电镜负染观察到病毒粒子呈椭圆或圆形外观,无囊膜的病毒粒子直径约110 nm～150 nm,有囊膜的成熟病毒粒子直径约150 nm～180 nm。PCR鉴定该毒株含有gE基因,其序列与GenBank登录的7株PRV同源性为97.7%～100%。用不同剂量病毒培养物接种家兔于24 h～72 h内全部死亡。研究表明,PRV-JF分离株对易感动物具有高致病性,为进一步开展该病毒流行病学、致病机理、疫苗免疫与诊断研究奠定了基础。

A virulent strain of pseudorabies virus （PRV） was isolated from the brain tissue of an infected piglet and adapted to cell culture through a series of passages and three plaque purifications in PCVl-free PK-15 cells. The isolate （PRV-JF） could induce typical cytopathogenic effect in cells and sustained a markedly increased titre after a series of passages （10^8.5 TCID50/mL at the 24 th passage）. The infectivity of virus could be neutralized with antiserum against PRV in vitro. Immunoperoxidase monolayer assay showed that virus antigens were distributed both in the infected cell nucleolus and cytoplasm. The viral particles have a round or elliptical shape under electron microscope, with a diameter of 110 nm-150 nm （non-enveloped） or 150 nm-180 nm （enveloped mature viruses）. The gE gene of the virus was sequenced and showed a nucleotide homology of 97.7 %-100 % with seven other PRV sequences published in GenBank. Experimental infection in rabbits caused animal death at 24 h-72 h post-inoculation. This study provided a basis for further research on epidemiology, pathogenicity, vaccination and diagnosis of the virus.