猪伪狂犬病病毒河南株的分离及鉴定

Isolation and identification of Henan isolates of pseudorabies virus

采用PCR方法对河南不同地市采集的疑似猪伪狂犬病病毒(PRV)感染病料进行PCR检测,PCR阳性的病料经处理后,接种ST细胞,分离到15株PRV。分离PRV传至第3代后,在ST细胞上均能出现较稳定的细胞病变,传代至第7代时病毒滴度在10^(5.6) TCID_(50)/0.1 mL^10~9 TCID_(50)/0.1 mL,且均能扩增出特异性gE基因片段(429bp)。将分离PRV接种6周龄雌性昆明小鼠,引起皮炎及小鼠先后死亡。以NY株为例进行理化特性试验,结果表明,对分析纯氯仿、胰酶敏感;对酸、碱及热有较强抵抗力,pH3.0盐酸处理1h、pH11.0 NaOH处理1h、56℃水浴处理1h,才能使其失活;对紫外线处理30min,仍具有感染性。经电镜观察,可看到大小均一、近似圆形的病毒粒子,大小为110~150nm,囊膜表面有呈放射状排列的纤突,表明所分离病毒均为PRV野毒株。

Samples suspiciously infected with pseudorabies virus（PRV） were collected from several regions in Henan and detected by polymerase chain reaction（PCR）. Fifteen PRV isolates were suc cessfully isolated by inoculating the PRV-positive samples into the swine testicular（ST） cells. The cells infected with these viruses displayed stable cytopathic effect after 3 passages,and the isolates reached to 10^5.6TCIDs0/0. 1 mL--109TCID50/0. 1 mL at the seventh generation. The specific gE gene fragment（429 bp） was amplified by PCR. The 15 isolates were highly pathogenic in Kunming mice,causing skin inflammation and death in all experimentally-infected mice at 120 h after chal- lenge. The NY strain was sensitive to chloroform and trypsin,but resistant to acid,alkali and hot, because it was killed until treated with pH3.0 acid or pHll. 0 NaOH for 1 h or heated at 80℃ for 1 h,and viral infectivity was not impaired by UV treating for 30 min. The virus was observed to be visible round with a diameter of 110--150 nm by electron microscopy,capsule surface has the fiber protrusion with radiate arrage. All these confirmed that the isolated viruses were PRV.