首例兽用活疫苗中污染副流感病毒5型的检测

The First Case of Contamination with Parainfluenza Virus 5 in Veterinary Live Vaccine

从某企业送检的猪伪狂犬活疫苗中检出疑似血凝性外源病毒污染,经病原学和分子生物学鉴定,证实该疫苗污染了PIV5,并对我国主要猪用活疫苗中污染PIV5情况进行了摸底检测。将样品经伪狂犬病毒特异性阳性血清中和后,接种Vero细胞连传5代,可出现细胞圆缩、聚集、颗粒增多等细胞病变。纯净性检测显示该分离株无细菌污染、支原体污染和外源病毒污染。将该分离毒株F10代培养物进行病毒含量和HA效价测定,分别为10^(7.0)TCID_(50)/mL和1∶256。以PIV5单克隆抗体作为一抗进行IFA染色,可在感染细胞的胞浆中观察到典型的绿色荧光;采用针对PIV5的L基因设计特异性引物,经RT-PCR可扩增出277 bp特异性片段,经测序后与GenBank中已发表的8株PIV5序列进行同源性比较,同源性为93.1%～97.4%,其中与我国分离的来自小熊猫的PIV5ZJQ-221株(登陆号:KF100034.1)同源关系最近,为97.4%;其次与犬源、猴源、牛源的PIV5同源性均在95.5%以上,与猪源的PIV5同源性为93.3%～95.9%,同源性均较高。基因序列系统进化树分析显示同样的结果。以上试验证明疫苗中污染的病毒为PIV5,并命名为PIV5/01株。针对国内猪用活疫苗中污染PIV5情况的初步摸底检测显示,PIV5污染率约为16.7%,提示应加强疫苗中污染PIV5的预防和控制。

Suspected hemagglutinating exogenous virus contamination was detected in a live procine Pseudorabies vaccine.It was confirmed by etiology and molecular biology that the vaccine was indeed contaminated by PIV5,and further detections were performed in major domestic live pig vaccine.The contaminated sample was first neutralized by Pseudorabies virus-specific positive serum,and subsequently inoculated with Vero cells for 5 generations.Cytopathic effects such as cell circle shrinkage,aggregation and granulation increase were observed.Purity test indicated that the isolate was free from bacterial contamination,mycoplasma contamination and exogenous virus.Virus content and HA titer of the F10generation culture of the isolate were measured,the results were 10^7.0TCID50/mL and1∶256 respectively.Typical green fluorescence was observed in the cytoplasm of infected cells by IFA staining with monoclonal antibody against PIV5.Meanwhile,the 277bp specific sequence fragment could be amplified by primers specially designed for the L gene of PIV5.The fragment was sequenced and compared with publised PIV5sequences of strain8in Genbank,the nucleotide homology is within 93.1%~97.4%,and the most closely related strain is PIV5ZJQ-221 isolated from lesser panda in China,which share up to97.4%homology with the isolate.With respect to PIV5from dog,monkey and bovine origin,the homology is above 95.5%.This isolate in particular shares quite high homolgy with pig-derived PIV5which range from 93.3 to 95.9%.The results of the phylogenetic tree analysis of gene sequence system draw the same conclusion.The tests above proved that the virus contaminated in the vaccine was PIV5,and named as strain PIV5/01.In addition,the preliminary tests for PIV5contramination among domestic swine live vaccines showed that the contamination rate of PIV5was about 16.7%,therefore here we remind that prevention and control of PIV5contamintion in vaccine should be strengthen.