免疫后发病仔猪中伪狂犬病毒的分离和鉴定

IDENTIFICATION AND CHARACTERIZATION OF A PSEUDORABIES VIRUS ISOLATED FROM A DEAD PIGLET BORN TO VACCINATED SOW

从2011年开始,全国多个省份发生了新生仔猪疑似伪狂犬病的典型症状。2012年下半年,我们从江苏省某伪狂犬病疫苗免疫猪场中发病仔猪的脑组织中分离到一株伪狂犬病毒(Pseudorabies virus,PRV),并进行了部分生物学特性分析。从发病死亡仔猪脑组织中,利用PCR技术针对PRV的gB基因和gE基因分别扩增出了两条特异性片段,经序列分析初步确定为伪狂犬病毒感染。随后,将病料上清接种Vero细胞,24 h后细胞发生变圆、空泡、合胞体等典型的细胞病变,将分离的病毒经过6轮蚀斑纯化后,用PRV抗体IFA检测显示,感染细胞产生阳性荧光反应。分离的病毒在Vero细胞上的生长滴度可以达到107.43TCID50/mL。将病毒接种小鼠很快出现了奇痒并最终死亡等伪狂犬症状,且比经典的PRV(S株)对小鼠的LD50低。对gE毒力基因序列的比对分析表明,该毒株与经典PRV强毒和疫苗株有明显差异。

The newborn pigs in many herds have suffered from a disease that is clinically similar to pseudorabies in China since 2011.In late 2012,the disease outbreak occurred in a herd in Jiangsu province that received PRV vaccination.Brain tissue of a dead piglet was collected and examined for PRV by PCR.Two pairs of primers specific for PRV gB and gE were designed and used to amplify the corresponding DNA segments.The resulting DNA segments were sequenced and shown to share very high homology with known PRV.A pseudorabies virus was isolated from this dead piglet.The homogenated brain tissue was inoculated to Vero cells.Classical cytopathogenic effects of herpesvirus appeared in Vero cells at 24 hours post-inoculation.The virus was cloned by six rounds of plaque purification in Vero cells.The infected cells showed positive reaction with PRV antibodies in IFA.Therefore,the isolated virus was confirmed to be PRV.The PRV isolate grew well in Vero cells with the titer up to 107.43 TCID50/mL.The mice infected with this isolate developed unbearable itching and died later.The LD50 of the isolate was lower than that of PRV S strain.Genetic analysis of gE gene revealed that the isolate was different from classical virulent strain and vaccine strain.