猪伪狂犬病病毒变异毒株HeNZK-2014的分离鉴定及gE基因序列分析

Isolation and Identification of Variant HeNZK-2014 of Pseudorabies Virus and Sequence Analysis of gE Gene

为了解猪伪狂犬病病毒(PRV)变异毒株的特点,本研究采集临床疑似PRV感染发病猪的淋巴结等组织样品进行PCR鉴定,选取仅PRV阳性的组织样品经研磨除菌后取上清接种于PK-15细胞进行病毒分离培养、蚀斑纯化、PCR和间接免疫荧光法(IFA)鉴定,采用Reed-Muench法测定PRV的TCID50,接种小鼠并观察临床症状,对纯化的PRV和死亡小鼠脑组织样品进行gE基因PCR扩增及测序分析。结果显示,PRV阳性病料接种于PK-15细胞24h后出现典型细胞病变(CPE),经3轮蚀斑纯化后PCR和IFA鉴定结果均为阳性,分离株命名为HeNZK-2014,其TCID50为10-9.77/0.1mL;以1×108个TCID50病毒悬液接种小鼠22h后可引起小鼠出现奇痒、撕咬、死亡等典型猪伪狂犬病症状,死亡小鼠脑组织样品PRV PCR检测结果为阳性;纯化病毒和死亡小鼠脑组织样品gE基因核苷酸序列同源性为100.0%,与GenBank中2011年以前登录的经典毒株位于不同分支,与2011年之后中国流行毒株位于同一分支,在第48和496位各有1个天冬氨酸(D)的插入,具有变异毒株的典型特征。本研究成功分离了1株PRV变异毒株,为进一步开展针对PRV变异毒株的疫苗及其防控研究奠定了基础。

In order to learn the situation of pig pseudorabies virus（PRV）variant in this study,tissue samples such as lymph nodes which were collected from clinical pigs with suspected PRV infection were identified by PCR.PRV positive sample were inoculated on PK-15 cells after grinding and degerming,with further experiment including virus isolation,plague purification,PCR and IFA identification,TCID50 confirmed by Reed-Muench method,inoculation test and observation of clinical symptoms in mice.The gEgene of purified PRV and brain tissue samples of dead mice were identified by sequencing analysis.The results showed that the virus grown on PK-15 cells could produce typical cytopathic effect（CPE）after 24h;After three rounds of plaque purification,the isolate was PRV positive identified by PCR and IFA,and nominated as HeNZK-2014;The TCID50 of the isolate was 10-9.77/0.1mL;The virus in 1×108 TCID50 inoculation was able to cause itching,tearing,death in infected mice,and PRV could be detected in tissues of dead mice;The molecular genetic variation analysis of gEgene by PCR amplification and clone sequencing indicated that the gE gene from brain tissue of infected mice shared 100.0% homology with HeNZK-2014,and located in a relatively independent branch with newly pandemic isolates in recent years after 2011,but was far from the classical strains before 2011,and both had two insertion of aspartic acid（D）at sites of 48 and 496amino acids,which were considered to be the typical characteristics of PRV variants.This study successfully isolated a PRV variant,which laid a foundation for further research on vaccine development,prevention and control against PRV variants.