湖南省部分地区猪伪狂犬病病毒的分离及主要毒力基因序列扩增与分析

Isolation and Genetic Analysis of Major Virulence Gene Sequence of Pseudorabies Virus in Some Areas of Hunan Province,China

为了解当前湖南省猪伪狂犬病病毒(PRV)病原特性及基因变异情况,研究于2017—2018年从湖南省长沙、株洲、岳阳和怀化等4个地区疑似发生猪伪狂犬病(PR)的8个猪场采集组织病料8份,经荧光定量PCR检测,其中5份为PRV阳性,将PRV阳性组织样品处理后接种于猪肾上皮细胞(PK15)并连续传代,最终成功分离到5株病毒,且进一步PCR鉴定证明分离的5株病毒均为PRV。其后对5株PRV TCID_(50)进行测定,同时对所有毒株的毒力或免疫相关基因(gB、gG、gH、gI、gL、gM、gE、TK和PK)进行扩增、测序及分析。根据地区来源将5株PRV分别命名为HuN-HH株、HuN-YY株、HuN-NX株、HuN-ZZ株和HuN-LY株,滴度分别为10^(-6.37)、10^(-7.12)、10^(-7.44)、10^(-7.62)和10^(-7.94) TCID_(50)/100μL。进一步序列分析结果发现研究分离的5株PRV的毒力或免疫相关基因核苷酸序列相对保守,与国内2012年以后流行的PRV变异株同源性最高,与国内流行的传统株和国外疫苗株同源性相对较低,值得注意的是,HuN-HH株gG基因有一段连续的氨基酸序列插入。以上研究结果表明,研究分离的PRV毒株均属于变异株,且部分毒株特定基因序列存在较大变异,但其影响还需要进一步探究。

The present study was conducted to investigate the pathogenic characteristics and genetic variation of porcine pseudorabies virus(PRV) in Hunan Province,eight tissue samples were collected from 8 pig farms suspected of pseudorabies in 4 areas of Hunan Province from 2017 to 2018.The results showed that 5 of 8 tissue samples were identified PRV-positive by real-time PCR.After tissue samples treatment,the supernatants were inoculated into Porcine kindly epithelial cells(PK15).Finally,five virus strains were successfully isolated,and identified as PRV by PCR assay.Additionally,the TCID_(50) of five strains were investigated,and the virulence or immune related genes(gB,gG,gH,gI,gL,gM,gE,TK and PK) of all strains were amplified,sequenced and analyzed.The five strains were named HuN-HH,HuN-YY,HuN-NX,HuN-ZZ and HuN-LY strains according to the regional origins.The titers of five PRV isolates obtained in this study were 10^(-6.37),10^(-7.12),10^(-7.44),10^(-7.62) and 10^(-7.94) TCID_(50)/100 μL,respectively.Further sequence analysis showed that the nucleotide sequences of virulence or immune related genes of the five PRV strains isolated in this study were relatively conservative,and shared highest homology with the PRV variant strains prevalent in China after 2012,while had relatively low homology with the Chinese PRV traditional strains before 2012 and PRV strains prevalent in foreign countries.The above results indicate that five PRV strains isolated in this study are variant strains,and some strains have large variation in specific gene sequences,but its impact needs to be further explored.