伪狂犬病病毒变异株UL3、UL46和UL50基因失活株的构建与生物学特性分析

Construction of UL3,UL46 and UL50 gene inactivation mutants of pseudorabies virus variant and their biological characteristics

为探究UL3、UL46和UL50基因对伪狂犬病病毒(pseudorabies virus, PRV)毒力的影响,本研究应用En Passant技术,分别对PRV变异株AH02LA的细菌人工染色体(bacterial artificial chromosome, BAC)的UL3、UL46和UL50起始密码子进行敲除,获得3株基因失活重组BAC:BAC^(PRV-UL3knock)、BAC^(PRV-UL46knock)和BAC^(PRV-UL50knock)。将BAC DNA与带有同源臂的gE/gI序列片段共转染猪睾丸(swine testis, ST)细胞,获得重组病毒:PRV-UL3^(knock)、PRV-UL46^(knock)和PRV-UL50^(knock)。生长动力学试验结果显示UL3、UL46和UL50突变失活株在感染12和36 h滴度显著低于PRV AH02LA亲本株,表明UL3、UL46和UL50可能介导病毒复制。小鼠的致病性试验显示,PRV-UL3^(knock)、PRV-UL46^(knock)和PRV-UL50^(knock)对小鼠的LD_(50)分别为10^(4.44)TCID_(50)、10^(4.5)TCID_(50)和10^(4.05)TCID_(50),与PRV AH02LA相似(LD_(50)为10^(4.91)TCID_(50)),暗示UL3、UL46和UL50基因可能对PRV毒力无显著影响。本研究分析了UL3、UL46和UL50基因对PRV毒力的影响,为PRV致病机制的研究提供了参考。

To investigate the effect of UL3,UL46 or UL50 gene on the virulence of pseudorabies virus,BACPRV-UL3 knock,BACPRV-UL46 knock and BACPRV-UL50 knock were generated by deletion of start codons in the UL3,UL46 or UL50 gene of bacterial artificial chromosome(BAC)of a virulent PRV isolates AH02 LA strainusing an En Passant technology.Three recombinant viruses were rescued by transfection of BACPRV-UL3 knock,BACPRV-UL46 knockor BACPRV-UL50 knock DNA and PCR fragments of the gE/gI gene with homologous sequence on ST cells,namely PRV-UL3^(knock),PRV-UL46^(knock)and PRV-UL50^(knock).Growth kinetics experiments showed that the titers of PRV-UL3^(knock),PRV-UL46^(knock)and PRV-UL50^(knock)were significantly lower than that of PRV AH02 LA parental strain at 12 and 36 hours post-infection,indicating that UL3,UL46 and UL50 gene might play an important role in viral replication.The pathogenicity test in mice showed that LD50 of PRVUL3^(knock),PRV-UL46^(knock)and PRV-UL50^(knock)were 104.44 TCID50,104.5 TCID50 and 104.05 TCID50,respectively,which were similar to that of PRV AH02 LA parental strain(LD50 was 104.91 TCID50).It suggests that UL3,UL46 and UL50 genes seemed to be of no significant effect on the virulence of PRV,which provides a reference for future research on the pathogenesis of PRV.