猪伪狂犬病病毒流行毒株gC基因的分子特征及其对小鼠的毒力试验

Molecular characterization of gC gene of pseudorabies virus epidemic isolates and their virulence test in mice

为了阐明猪伪狂犬病病毒(PRV)流行毒株gC基因的分子特征及其对小鼠的毒力,对来自不同年代分离的6株PRV流行毒株进行了gC基因分子变异特征分析,通过接种BALB/c小鼠检验毒株的毒力。对临床病例分离的6株PRV和30个Gen Bank下载的gC基因序列进行分析表明,这6株PRV与2012年以后流行的毒力增强的变异毒株处于同一分支;这些毒株的氨基酸同源性为98.7%~100%,而与经典的PRV-SC毒株的氨基酸同源性仅为93.2%~94.0%。PRV毒株g C蛋白第52~70位氨基酸为高变区,尤其第64~70位出现7个氨基酸(AAASTPA)插入。对gC蛋白糖基化位点分析表明,PRV变异毒株较经典毒株增加了6个O-连接糖基化位点,且gC蛋白B细胞表位出现3处突变,分别为P69R、P80Q和N83G。gC蛋白上硫酸乙酰肝素结合域(HBD)出现2处突变,分别为HBD1第5位氨基酸由经典毒株的P突变为Q,HBD3的第3位氨基酸由Y突变为C。用分离的6株PRV接种BALB/c小鼠试验显示,PRV-HRB和PRV-SH毒株半数致死量(LD50)均为1×10^(3.21)/mL,PRV-DL、PRV-JL及PRV-GZL的LD50为1×10^(3.75)/mL,PRV-JF的LD50为1×10^(4.32)/mL。上述结果阐明了PRV流行毒株g C基因的分子特征及其对小鼠的毒力,为该病毒的致病机制研究提供了科学依据。

To elucidate the molecular characterization of the gC gene of pseudorabies virus（PRV） epidemic isolates and their virulence in mice,six PRVisolates from clinical cases at different areas were isolated to be used for the genetic variation analysis and virulence test in BALB/c mice. The gC amino acid sequences of the six PRV isolates and other thirty sequences of PRY strains available in GenBank were used for analysis and comparison. The results suggested that the six isolates and PRV variants isolated in China since 2011 belonged to the same branch,and shared from 98. 7% to 100% identity with PRV variants,but only from 93.2% to 94.0% identity with the classic strain PRV-SC.A hypervariable region located in No. 52--70 aa was found on the gC protein for the six PRV isolates, especially the insert sequences of seven amino acids（^64AiASTPAT^70）.The analysis of the gC protein glycosy-lation sites showed that six O-linked glycosylation sites were added compared with the classic strain PRV-SC. Furthermore,one mutation of P69R and two mutations of P80@ and N83G were found in the B cell epitopes of gC,respectively. Meanwhile,two mutations were found in the heparin binding domain（HBD）,of which the fifth amino acid was mutated into Q,and the third amino acid was mutated into C. BALB/c mice were inoculated with the six PRVisolates to evaluate their virulence. The results indicated that the LD50 of PRV-HRB and PRV-SH isolates was 1 X I0321/mL,PRV-DL,PRV-JL and PRV-GZL were 1×10^3.75/mL in LD50, while PRV-JF was 1× 10^4.32/mL in LDs0. This study result illuminated the correlation between the molecular characterization of the gC gene of PRV new epidemic isolates and their virulence in mice,which provided a scientific basis for studying the pathogenesis of the virus.