猪伪狂犬病病毒四川株的分离鉴定及UL43基因序列分析

Identification of swine pseudorabies virus isolated from Sichuan and sequence analysis of UL43 gene

从四川某猪场发病仔猪体内分离到1株病毒,该毒株能在Vero、MDBK、PK15、ST、MDCK、BHK21、Marc145、鸡胚成纤维细胞(CEF)上增殖并产生细胞病变。通过蚀斑克隆对其进行纯化,克隆毒株在Vero细胞上为3.0×107TC ID50/0.1mL。该病毒在Vero细胞上连传15代,其TCID50变化很小。病毒对5-溴脱氧尿核苷、氯仿敏感。用该病毒接种家兔和仔猪均出现典型的伪狂犬病症状,gE-ELISA检测接种分离毒的仔猪血清,伪狂犬病毒(PRV)抗体阳性。电镜观察可见直径110～140 nm的典型疱疹病毒粒子。上述结果表明分离株为PRV,并命名为SE株。根据已发表的UL43序列设计1对引物,以分离毒株基因组DNA为模板进行PCR扩增,对目的产物进行克隆及测序分析,结果与GenBank收录的其他PRV毒株(Becker、Ea)的UL43核苷酸序列同源性为98.8%,氨基酸同源性分别为95.2%、97.3%。

One virus isolate was got from brains and visceral organ of piglets.The typical cytopathic effect(CPE) could be seen when the isolate was cultured on different kinds of cells(Vero,MDBK,PK15,ST,MDCK,BHK21,Marc145 and CEF) and its TCID50 was 3.0×10-7/0.1mL on Vero cells.The isolate was sensitive to 5-bromodeoxyuridine and chloroform.Typical clinical signs could be observed when the pigs and rabbits were infected with the isolate.The antibodies to pseudorabies virus(PRV) were positive in the sera from the piglets infected with the isolate by gE-ELISA.Electron microscopical observation showed a typical diameter of 110~140 nm herpes virus particles.The isolate was demonstrated to be PRV,and named as SE strain.According to the UL43 gene sequence of PRV in GenBank,a pair of primers was designed.And then the PCR product of the UL43 gene was cloned and sequenced.The sequencing analysis of the gene wasperformed using the DNAStar software.The results demonstrated that compared with the UL43 genes of the PRV strains Becker and Ea published in GenBank,the identity of the nucleotide sequences was 98.8%,and the similarities of amino acid sequences were 95.2% and 97.3%,respectively.