犬源伪狂犬病毒BJ/RD株的分离鉴定及gE基因分析

摘要

Typical pseudorabies-like itching sign was observed in a dog that had no clear route of transmission. Samples of sympathetic trunk, cervical spinal cord, trigeminal nerve and brainstem were tested using PRV-specific PCR. The PRV DNA fragments were detected in these tissues. Then brainstem was homogenized and inoculated into Vero cell cultures. At 72 h post inoculation, PRV specific CPE was showed in Vero cells, and the results of immunohistochemical test and PCR were positive. The results indicated that the isolated virus was PRV and designated as BJ/RD strain. The gE gene of the BJ/RD strain was sequenced and compared with the known sequences in GenBank. The BJ/RD strain shared 100%nucleotide homology with canine BJ/YT strain from Beijing and porcine HB/HD strain from Hebei province, and 99.9%nucleotide homology with HB/HS, HB/BD and HB/LF strains.%本研究对1例具有剧烈搔痒症状，但无明确传播途径的病犬的组织进行伪狂犬病毒（Pseudorabies virus，PRV）特异性PCR检测，从脑干、三叉神经、交感神经和颈段脊髓中扩增出预期大小的DNA片段。将PCR为阳性的脑干组织病料接种Vero细胞，培养72 h后细胞发生圆缩、脱落等病变。对出现稳定病变的细胞培养物进行PRV PCR检测获得预期大小的DNA片段，用PRV mAb进行免疫组化染色，部分细胞中出现大量棕褐色阳性颗粒，从而判定感染细胞中存在PRV，将此分离株命名为BJ/RD株。根据GenBank登录的PRV（JF797219）序列设计引物，扩增gE基因，并对扩增产物进行序列测定与分析，结果显示， BJ/RD株与此前分离的犬源毒株BJ/YT及2012年河北猪群分离株HB/HD的gE基因序列完全相同（核酸同源性为100%），与2012年河北地区分离株HB/HS、HB/BD、HB/LF核苷酸同源性99.9%，而与国内外其他猪源PRV分离株gE基因序列有一定差异。