猪群混合感染伪狂犬病毒及乙型脑炎病毒的鉴定与分离

DUAL INFECTION OF SWINE PSEUDORABIES VIRUS AND JAPANESE ENCEPHALITIS VIRUS IN PIGS

云南省文山市某规模化猪场2015年7-8月频繁发生以种公猪一侧睾丸肿大,母猪流产、产死胎、木乃伊胎及弱仔为主要临床症状的繁殖障碍疫情.为了查明病因,对送检流产胎猪进行病理解剖,采集胎猪及胎盘病料进行猪瘟病毒、猪繁殖与呼吸综合征病毒欧洲型及美洲型毒株、猪细小病毒、伪狂犬病毒、乙型脑炎病毒、猪流产衣原体、布氏杆菌及弓形虫PCR和real-time PCR检测,同时应用猪肾原代细胞及BHK-21传代细胞进行病毒分离鉴定以及细菌分离鉴定.检测结果显示,从流产胎猪内脏器官、大脑及胎盘组织扩增出乙型脑炎病毒prM基因及伪狂犬病毒gE基因目的条带.序列分析结果显示,扩增的乙型脑炎病毒prM基因序列与NCBI GenBank中的参考毒株（Accession no.AB594829.1）序列同源性达99%;伪狂犬病毒gE基因核苷酸序列与NCBI GenBank中的参考毒株（Accession no.KT936475.1）序列同源性达100%.应用BHK-21传代细胞盲传分离获得1株可致细胞病变（cytopathic effect,CPE）的病毒株,鉴定为乙型脑炎病毒,并命名为JEV-2015-WS-YN.由此推测,引起此次猪群繁殖障碍极有可能为日本乙型脑炎病毒和伪狂犬病毒共同感染造成.

A reproductive disease with clinical manifestations of boar testicle swelling, sow abortion, stillbirth, mummy and weak off spring occurred on a large-scale pig farm from July to August of 2015 in city of Wenshan, Yunnan province.In order to identify the etiological agent（s）, CSFV, PRRSV （American/EU type/）, PPV, PRV, JEV,Chlamydia psittaci,Toxoplasma gondii,Brucella suis were examined using PCR, real-time PCR and bacterial isolation. As a result, 600 bp fragments of PrM gene of JEV and 810 bp fragments of gE gene of PRV were amplified in RT-PCR or PCR. Sequence analysis showed that the homology of nucleotide sequences of the amplicons was 99% with the reference strains of JEV （Accession：AB594829.1） and 100% with PRV （Accession：KT936475.1） from GenBank. In addition, primary pig kidney cells and BHK21 cells were used for virus isolation. One virus isolate of JEV was obtained from aborted fetal tissue and designated as JEV-2015-WS-YN. Although PRV detection in PCR was positive, isolation of PRV failed after 5 blind passages in primary pig kidney cells and BHK21 cells. According to the animal pathogenicity, epidemiology and etiology analysis, this reproductive failure was most likely caused by dual infection of JEV and PRV. After immunization with Japanese encephalitis attenuated live vaccine （SA14-14-2 strain） and pseudorabies gene deletion vaccine （Bartha K61 strain）, the reproductive problem was prevented and controlled successfully in pig herds.