高致病性猪繁殖与呼吸综合征病毒SHxx13/2013株的分离与鉴定

摘要

In early 2013, an acute disease characterized by high fever and respiratory problems in piglets outbroke on a pig farm in Shanghai and caused many deaths. To investigate the causative agent, a total of 15 serum samples were collected and viruses were examined in RT-PCR. Eleven out of of 15 clinical samples showed strong Porcine reproductire and respiratory syndrone virus (PRRSV) signal and the same PCR fragments as compared with highly pathogenic PRRSV (HP-PRRSV). One PRRSV strain was isolated from a PCR positive sample and designated as SHxx13/2013 strain. The SHxx13/2013 strain induced cytopathic effect (CPE) on MarC-145 cells at the first passage, which shared similar plaque morphology and growth kinetics with HuN4 strain. The complete genome of SHxx13/2013 strain was 15 319 bp. The deletion of total 30 amino acids occurred at positions 482 and 534-562 in Nsp2 gene, which was a common feature of HP-PRRSV. The complete genome of the SHxx13/2013 strain was 99.6%identity to that of HuN4 strain, indicating that it was close to HuN4. The results concluded that HP-PRRSV was the causative agent in pig population in Shanghai.%2013年初上海某猪场保育猪群中出现高热、呼吸障碍等临床表现，导致大部分发病猪死亡，为了确定此次猪群中爆发疫病的病原，本研究从发病猪群中随机采集了15份血液样品，利用RT-PCR方法对本次疫病病原进行了检测。结果显示，有11份临床样品呈现猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus, PRRSV）强阳性，且与高致病性猪繁殖与呼吸综合征病毒（Highly pathogenic Porcine reproductive and respiratory syndrome virus, HP-PRRSV）对照大小相一致。随后从阳性样品中选取SHxx13/2013在Marc-145细胞中进行病毒的分离与鉴定，结果显示SHxx13/2013在细胞接种后第1代即出现明显的细胞效应（cytopathic effect, CPE），其病变特征与高致病性PRRSV强毒HuN4株一致。对SHxx13/2013第5代细胞分离毒进行RT-PCR和IFA等特异性鉴定，结果显示该毒株为PRRSV分离株，其蚀斑形态和生长特性与强毒HuN4株相似。分段克隆该毒株全长基因进行测序和序列分析，结果显示，新分离的流行毒SHxx13/2013株全长基因组为15319 bp，其Nsp2基因特征与HP-PRRSV一致，在第482位和第534~562位发生两处共30个氨基酸的不连续缺失，且该毒株与高致病性PRRSV代表毒株HuN4亲缘关系较近，全长基因的核苷酸同源性为99.6%。本研究结果表明新分离的SHxx13/2013株属于高致病性PRRSV分离株，据此我们推测今年年初上海某猪场爆发的疫情主要是由HP-PRRSV感染所致。