将RT-PCR检测为猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus, PRRSV）阳性的肺组织碾磨、过滤后接种MARC-145细胞，在细胞上连续传代，上清传至第二代后能产生细胞病变。通过RT-PCR方法可以检测到释放在细胞上清中PRRSV RNA。间接免疫荧光试验也能检测细胞内PRRSV N蛋白的表达，表明该PRRSV毒株成功地在MARC-145细胞中分离，命名为PRRSV NN1396株。对NN1396分离株GP5基因进行克隆测序分析，结果表明该毒株与NCBI登录的PRRSV GP5核苷酸同源性为62.9%~98.2%，与PRRSV原型毒株VR-2332的同源性为87.9%，与高致病性毒株PRRSV JXA1同源性为98.0%，通过GP5的遗传进化分析发现所分离的PRRSV为北美型PRRSV，且与高致病性PRRSV分布在同一个小分支上；对部分nsp2基因的测序、比对结果表明，该部分基因与NCBI登录的参考序列VR-2332氨基酸同源性为73.0%，与JXA1的同源性为94.8%。NN1396分离株Nsp2蛋白含有高致病性PRRSV特异的1+29个氨基酸的缺失，此外，在缺失29个氨基酸区域的上游，含有连续19个氨基酸缺失。该PRRSV毒株的分离和Nsp2蛋白部分序列的缺失鉴定，为下一步研究Nsp2部分氨基酸缺失对病毒生物学特性影响奠定基础。%The lung tissues with Porcine reproductive and respiratory syndrome virus(PRRSV) RT-PCR positive results were homogenized and inoculated into MARC-145 cells for virus isolation. PRRSV typical CPE was observed at the second passage. PRRSV RNA was detected in supernatants using RT-PCR. PRRSV N protein was confirmed in inoculate MARC-145 cells. The obtained PRRSV was designated as NN1396. Hypervariable region of GP5 protein was sequenced and analyzed. The GP5 of NN1396 strain shared 62.9%-98.2%identities with other PRRSV strains. Phylogenetic analysis showed that NN1396 strain belonged to North American genotype and HP-PRRSV. Sequence analysis of partial nsp2 gene showed that NN1396 strain had 81.4%amino acid identity with VR-2332 strain and 96.5%with JXA1. More importantly, NN1396 strain contained a deletion of 19 aa in nsp2 coding region , which was located upstream of 29 aa deletion. Characterization of PRRSV NN1396 strain was useful to study the effect of partial gene deletion of nsp2 on its biological activities.
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