一株重组类NADC30猪繁殖与呼吸综合征病毒的分离鉴定及基因组分析

Isolation, Identification and Genome Analysis of a Recombinant NADC30-like Porcine Reproductive and Respiratory Syndrome Virus

关键词:  类NADC30猪繁殖与呼吸综合征病毒  分离鉴定  基因重组：细胞嗜性

Key words:  NADC30-like PRRSV  isolation and identification  gene recombination  cellular tropism

摘要:

为分离上海地区猪繁殖与呼吸综征病毒（PRRSV）并分析其分子遗传进化关系，对来自该地区某猪场疑似PRRSV感染病猪的2份肺样进行RT-PCR检测，阳性样品接种猪肺泡巨噬细胞(PAMs)，将盲传3代后出现细胞病变的PAMs经间接免疫荧光鉴定，通过RT-PCR扩增分离株全基因组序列，测序后与GenBank登录的参考株全基因组序列进行同源性及遗传进化分析，分析了分离株Nsp2、GP5氨基酸序列的遗传进化关系，并对分离株做了重组分析和细胞嗜性试验。结果显示，成功分离出1株PRRSV，命名为SH2019，其全基因组开放阅读框大小为14677 bp，分离株与NADC30的核苷酸同源性最高，属于目前流行于中国的类NADC30 PRRSV，其Nsp2存在131个氨基酸缺失的分子特征；分离株是以谱系1毒株为主要亲本，以谱系3、谱系5或谱系8毒株为次要亲本，在12601~12901 nt(ORF2~ORF4)发生了基因重组的重组毒株，分离株不能感染Marc-145细胞。本研究分离鉴定一株重组类NADC30 PRRSV，可为该地区猪繁殖与呼吸综合征的防控提供参考。

Abstract:

To isolate the porcine reproductive and respiratory syndrome virus (PRRSV) in Shanghai area and analyze its molecular genetic evolution relationship, two lung samples for RT-PCR detection were collected from the suspected PRRSV-infected pigs in a pig farm of this area, and positive samples were inoculated with porcine alveolar macrophages (PAMs), the PAMs with cytopathic effect after 3 generations of blind passage were identified by indirect immunofluorescence assay, the whole genome sequence of the isolate was amplified by RT-PCR and sequenced, and the homology analysis was performed with the complete genome sequence of the reference strain registered in GenBank, the homology and phylogenetic analysis of the amino acid sequence of the isolate Nsp2 and GP5 were analyzed and the recombination event analysis was carried out，and cellular tropism test was investigated. The results showed that 1 strain PRRSV was successfully isolated and named SH2019, the isolate has the highest nucleotide homology with NADC30, it belongs to the NADC30-like PRRSV that is currently popular in China, and there is a molecular feature of 131 amino acid deletions in Nsp2, the isolate is a recombinant strain that takes the strain of lineage 1 as the main parent, and the strain of lineage 3, lineage 5 or lineage 8 as the secondary parent, with genetic recombination at 12601~12901 nt, the isolate couldn'' t infect Marc-145 cells. A recombinant NADC30-like PRRSV was isolated and identified in this study, which might provide reference for the prevention and control of PRRS in the region.