1株多毒株重组PRRSV的分离鉴定与遗传进化分析  

Isolation,Identification and Genetic Evolution Analysis of a Multi-strain Recombinant PRRSV

作　　者：元娜[1,2] 邵明珠 吕凤霞 窦丽娜 李向清 赵宝凯 董世山 YUAN Na;SHAO Mingzhu;LYU Fengxia;DOU Lina;LI Xiangqing;ZHAO Baokai;DONG Shishan(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071000,China;Beijing Vica Biotechnology Co.,Ltd.,Beijing 100085,China;Henan Muxiang Animal Pharmaceutical Co.,Ltd.,Zhengzhou 450000,China)

机构地区：[1]河北农业大学动物医学院,河北保定071000 [2]北京大伟嘉生物技术股份有限公司,北京海淀100085 [3]河南牧翔动物药业有限公司,河南郑州450000

出　　处：《中国兽医杂志》2025年第3期58-65,共8页Chinese Journal of Veterinary Medicine

摘　　要：为了分析猪繁殖与呼吸综合征病毒(PRRSV)遗传变异特性,本试验对来自山西省某猪场疑似感染PRRSV的母猪血清和仔猪肺脏组织样品进行PRRSV实时荧光定量逆转录PCR(qRT-PCR)检测,阳性肺脏组织样品研磨液接种于猪肺泡巨噬细胞(PAMs),将出现细胞病变的PAMs进行有限稀释法纯化后用间接免疫荧光(IFA)鉴定,利用逆转录PCR(RT-PCR)分段扩增分离毒株全基因组序列,并用分子生物学软件进行开放阅读框5(ORF5)基因和全基因组的同源性和遗传进化分析、非结构蛋白2(Nsp2)氨基酸特征及全基因组序列的重组分析。结果显示,qRT-PCR检测样品总阳性率为80%(16/20),从PRRSV阳性仔猪肺脏组织中分离获得1株PRRSV,命名为SXZY202301;分离毒株基因组全长15 021 bp,与NADC30毒株的核苷酸同源性达到90.4%,在遗传进化树中处于同一分支,属于Sublineage 1.8;ORF5基因与IA/2014/NADC34毒株的ORF5基因高度相似,同源性达到了95.1%,在遗传进化树中处于同一分支,属于Sublineage 1.5;分离毒株与类NADC30毒株Nsp2氨基酸序列具有相同的131个氨基酸缺失的典型特征;重组分析结果显示,SXZY202301存在6个重组位点,是以类NADC30毒株为主要亲本,以JXA1毒株和类NADC34毒株为次要亲本的多毒株重组类毒株。本试验为进一步研究PRRSV的重组和演化提供参考。To analyze the genetic variation characteristics of porcine reproductive and respiratory syndrome virus(PRRSV),serum samples from sows and lung tissue samples from piglets suspected of being infected with PRRSV at a pig farm in Shanxi Province were tested using real-time fluorescence quantitative reverse transcription PCR(qRT-PCR).Positive lung tissue samples were inoculated into porcine alveolar macrophages(PAMs).After purification by limiting dilution,the PAMs showing cytopathic effects were identified by indirect immunofluorescence assay(IFA).Full-length genome sequencing of the isolated strain was performed by segmental reverse transcription PCR(RT-PCR)amplification,followed by homology and genetic evolution analysis of the open reading frame 5(ORF5)gene and full genome sequence using molecular biological software.Amino acid features of nonstructural protein 2(Nsp2)and recombination analysis of the full genome sequence were also conducted.The results showed that the overall positivity rate of the qRT-PCR was 80%(16/20).One PRRSV strain was isolated from the lung tissue of a PRRSV-positive piglet,named SXZY202301.The genome of this isolated strain was 15021 bp in length,with a nucleotide homology of 90.4%to the NADC30 strain.It clustered in the same branch of the phylogenetic tree,belonging to Sublineage 1.8.The ORF5 gene of the isolated strain showed a high similarity(95.1%)to the ORF5 gene of the IA/2014/NADC34 strain and clustered in the same branch of the phylogenetic tree,belonging to Sublineage 1.5.The Nsp2 amino acid sequence of the isolated strain exhibited the typical characteristic of 131 amino acid deletions,which is similar to that of the NADC30 strain.Recombination analysis revealed that SXZY202301 had six recombination sites and was a multi-strain recombinant virus,primarily derived from the NADC30-like strain and secondarily from the JXA1 strain and NADC34-like strain.This study provides valuable reference for further research on PRRSV recombination and evolution.

关 键 词：猪繁殖与呼吸综合征病毒(PRRSV) 分离鉴定 测序分析 遗传进化 重组 

分 类 号：S855.3[农业科学—临床兽医学]