三谱系重组猪繁殖与呼吸综合征病毒株分离鉴定与基因组特征分析  

Isolation,Identification and Genetic Variation of a Three-Lineage Strain Recombined Porcine Reproductive and Respiratory Syndrome Virus

作　　者：李欣蕾 孙久英 杨程 程宁 王凯月 王欢欢 程雪娇 赵健 孙英峰 LI XinLei;SUN JiuYing;YANG Cheng;CHENG Ning;WANG KaiYue;WANG HuanHuan;CHENG XueJiao;ZHAO Jian;SUN YingFeng(College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300384;Tianjin Nongken Kangjia Ecological Breeding Co.,Ltd,Tianjin 300384;Tianjin Zhongsheng Challenge Biotechnology Co.,Ltd,Tianjin 300380)

机构地区：[1]天津农学院动物科学与动物医学学院,天津300384 [2]天津农垦康嘉生态养殖有限公司,天津300384 [3]天津市中升挑战生物科技有限公司,天津300380

出　　处：《中国农业科学》2024年第24期4978-4989,共12页Scientia Agricultura Sinica

基　　金：天津市科技支撑计划重点项目(22YFZCSN00100);天津市优秀农业科技特派员项目(22zycgsn00570);天津市科技计划项目(22YDTPJC00420)。

摘　　要：【目的】通过对天津地区某疑似猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)发病断奶仔猪肺脏组织进行病毒分离鉴定以及全基因组分子特征分析,为猪场疫病防控提供基础数据。【方法】采集发病猪肺组织检测并将PRRSV核酸阳性样品接种于猪肺泡巨噬细胞(porcine alveolar macrophages,PAMs),采用有限稀释法纯化分离病毒,对已纯化的病毒进行间接免疫荧光试验(indirect immunofluorescence assay,IFA)鉴定,利用重叠引物RT-PCR分段扩增并克隆测序获得全基因组序列,并通过生物学分析软件分别对分离毒株的核苷酸与氨基酸序列进行同源性、遗传演化与重组事件分析。【结果】成功分离1株PRRSV毒株,将其命名为N1;该毒株全基因组全长大小为15016 bp(不含polyA尾),核苷酸同源性比对分析显示与谱系8.3代表毒株JXA1全基因组同源性最高,为90.0%,与其他谱系各代表毒株的全基因组同源性为85.0%—90.0%,其中ORF2a、ORF2b、3′UTR区域同源性与谱系3(QYYZ-like)毒株最高达93.7%,与谱系1.8(NADC30-like)毒株在ORF5-ORF7区域同源性最高达96.2%,其余区域均与谱系8.3(JXA1-like)毒株有高度同源性,同时N1毒株的Nsp2区域具有与谱系1.8(NADC30-like)毒株特征一致的131(111+1+19aa)个不连续氨基酸缺失特征;GP5蛋白的预测毒力位点及主要中和抗原表位与谱系1(NADC34-like、NADC30-like)PRRSV毒株相同,但位于N^(33)、N^(44)和N^(51)的3个N-糖基化位点存在一定程度突变,这些潜在的糖基化位点突变很可能造成病毒致病力差异,并导致病毒免疫逃逸发生;根据ORF5及全基因组序列构建的遗传进化树基因分型显示,基于N1毒株ORF5基因遗传进化树与谱系1.8(NADC30-like)毒株亲缘较近,处于同一分支,但基于N1毒株全基因组遗传进化树与谱系8.3(JXA1-like)毒株处于同一分支,提示该毒株存在重组可能性;通过Simplot和RDP4.0等多种生物信息学软【Objective】This study aimed to provide basic data for prevention and control of swine diseases in pig farms through analysis about molecular characterization of PRRSV isolated from the lungs of diseased piglets in pig farm in Tianjin.【Method】Lung tissues of diseased piglets were collected,and PRRSV nucleic acid positive samples were inoculated into porcine alveolar macrophages (PAMs) for virus isolation and identification.The virus was purified by limited dilution method and identified by indirect immunofluorescence assay (IFA).Then,it was segmented amplification using overlapping primers of RT-PCR,and whole genome sequence was obtained by cloning.The nucleotide and amino acid sequences of N1 strain were analyzed for homology,genetic evolution,and recombination events using biological analysis software.【Result】The PRRSV strain,named N1,was successfully isolated with the whole genome length of 15016 bp,excluding polyA tail.The homology analysis based on whole genome sequences showed that the homologous of N1 strain was 85.0%-90.0%to other lineage PRRSV strains,with the highest homology of 90.0%to lineage 8.3,the representative strain of JXA1,and the N1 isolate shared 93.7%identity with lineage 3 (QYYZ-like) in the ORF2a,ORF2b,and 3'UTR regions,and shared 96.2%identity with in the ORF5-ORF7 regions with lineage 1.8 (NADC30-like),while the rest of the regions had a high degree of homology with sublineage 8.3 (JXA1-like).It had similar 131 (111+1+19aa) discontinuous amino acid deletion pattern with lineage 1.8 (NADC30-like) in Nsp2-coding region.The virulence sites and major neutralizing antigenic epitopes of GP5 protein were identical to lineage 1 (NADC34-like,NADC30-like),but three N-glycosylation sites locating at N~(33),N~(44) and N~(51) were mutated to a certain extent compared with the other representative strains.These potential glycosylation sites were likely to contribute to the differences in viral virulence and led to the occurrence of viral immune escape.According to the genetic phylogenetic t

关 键 词：猪繁殖与呼吸综合征病毒 分离鉴定 重组 遗传演化分析 

分 类 号：S852.651[农业科学—基础兽医学]