一株多谱系重组NADC34-like-PRRSV的分离鉴定及其基因组特征分析  

Isolation,Identification and Genomic Characterization of A Multi-lineage Recombinant NADC34-like PRRSV

作　　者：张兴民 张婧[1,2] 虞凌雪 王健 王方洲 李娇阳 李国秀 李冬 马雪青[1,2] 白兴文 龚真莉[1,2] 卢曾军 曾巧英[3] 孙普 ZHANG Xingmin;ZHANG Jing;YU Lingxue;WANG Jian;WANG Fangzhou;LI Jiaoyang;LI Guoxiu;LI Dong;MA Xueqing;BAI Xingwen;GONG Zhenli;LU Zengjun;ZENG Qiaoying;SUN Pu(Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China;Gansu Province Research Center for Basic Disciplines of Pathogen Biology,Lanzhou 730046,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730000,China;Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200441,China)

机构地区：[1]中国农业科学院兰州兽医研究所兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室,兰州730000 [2]甘肃省病原生物学基础学科研究中心,兰州730046 [3]甘肃农业大学,兰州730070 [4]中国农业科学研究院上海兽医研究所,上海200441

出　　处：《病毒学报》2024年第5期1062-1075,共14页Chinese Journal of Virology

基　　金：国家生猪技术创新中心牧原项目(项目号:NCTIPMY23004),题目:甘肃农村中小规模和散养户猪场的疫病实验室诊断及防治技术培训;甘肃省科技计划项目(项目号:20JR10RA022),题目:Lnc-000397抑制PRRSV复制的功能研究;兰州市科技计划项目(项目号:2023-1-44),题目:猪繁殖与呼吸综合征病毒N蛋白抗体竞争ELISA试剂盒的研制及推广应用。

摘　　要：本研究2021年9月从甘肃规模化猪场采集疑似感染猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)的仔猪肺脏组织和血清,经(Open Reading Frame 5,ORF5)基因分型RT-PCR扩增测序鉴定,阳性样品进行病毒分离。经间接免疫荧光试验和感染细胞病理电镜切片鉴定分离毒株,采用RTPCR方法对PRRSV分离株进行全基因组序列扩增,利用生物学软件分析其遗传演化特征,同时也对该场107份猪血清样本检测。研究结果显示,ORF5基因分型RT-PCR扩增测序结果表明,该猪场为NADC34-like-PRRSV感染;间接免疫荧光显示能够与PRRSV N蛋白单克隆抗体(SR30)反应,电镜观察到感染细胞中直径大小为50-80 nm病毒粒子,其基因组全长大小为15078 bp,将分离株命名为PRRSV GSJT/9/2021株。分离株与不同谱系代表株VR-2332、JXA1、HuN4、NADC30和NADC34的全长核苷酸序列同源性分别为85.2%、86.4%、86.5%、88.7%和85.9%;ORF5基因序列与代表毒株ORF5基因序列及编码氨基酸序列的相似性分别为83.7%~94.9%与84.6%~95.5%,其中与IA/2014/NADC34毒株的核苷酸和氨基酸同源性较高分别为94.9%和95.5%;基于GSJT/9/2021全基因组和ORF5基因分别构建进化树对分离株进行分型,结果显示GSJT/9/2021的全基因组与类NADC30毒株亲缘关系较近,属于同一个分支,而ORF5基因遗传进化树表明GSJT/9/2021毒株与类NADC34毒株亲缘关系最近;ORF5氨基酸序列分析结果显示,ORF5的变异程度最大,GP5蛋白中4个糖基化位点与国内流行的NADC34毒株相比额外增加一个糖基化位点第57位,在诱骗表位37-45 aa中第38、39位出现独特突变(H38Q,L39F);Nsp2氨基酸序列分析结果显示,Nsp2蛋白存在“111+1+19a”(323-433位、483位、504-522位)缺失模式,与NADC30毒株缺失模式一致;重组分析表明GSJT/9/2021株由NADC30-like PRRSV、HUN4与IA/2014/NADC34之间发生重组变异而来,重组位点分别位于Nsp4、Nsp9、GP2a和M的氨基端。107份In this study,lung tissue and serum samples of piglets suspected to be infected with PRRSV associated abortion were collected from large-scale pig farms in Gansu Province,China in September 2021.After ORF5 gene characteristic RT-PCR detection and sequencing,the virus was isolated with PAM cells.The strain was identified and isolated by indirect immunofluorescence assay and pathological electron microscopy sections of infected cells.The whole genome sequence of PRRSV isolates was amplified by RT-PCR.The genome characteristics and recombination were compared and analyzed with biological software.The results showed that the nucleotide and amino acid homology between the ORF5 gene and that of NADC34 strain was 94.9%and 95.5%,respectively.Indirect immunofluorescence showed that the virus could react with PRRSV N protein monoclonal antibody(SR30).Virions with diameter of 50-80 nm and total genome size of 15078 bp were observed in infected cells under electron microscopy.The isolate was named PRRSV GSJT/9/2021strain.The nucleotide homology between the isolates and representative strains VR-2332,JXA1,HuN4,NADC30 and NADC34 were 85.2%,86.4%,86.5%,88.7%and 85.9%,respectively.Phylogenetic trees were constructed based on the whole genome of GSJT/9/2021 and ORF5 gene respectively to type the isolates.The results showed that the whole genome of GSJT/9/2021 was closely related to NADC30-like strains and belonged to the same cladistic branch.The phylogenetic tree of ORF5 gene showed that GSJT/9/2021 was closely related to NADC34-like strain.ORF5 and Nsp2 showed the highest degree of variation in the genome of the isolates.Four glycosylation sites in GP5 protein showed an additional glycosylation site 57th compared with NADC34 strain prevalent in China,and unique mutations(H38Q,L39F)appeared at the 38th and 39th spots of decoy epitopes 37-45 aa.131 discontinuous amino acids(323-433,483,504-522)were absent in Nsp2protein.Recombination analysis showed that GSJT/9/2021 strain was derived from a complex recombination mutation between

关 键 词：猪繁殖与呼吸综合征病毒 多谱系重组毒株 分离鉴定 基因组特征 细胞嗜性 

分 类 号：S855.3[农业科学—临床兽医学]