贵州1株G9P[23]型猪A群轮状病毒的分离鉴定及基因组序列分析  

Isolation,identification,and genome sequence analysis of a strain of G9P[23]porcine group A rotavirus from Guizhou

作　　者：柳佳佳 梁海英[1] 曾智勇[1] 汤德元[1] 王彬[1] 叶泥 边孟婷 黄书 田红利 潘向英 LIU Jiajia;LIANG Haiying;ZENG Zhiyong;TANG Deyuan;WANG Bin;YE Ni;BIAN Mengting;HUANG Shu;TIAN Hongli;PAN Xiangying(College of Animal Science,Guizhou University,Guiyang550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025

出　　处：《中国兽医学报》2024年第5期900-906,913,共8页Chinese Journal of Veterinary Science

基　　金：贵州省科技支撑计划资助项目(黔科合支撑[2021]一般162项目)。

摘　　要：为了解贵州省G9型猪轮状病毒(PoRV)流行毒株的基因组特征和遗传进化关系,本试验对送检腹泻仔猪的肠内容物和粪样进行病原检测,选取G9型PoRV阳性样品通过Vero细胞进行分离培养及病毒鉴定,并对分离毒株进行基因组遗传变异分析。结果显示,阳性样品经胰酶预处理后接种至Vero细胞,盲传至第15代出现稳定CPE,前期细胞变圆后固缩、脱落,后期胞浆界限不清,出现拉网现象,用特异性引物对细胞培养物进行RT-PCR鉴定,成功扩增341 bp的目的条带。细胞培养物的间接免疫荧光鉴定显示有特异性绿色荧光反应,电镜观察下可见晶格状排列的无囊膜病毒粒子,大小约70 nm,表明成功分离到1株PoRV,将之命名为GZZY2021。病毒增殖曲线结果显示,用GZZY2021株感染细胞后,0~12 h病毒含量最低,12~30 h病毒增殖速度最快,30~48 h病毒增殖速度较平缓,48 h病毒滴度达到峰值(10-5.60/0.1 mL),而后进入稳定期。全基因组克隆测序分析结果显示,GZZY2021分离株基因组全长为18 506 bp, 11个基因最相似序列同源性均>95.00%,VP1、VP2、VP4、VP7、NSP1、NSP3、NSP4基因与猪源毒株同源性最高,VP3基因与大熊猫源毒株相似性最高,VP6、NSP2、NSP5基因与人源毒株同源性最高,提示分离株为三元重配毒株,完整的基因型为G9-P[23]-I1-R1-C1-M1-A8-N1-T1-E1-H1。VP4、VP7基因与同型参考毒株相比,分别发生5、3处独有氨基酸位点变异;与NX疫苗株的中和表位分别存在22、4处氨基酸位点差异。重组分析结果显示,GZAS2020株的NSP5基因是以PTR(FJ422141.1)为主要亲本毒株,A411(EF990694.1)为次要亲本毒株重组产生的毒株,重组断点位于121和321 bp。综上所述,本研究应用Vero细胞成功分离到1株G9P[23]型基因重配PoRV,不但可对持续监测贵州省PoRV的流行动态趋势提供研究数据,而且对筛选该领域的候选疫苗株以及该病的深入研究打下基础。To understand the genomic characteristics and genetic evolution relationship of G9 porcine rotavirus(PoRV)epidemic strain in Guizhou Province,the pathogen of intestinal contents and fecal samples of diarrhea piglets was detected,and the positive samples of G9 PoRV were isolated and cultured by Vero cells,and the genetic variation of the isolated strain was analyzed.The results showed that the positive samples were inoculated into Vero cells after pre-treatment with trypsin,and stable CPE appeared after blind transmission to the 15th generation.After the cells were rounded in the early stage,they contracted and fell off,and the cytoplasm boundary was unclear in the later stage,which led to the net was pulled.The specific primers were used to identify the cell culture by RT-PCR,and the target band of 341 bp was successfully amplified.Indirect immunofluorescence identification showed specific green fluorescence reaction,and electron microscope observation showed that there were lattice-arranged encapsulated virus particles with a size of about 70nm.A strain of PoRV was successfully isolated and named GZZY2021.The growth curve showed that the virus content of GZZY2021strain was the lowest in 0-12h,and the virus proliferation rate was the fastest in 12-30h,and the virus proliferation rate was gentle in 30-48h,and the virus titer reached the peak at 48h(10-5.60/0.1mL),and then entered a stable period.Genome cloning and sequencing analysis showed that the whole genome of GZZY2021 isolate was 18506bp,and the homology of the most similar sequences of 11genes was more than 95.00%.The genes VP1,VP2,VP4,VP7,NSP1,NSP3and NSP4have the highest homology with pig-derived strains,while VP3had the highest homology with giant panda-derived strains.The VP6,NSP2and NSP5genes have the highest homology with human strains,suggesting that the isolated strain is a ternary recombinant strain,and the complete genotype is G9-P[23]-I1-R1-C1-M1-A8-N1-T1-E1-H1.Compared with reference strains of the same type,VP4and VP7genes have five unique amin

关 键 词：猪A群轮状病毒 分离鉴定 序列分析 

分 类 号：S852.65[农业科学—基础兽医学]