猪轮状病毒G9P[7]型云南株的分离鉴定及VP4和VP7基因测序分析  

Isolation and Identification of Porcine Rotavirus Type G9P[7]in Yunnan and Sequencing Analysis of VP4 and VP7 Genes

作　　者：王修武 邓可辉 马沐林 关金连 韩淑杰 郭智轩 孙守湖 贺东生 WANG Xiu-wu;DENG Ke-hui;MA Mu-lin;GUAN Jin-lian;HAN Shu-jie;GUO Zhi-xuan;SUN Shou-hu;HE Dong-sheng(Zhaoqing Branch of Guangdong Provincial Laboratory of Lingnan Modern Agricultural Science and Technology,Zhaoqing,Guangdong,526238,China;Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control,College of Veterinary Medicine,South China Agricultural University,Guangzhou,Guangdong,510640,China)

机构地区：[1]岭南现代农业科学与技术、广东省实验室肇庆分中心,广东肇庆526238 [2]华南农业大学兽医学院、广东省动物源性人兽共患病预防与控制重点实验室,广东广州510640

出　　处：《动物医学进展》2024年第8期1-7,共7页Progress In Veterinary Medicine

基　　金：广东省科技计划项目(P20211154-0302)。

摘　　要：为确定云南省某猪场仔猪腹泻的病原,对送检腹泻病仔猪小肠组织样品进行猪急性腹泻综合征冠状病毒(SADS-CoV)、猪流行性腹泻病毒(PEDV)、猪丁型冠状病毒(PDCoV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(PoRV)RT-PCR检测,将PoRV检测阳性样品处理后接种至MA-104细胞进行PoRV分离;对分离毒株进行间接免疫荧光、电镜观察、胶体金检测和VP4、VP6、VP7基因测序及遗传进化分析。结果显示,仔猪肠道组织样品经终浓度20μg/mL的胰酶在37℃孵育2 h,能在MA-104细胞上增殖传代,第3代出现稳定的细胞病变,盲传20代,每隔5代进行PoRV的检测;间接免疫荧光试验可见特异性荧光,胶体金和RT-PCR检测均为PoRV阳性,电镜观察可见病毒粒子直径约为65 nm,呈车轮状,符合PoRV粒子典型形态特征,将该分离株命名为PoRV YN-A株。基因同源性比较和遗传进化树分析显示,YN-A株的VP6基因序列与国内A群GD株的同源性为99.93%,VP7与中国G9型NJ2012株的同源性为99.12%,VP4与美国P[7]型OSU株的同源性为98.91%,确定该分离株为猪A群轮状病毒G9P[7]型。YN-A株VP7序列在9个氨基酸位点存在点突变,即aa9(I→V)、aa16(V/T→I)、aa44(A→V)、aa100(D/E→N)、aa212(T/A→K)、aa221(N→S)、aa225(V→A)、aa271(I→V)、aa267(D→E)。首次从云南地区的腹泻仔猪小肠组织中成功分离到1株G9P[7]型的新基因型PoRV毒株,为PoRV的致病性、分子生物学特性研究及PoRV G9优势基因型新疫苗研发奠定了基础。In order to determine the pathogen of piglet diarrhoea in a pig farm in Yunnan province,the small intestine tissue samples of piglets sent for diarrhoeal detection were tested for swine acute diarrhea syndrome corona virus(SADS-CoV),porcine epidemic diarrhoea virus(PEDV),porcine deltacoronavirus(PDCOV),porcine infectious gastroenteritis virus(TGEV)and porcine rotavirus(PoRV)by RT-PCR,and the positive samples were inoculated into MA-104 cells for PoRV isolation;the isolates were subjected to indirect immunofluorescence,electron microscopy,colloidal gold detection,sequencing of VP4,VP6 and VP7 genes and their genetic evolution analysis.The results showed that a PoRV stain grew and proliferated sustainably on MA-104 cells while the pig intestinal tissue samples were treated with trypsin at a final concentration of 20μg/mL for 2 h at 37℃.The cytopathic lesions appeared from the 3rd viral passage in cell culture to the 20th passage.PoRV was detected every 5 generations;specific fluorescence was shown in the indirect immunofluorescence test;colloidal gold and RT-PCR tests were positive for PoRV;This PoRV particle has the size of 65 nm with a wheel-like structure in electro-microscopy test that is consistent with the typical morphological characteristics of PoRV particles.Genetic homology comparison and genetic evolutionary tree analysis showed that the VP6 gene sequence of YN-A strain was 99.93%homologous to the domestic Group A GD strain,VP7 was 99.12%homologous to the Chinese G9 type NJ2012 strain,and VP4 showed 98.91%homology with the US P[7]OSU strain,identifying the isolate as porcine group A rotavirus type G9P[7].YN-A strain VP7 sequence had 9 amino acid site mutations,namely aa9(I→V),aal6(V/T→I),aa44(A→V),aa100(D/E→N),aa212(T/A→K),aa221(N→S),aa225(V→A),aa271(I→V),and aa267(D→E).In this study,a new genotype of G9P[7]type was successfully isolated from the small intestine of diarrhoeic piglets in Yunnan for the first time,which will provide a reference for the research on the pathogenicity and

关 键 词：猪A群轮状病毒 分离鉴定 G9基因型 基因测序 电镜观察 

分 类 号：S852.65[农业科学—基础兽医学]