摘要
为了对患腹泻疾病的犊牛进行病原学鉴定,并进一步丰富我国牛轮状病毒(BRV)的流行病学数据,本研究采用RT-PCR方法对8份腹泻犊牛的粪便样品进行BRV VP7基因检测,结果显示有1份样品为阳性。将该阳性病料样品接种Marc-145细胞。对产生细胞病变(CPE)的阳性样品连续传5代后,经间接免疫荧光试验(IFA)和电镜观察,结果表明从粪便样品中分离的病毒为BRV,命名为DZ株。对DZ株的11个基因节段进行RTPCR扩增、测序、拼接后对11个基因节段进行同源性及分型分析;构建分离病毒VP7、VP4基因进化树,分析其遗传进化关系及其氨基酸序列的变异情况。结果显示,DZ株基因组10个基因节段分别与来源于牛(VP2、VP7、NSP1、NSP3)、犬(VP3、NSP5)、羔羊(NSP2)、马(VP6)、猕猴(VP1)、人(NSP4)以及人-牛基因重配(VP4)的轮状病毒株。其中VP1基因与猕猴轮状病毒(RRV)的同源性最高,为81.3%,但低于VP1基因分型的临界值83%,因此DZ株的VP1为新出现的基因型,命名为R17;分离株NSP3基因与法国RF株NSP3基因的同源性高达98%,DZ株与RF株的NSP3属于同一基因型,命名为T18型。所以,本研究分离的BRV DZ株的基因型为G6-P5-I2-R17-C2-M2-A3-N2-T18-E2-H5。VP7和VP4基因遗传进化分析结果显示,DZ株VP7基因来源于韩国KJ69-1株,VP4基因来源于美国疫苗株RotaTeq-SC2-9。与同源性最高的病毒株相比,DZ株VP4、VP7蛋白氨基酸序列分别发生了9处和8处突变。这些突变可能会引起VP7和VP4蛋白的抗原性和组织嗜性变化,进而影响病毒的免疫原性、宿主嗜性以及致病性。综上所述,DZ株是多宿主来源的基因重配病毒株,且主要抗原蛋白VP7和VP4发生了较大变异。本研究为我国BRV遗传进化及其分子流行病学和疫苗的研究奠定了实验基础。
In order to identify the pathogen of calves suffering from diarrhea,and to further enrich the epidemiological data of bovine rotavirus(BRV)in China.In this study,the RT-PCR method was used to detect the BRV VP7 gene in 8 stool samples of diarrhea calves,the results showed that one sample was positive.The positive sample was inoculated into Marc-145 cells.After 5 consecutive generations of positive samples that produced cytopathic lesions(CPE),the indirect immunofluorescence test(IFA)and electron microscope showed that the virus isolated from the samples was determined to be bovine rotavirus(BRV),named as DZ strain.The 11 gene segments of the DZ strain were amplified by RT-PCR,sequenced,and the 11 gene segments were analyzed for homology and typing;the isolated virus VP7 and VP4 gene evolutionary trees were constructed to analyze the genetic evolution relationship and the variation of amino acid sequence.The results showed that the 10 gene segments of the DZ strain genome were related to those derived from cattle(VP2,VP7,NSP1,NSP3),dogs(VP3,NSP5),lambs(NSP2),horses(VP6),rhesus monkeys(VP1),humans(NSP4)and human-bovine gene reassortment rotavirus strains(VP4).VP1 gene and rhesus rotavirus strain(RRV)has the highest homology of 81.3%,which is lower than the critical value of VP1 genotyping of 83%.Therefore,the VP1 of the DZ strain is a new genotype,named as R17.The homology of NSP3 gene of the isolated strain and the French RF strain was up to 98%,and the NSP3 gene of the DZ strain and the RF strain belong to the same genotype,named T18.Therefore,the genotype of the BRV DZ strain isolated in this study is G6-P5-I2-R17-C2-M2-A3-N2-T18-E2-H5.The results of genetic evolution analysis of VP7 and VP4 genes showed that the VP7 gene of the DZ strain was derived from the Korean KJ69-1 strain,and the VP4 gene was derived from the American vaccine RotaTeq-SC2-9 strain.Compared with the virus strain with the highest amino acid sequence homology,the VP4 and VP7 proteins of the DZ strain had the 9 and 8 mutations,respectively.Th
作者
曹存
于德斌
徐晓静
于力
常继涛
CAO Cun;YU De-bin;XU Xiao-jing;YU Li;CHANG Ji-tao(Division of Livestock Infections Disease,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;Research and Development Department,Harbin Veken Biotechnology Co.,Ltd.,Harbin 150069,China;College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010010,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2021年第9期939-945,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
内蒙古自治区科技重大专项“牛腹泻疾病防控关键技术研发与标准化操作规程示范推广”(2020ZD0006)。
关键词
牛轮状病毒
基因序列分析
重配株
分离鉴定
bovine rotavirus
gene sequence analysis
reassortant strain
isolation and identification
