摘要
为了确定2020年河南某猪场水疱性疾病的病因,通过提取发病猪的水疱皮样品的RNA,用RT-PCR方法初步检测,经BHK-21细胞分离培养病毒,进一步用间接免疫荧光试验(IFA)鉴定;利用蚀斑形成试验及病毒一步生长曲线,分析病毒分离株的增殖特性;RT-PCR方法扩增全基因组序列,进而构建基于P1基因的系统进化树。RT-PCR鉴定结果表明,猪场水疱性疾病的样品中只有A型塞内卡病毒(SVA)核酸呈阳性;细胞分离和IFA鉴定结果表明,成功分离到的1株病原为SVA,并命名为SVA/HN/2020,该分离株能够与SVA免疫家兔多抗发生特异性反应;蚀斑试验和病毒一步生长曲线结果表明,该毒株能够在BHK-21细胞层上形成蚀斑,并在感染后的第16小时病毒滴度达到最高为1×10^(9.5)TCID_(50)/100μL;全基因组测序结果表明,该毒株基因组全长7279 bp(不含Poly A),开放阅读框为6546 bp,可编码含2181个氨基酸残基的多聚蛋白;其中P1基因大小为2574 bp,其核苷酸序列分析显示,该分离株与在我国分离的SVAHB/CH/2016和SVA CH/01/2015的同源性最高,为99.6%~99.7%,而与2018-2020年间从广东、广西、河南、河北等地的分离毒株的同源性为95.6%~97.0%。结论:本研究成功分离到1株SVA分离株,为进一步研究SVA致病机制及防控技术研发奠定了基础。
In order to determine the causal agents of vesicular disease occurred in pig farm in Henan in 2020,RNA was extracted from the vesicle skin samples of diseased pigs,and was preliminarily identified by RT-PCR.The virus strain was isolated and cultured in BHK-21 cells,and was further identified by indirect immunofluorescence assay(IFA).The proliferation characteristics of the virus strains were analyzed by the plaque formation test and determination of one-step virus growth curve.Whole genome sequence of the virus strain was amplified by RT-PCR,and the phylogenetic tree based on P1 gene was constructed.RT-PCR showed that the Senecavirus A(SVA)nucleic acid was only positive in the samples of vesicular disease in pig farms.The cell isolation and IFA identification showed that successful isolated pathogen was a SVA and was named SVA/HN/2020.The cell isolation and IFA showed that the SVA was reacted with rabbit anti-SVA antibody specifically.Plaque formation experiments and one-step virus growth curve showed that this strain could form plaques on the BHK-21 cell layer,and the virus titer was highest at 16 h infection,which was 1×10^(9.5)TCID_(50)/100μL.Whole genome sequencing results showed that the full-length genome of this virus strain was 7279 bp(excluding poly A),and the open reading frame was 6546 bp,and the open reading frame encoded a polyprotein of 2181 amino acids.Among whole genome,the P1 gene was 2574 bp,and the nucleotide sequence analysis showed that this isolate had high homology with HB/CH/2016 and CH/01/2015 from China,and far from the isolates from the Guangdong,Guangxi,Henan and Hebei during 2018 to 2020 in China.This study had successfully isolated a SVA,which built a foundation for further research on the pathogenesis of SVA and development of prevention and control technology for SVA.
作者
郑敏
杨帆
曹伟军
张伟
唐丽杰
郑海学
ZHENG Min;YANG Fan;CAO Wei-jun;ZHANG Wei;TANG Li-jie;ZHENG Hai-xue(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;State Key Laboratory of Veterinary Etiological Biology/National Foot-and-Mouth Disease Reference Laboratory/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2022年第10期1281-1287,共7页
Chinese Veterinary Science
基金
国家重点研发计划项目(2021YFD1800302,2021YFD1800305)
甘肃省科技重大专项(19ZD2NA001,21ZD3NA001)
关键词
A型塞内卡病毒
分离鉴定
遗传进化分析
Senecavirus A
isolation and identification
genetic evolutionary analysis
作者简介
郑敏(1994-),女,河南信阳人,硕士生,研究方向为病毒分子学,E-mail:Zhengmin105251@163.com;通讯作者:唐丽杰,教授,主要从事分子病毒学方向研究,E-mail:tanglijie@163.com;通讯作者:郑海学,研究员,主要从事口蹄疫及动物新发疫病防控研究,E-mail:zhenghaixue@caas.cn。
