血清4型禽腺病毒浙江株的分离鉴定及其全基因组序列分析  

Isolation, identification and whole genome sequence analysis of serotype 4 fowl adenovirus Zhejiang strain

作　　者：李夏 夏文君 毛鍶超 卢舒婷 莫开昆 廖敏[1] 周继勇[1] 郑肖娟[1] LI Xia;XIA Wenjun;MAO Sichao;LU Shuting;MO Kaikun;LIAO Min;ZHOU Jiyong;ZHENG Xiaojuan(Key Laboratory of Animal Virology,Ministry of Agriculture and Rural Affairs,College of Animal Sciences,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学动物科学学院农业农村部动物病毒学重点实验室

出　　处：《浙江大学学报（农业与生命科学版）》2019年第5期635-646,共12页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家重点研发计划(2016YFD05000800);国家现代农业产业技术体系建设项目(CARS-40-K13)

摘　　要：对2015年浙江省某鸡场疑似心包积液-肝炎综合征的患病鸡进行病原检测和分离鉴定,并对分离到的禽腺病毒全基因组进行遗传进化分析。采用聚合酶链式反应(polymerase chain reaction,PCR)扩增结合核酸测序分析确定其病原为血清4型禽腺病毒(FAdV4);通过鸡胚接种和原代鸡胚肾(chicken embryo kidney,CEK)细胞多次传代培养,获得FAdV4细胞适应毒株ZJ2015。血凝实验显示:ZJ2015株不能凝集小鼠、大鼠、鸡和绵羊的红细胞,间接免疫荧光分析表明其半数组织培养感染剂量(50%tissue culture infective dose,TCID50)为2.0×10^6mL^-1。以0.2 mL/枚剂量接种鸡胚,该毒株能100%致死鸡胚,且致死鸡胚胚体潮红、肝肿大、出血等。将FAdV4全基因组分11段分别进行PCR扩增和测序,获得跨越ZJ2015毒株的全基因组序列(GenBank登录号:MF521611.1),对其全基因组、hexon和fiber-2基因的氨基酸进行遗传进化分析显示:ZJ2015毒株与近几年国内流行的FAdV4强毒株处于同一个分支上,同源性达到99.87%以上;与ON1、KR5、MX-SHP95等较早毒株相比,ZJ2015具有1966 bp的大段缺失及Fiber-2蛋白上多个位点的突变。浙江毒株ZJ2015的分离可为进一步开展FAdV4的诊断防控技术和毒力变异机制的研究奠定基础。Fowl adenovirus causing the pericardial effusion-hepatitis syndrome was identified from a chicken farm in Zhejiang Province in 2015,and its whole genome was sequenced to explore its genetic evolution.The viral nucleic acid was amplified by polymerase chain reaction(PCR)and further sequenced to confirm that the serotype 4 fowl adenovirus(FAdV4)is the main causative pathogen.A cell-adapted FAdV4 strain ZJ2015 was successfully obtained by multiple passages using chicken embryos and then primary chicken embryo kidney(CEK)cells.Hemagglutination assay revealed that the ZJ2015 strain could not agglutinate red blood cells of mice,rats,chickens and sheep,and the 50%tissue culture infective dose(TCID50)was 2.0×10^6 mL^-1 by indirect immunofluorescent assay.The strain could kill chicken embryos at a dose of 0.2 mL causing the flushing,hepatomegaly,hemorrhage,etc.The 11 segments covering the FAdV4-ZJ2015 whole genome was amplified by PCR and further sequenced to obtain the whole genome sequences(GenBank ID:MF521611.1).Evolution analysis based on the whole genome,Hexon and Fiber-2 protein showed that the ZJ2015 strain was on the same branch as the prevailing FAdV4 strains in China,with the homology over 99.87%.Different from the earlier strains such as ON1,KR5 and MX-SHP95,the ZJ2015 strain was found to have a 1966 bp deletion in the whole genome and multiple mutations in the Fiber-2 protein.In a word,the isolation of ZJ2015 strain from Zhejiang Province provides a basis for further studies on diagnostics and disease control,as well as the mechanism of virulence variation of FAdV4.

关 键 词：血清4型禽腺病毒 分离鉴定 全基因组测序 hexon基因 fiber-2基因 

分 类 号：S855.3[农业科学—临床兽医学]