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

doi:10.3785/j.issn.1008-9209.2019.03.041

浙江大学学报（农业与生命科学版）

2019, Vol. 45

Issue (5): 635-646 DOI: 10.3785/j.issn.1008-9209.2019.03.041

动物科学与动物医学

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

李夏(

),夏文君,毛鍶超,卢舒婷,莫开昆,廖敏,周继勇,郑肖娟(

)

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，杭州 310058

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

Xia LI(

),Wenjun XIA,Sichao MAO,Shuting LU,Kaikun MO,Min LIAO,Jiyong ZHOU,Xiaojuan ZHENG(

)

Key Laboratory of Animal Virology, Ministry of Agriculture and Rural Affairs, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China

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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2.0×10⁶ mL^－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1 966 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。

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

Abstract:

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 2.0×10⁶ 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 1 966 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.

Key words: serotype 4 fowl adenovirus isolation and identification whole genomic sequencing hexon gene fiber-2 gene

收稿日期: 2019-03-04 出版日期: 2019-12-05

CLC:

S 855.3

基金资助: 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)

通讯作者: 郑肖娟 E-mail: 1224946704@qq.com;zhengxiaojuan@zju.edu.cn

作者简介: 李夏（https://orcid.org/0000-0001-6056-1828），E-mail：1224946704@qq.com

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引用本文:

李夏,夏文君,毛鍶超,卢舒婷,莫开昆,廖敏,周继勇,郑肖娟. 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[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(5): 635-646.

Xia LI,Wenjun XIA,Sichao MAO,Shuting LU,Kaikun MO,Min LIAO,Jiyong ZHOU,Xiaojuan ZHENG. Isolation, identification and whole genome sequence analysis of serotype 4 fowl adenovirus Zhejiang strain. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(5): 635-646.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2019.03.041 或 http://www.zjujournals.com/agr/CN/Y2019/V45/I5/635

表1 鸡常见病原的鉴别检测引物

表2 跨越FAdV4-ZJ2015株全基因组的11个片段的扩增引物

图1 肝组织及鸡胚尿囊液中病毒核酸的鉴别检测A、B.鸡肝组织样品（M：100 bp DNA梯状标志物；1～4：4只鸡肝组织样品；P1、P2、P3：分别为病毒接种SPF鸡胚至第1、2、3代的尿囊液）；C、D.尿囊液样品（M：100 bp DNA梯状标志物；1～3：分别为第1、2、3代尿囊液接种CEK细胞的上清液；4：各种检测病毒的阳性对照；5：阴性对照；此外，EDSV没有阳性对照，4、5均为阴性对照）。

图2 FAdV4-ZJ2015感染CEK细胞的间接免疫荧光检测

图3 FAdV4-ZJ2015株的鸡胚致病性实验和肝组织中Fiber-2蛋白的表达A.未感染（mock）和FAdV4-ZJ2015第7代（P7）、第11代（P11）细胞适应毒感染鸡胚的胚体（a、b）、胚体剖解（c）及其肝组织（d）；B.以Fiber-2蛋白鼠多抗对未接毒（mock）及不同代次（P3/P7/P8/P9/P11）细胞毒接种鸡胚的肝组织中病毒蛋白表达的蛋白质印迹法检测。

图4 分段PCR扩增法对FAdV4-ZJ2015全基因组的扩增结果M：1 kb DNA标志物；F1～F11：跨越FAdV4基因组全长的11个PCR扩增片段。

图5 基于ZJ2015全基因组核苷酸水平的系统进化分析

表3 基于ZJ2015全基因组核苷酸水平的同源性比对

图6 基于Hexon（A）和Fiber-2（B）蛋白的氨基酸水平系统进化分析

1	HESS M. Detection and differentiation of avian adenoviruses: a review. Avian Pathology, 2000,29(3):195-206.
2	MEULEMANS G, BOSCHMANS M, BERG T P, et al. Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses. Avian Pathology, 2001,30(6):655-660.
3	GANESH K, RAGHAVAN R. Hydropericardium hepatitis syndrome of broiler poultry: current status of research. Research in Veterinary Science, 2000,68(3):201-206.
4	XIA J, YAO K C, LIU Y Y, et al. Isolation and molecular characterization of prevalent fowl adenovirus strains in southwestern China during 2015—2016 for the development of a control strategy. Emerging Microbes & Infections, 2017,6(11):e103.
5	牛登云,沈元,王蕊,等.2015年我国Ⅰ群禽腺病毒分子流行病学调查.中国家禽,2016,38(9):65-68. NIU D Y, SHEN Y, WANG R, et al. Molecular epidemiological survey of group Ⅰ avian adenovirus in China in 2015. Chinese National Poultry, 2016,38(9):65-68. (in Chinese)
6	李佳,文鹏程,韩振海,等.新发鸡心包积液综合征研究进展.中国兽医杂志,2016,52(8):65-67. LI J, WEN P C, HAN Z H, et al. Research progress on new-onset chicken effusion syndrome. Chinese Journal of Veterinary Medicine, 2016,52(8):65-67. (in Chinese)
7	ROSENBERGER J K, ECKROADE R J, KLOPP S, et al. Characterization of several viruses isolated from chickens with inclusion body hepatitis and aplastic anemia. Avian Diseases, 1974,18(3):399-409.
8	HOWELL J, MACDONALD D W, CHRISTIAN R G. Inclusion body hepatitis in chickens. Canadian Veterinary Journal, 1970,11(5):99-101.
9	李海英,尹燕博,郭妍妍,等.12株肉仔鸡包涵体肝炎病毒的分离和PCR鉴定.中国兽医科学,2010，40(7):722-727. LI H Y, YIN Y B, GUO Y Y, et al. Isolation and PCR identification of 12 strains of inclusion body hepatitis virus from clinical broilers. Chinese Veterinary Medicine, 2010，40(7):722-727. (in Chinese with English abstract)
10	LI Y, FU J Y, CHANG S, et al. Isolation, identification, and hexon gene characterization of fowl adenoviruses from a contaminated live Newcastle disease virus vaccine. Poultry Science, 2017,96(5):1094-1099.
11	KIM M S, LIM T H, LEE D H, et al. An inactivated oil-emulsion fowl adenovirus serotype 4 vaccine provides broad cross-protection against various serotypes of fowl adenovirus. Vaccine, 2014,32(28):3564-3568.
12	MASOOD A, REHAN A, HAYAT C S, et al. Comparative immune response of formalin inactivated and binary ethyleneimine inactivated Angara disease vaccines. Pakistan Journal of Biological Sciences, 2000,3(8):1313-1314.
13	李京帅,李银聚,张春杰,等.禽腺病毒血清4型河南株的分离鉴定及对鸡胚致病性的研究.中国兽医科学,2018,48(4):479-483. LI J S, LI Y J, ZHENG C J, et al. Isolation and identification of fowl adenovirus serotype 4 and pathogenicity in chicken embryonated eggs. Chinese Veterinary Medicine, 2018,48(4):479-483. (in Chinese with English abstract)
14	王艳,高亚东,郭程程,等.一株禽腺病毒的分离鉴定及生物学特性研究.黑龙江畜牧兽医,2018(7):124-127. WANG Y, GAO Y D, GUO C C, et al. Isolation, identification and biological characteristics of avian adenovirus. Heilongjiang Animal Husbandry and Veterinary, 2018(7):124-127. (in Chinese)
15	杨晓伟,杨万秋,陆婧,等.禽腺病毒Ⅰ群血清4型的分子鉴定及其卵黄抗体的防控效果.畜牧与兽医,2018(1):113-117. YANG X W, YANG W Q, LU J, et al. Molecular identification of fowl adenovirus group Ⅰ (type 4) and control effects of the egg yolk antibody. Animal Husbandry and Veterinary, 2018(1):113-117. (in Chinese with English abstract)
16	梁广成.血清4型禽腺病毒的分离、致病性及其抗体间接免疫荧光方法的初步建立.江苏，扬州：扬州大学,2017:28-45. LIANG G C. Isolation, pathogenicity and indirect immunofluorescence of serum type 4 avian adenovirus. Yangzhou， Jiangsu: Yangzhou University, 2017:28-45. (in Chinese with English abstract)
17	刘延珂,万文妍,王贝贝,等.高致病性禽腺病毒4型中国流行株在LMH细胞中的增殖规律.畜牧与兽医,2018,50(3):82-86. LIU Y K, WANG W Y, WANG B B, et al. Replication pattern of highly pathogenic FAdV-4 Chinese epidemic strain in LMH cells. Animal Husbandry and Veterinary, 2018,50(3):82-86. (in Chinese with English abstract)
18	WANG P, ZHANG J J, WANG W K, et al. A novel monoclonal antibody efficiently blocks the infection of serotype 4 fowl adenovirus by targeting fiber-2. Veterinary Research, 2018,49(1):29.
19	RUAN S F, ZHAO J, YIN X M, et al. A subunit vaccine based on fiber-2 protein provides full protection against fowl adenovirus serotype 4 and induces quicker and stronger immune responses than an inactivated oil-emulsion vaccine. Infection Genetics & Evolution, 2018(61):145-150.
20	GUAN R, TIAN Y M, HAN X X, et al. Complete genome sequence and pathogenicity of fowl adenovirus serotype 4 involved in hydropericardium syndrome in Southwest China. Microbial Pathogenesis, 2018,117:290-298.
21	缪从容,吴贤福.几种鸡胚原代细胞的制备方法.中国兽药杂志,1997,31(1):16-18. MIAO C R, WU X F. Methods for the production of several primary chicken embryo cells. Chinese Journal of Veterinary Medicine, 1997,31(1):16-18. (in Chinese with English abstract)
22	PIZZI M. Sampling variation of the fifty percent end-point, determined by the Reed-Muench (Behrens) method. Human Biology, 1950,22(3):151-190.
23	ZHAO J, ZHONG Q, ZHAO Y, et al. Pathogenicity and complete genome characterization of fowl adenoviruses isolated from chickens associated with inclusion body hepatitis and hydropericardium syndrome in China. PLoS One, 2015,10(7):e133073.
24	陈玲,张兵,宋亚芬,等.禽腺病毒的分离鉴定及LMH细胞适应毒的生物学特性研究.中国兽药杂志,2018,52(6):1-6. CHEN L, ZHANG B, SONG Y F, et al. Isolation and identification of a fowl adenovirus and the biological characteristics of LMH-adapted virus strain. Chinese Journal of Veterinary Drug, 2018,52(6):1-6. (in Chinese with English abstract)
25	陈天骜.血清4型禽腺病毒GX2013毒株的生物学特性鉴定和检测方法的建立.江苏，扬州:扬州大学,2016. CHEN T A. Identification of biological characteristics of fowl adenovirus serotype 4 (FAdV-4) GX2013 strain and establishment of detection methods for FAdV-C. Yangzhou, Jiangsu: Yangzhou University, 2016. (in Chinese with English abstract)
26	冯茹,高宇瑾,张梦荷,等.Ⅰ群禽腺病毒4型陕西分离株33K、Fiber-1、Fiber-2、pⅢa基因的克隆及序列分析.动物医学进展，2018,39(4):7-13. FENG R, GAO Y J, ZHANG M H, et al. Cloning and sequence analysis of 33K, Fiber-1, Fiber-2 and pⅢa genes of fowl adenovirus type 4 isolated from Shaanxi Province. Progress in Veterinary Medicine, 2018,39(4):7-13. (in Chinese with English abstract)
27	王娟,刘亮亮,李慧昕,等.禽腺病毒血清4型的分离鉴定及遗传演化分析.中国兽医科学,2017,47(6):755-761. WANG J, LIU L L, LI H X, et al. Isolation, identification and phylogenetic analysis of fowl adenovirus serotype 4. Chinese Veterinary Science, 2017,47(6):755-761. (in Chinese with English abstract)
28	LI L T, LUO L, LUO Q P, et al. Genome sequence of a fowl adenovirus serotype 4 strain lethal to chickens, isolated from China. Genome Announcements, 2016,4(2):e116-e140.
29	PAN Q, WANG J, GAO Y L, et al. The natural large genomic deletion is unrelated to the increased virulence of the novel genotype fowl adenovirus 4 recently emerged in China. Viruses, 2018,10(9):494.

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