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

doi:10.3785/j.issn.1008-9209.2019.03.041

Journal of Zhejiang University (Agriculture and Life Sciences)

2019, Vol. 45

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

Animal sciences & veterinary medicine

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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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

Received: 04 March 2019 Published: 05 December 2019

CLC:

S 855.3

Corresponding Authors: Xiaojuan ZHENG E-mail: 1224946704@qq.com;zhengxiaojuan@zju.edu.cn

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	Xia LI
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	Kaikun MO
	Min LIAO
	Jiyong ZHOU
	Xiaojuan ZHENG

Cite this article:

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.

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

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

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

Table 1 Detection primers for common pathogens in chickens

Table 2 Primers to amplify 11 fragments covering the whole genome of FAdV4-ZJ2015 strain

Fig. 1 Identification of viral nucleic acids in liver tissues and allantoic fluid of chicken embryosA and B: Liver tissue samples from chickens (lane M: 100 bp DNA ladder marker; lanes 1-4: Liver tissues from four chickens; P1, P2, and P3: Allantoic fluid of SPF chicken embryos inoculated with FAdV4 for 1-3 passages, respectively). C and D: Allantoic fluid samples from chickens (lane M: 100 bp DNA ladder marker; lanes 1-3: Supernatants of CEK cells inoculated with the allantoic fluid of the P1-P3 passages, respectively; lane 4: Positive control for each virus; lane 5: Negative control; no positive control for EDSV, and the lanes 4 and 5 are negative controls.

Fig. 2 Indirect immunofluorescence assay of CEK cells infected with FAdV4-ZJ2015 strain

Fig. 3 Pathogenicity of FAdV4-ZJ2015 strain on chicken embryo and expression of Fiber-2 protein in the liver tissuesA. Embryoid body (a, b), chicken embryo, embryo body section (c) and liver tissue (d) of uninfected (mock) and infected with the supernatants of the P7 and P11 passages of cell-adapted FAdV4-ZJ2015 strain; B. Expression detection of the Fiber-2 protein in liver tissues of uninfected (mock) and infected with serial passages (P3/P7/P8/P9/P11) of FAdV4-ZJ2015 by anti-Fiber-2 polyclonal antibody by Western blotting.

Fig. 4 Amplification result of the whole genome of FAdV4-ZJ2015 by segmented PCR amplificationM: 1 kb DNA marker; F1-F11: PCR products of the 11 amplified fragments cross the full length of FAdV4 genome.

Fig. 5 Phylogenetic analysis based on the whole genome of ZJ2015 strain

Table 3 Homology alignment based on the whole genome of ZJ2015 strain

Fig. 6 Phylogenetic analysis based on amino acid levels of Hexon (A) and Fiber-2 (B) proteins


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