Molecular epidemiology of chicken anaemia virus in sick chickens in China from 2014 to 2015

RESEARCH ARTICLE Molecular epidemiology of chicken anaemia virus in sick chickens in China from 2014 to 2015 Shuai Yao1,2, Tianbei Tuo1,2, Xiang Gao2, Chunyan Han2,3, Nana Yan2, Aijing Liu2, Honglei Gao2, Yulong Gao2, Hongyu Cui2, Changjun Liu2, Yanping Zhang2, Xiaole Qi2, Altaf Hussain ID2, Yongqiang Wang2*, Xiaomei Wang1,2* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 College of Veterinary Medicine, Northeast Agricultural University, Harbin, China, 2 Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China, 3 College of Wildlife Resource, Northeast Forestry University, Harbin, China * (YQW); (XMW) Abstract OPEN ACCESS Citation: Yao S, Tuo T, Gao X, Han C, Yan N, Liu A, et al. (2019) Molecular epidemiology of chicken anaemia virus in sick chickens in China from 2014 to 2015. PLoS ONE 14(1): e0210696. https://doi. org/10.1371/journal.pone.0210696 Editor: Yongchang Cao, Sun Yat-Sen University, CHINA Received: January 17, 2018 Accepted: January 1, 2019 Published: January 18, 2019 Copyright: © 2019 Yao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chicken anaemia virus (CAV), a member of the genus Gyrovirus, is the etiological agent of chicken infectious anaemia. CAV infects bone marrow-derived cells, resulting in severe anaemia and immunosuppression in young chickens and a compromised immune response in older birds. We investigated the molecular epidemiology of CAV in sick chickens in China from 2014 to 2015 and showed that the CAV-positive rate was 13.30%, in which mixed infection (55.56%) was the main type of infection. We isolated and identified 15 new CAV strains using different methods including indirect immunofluorescence assay and Western Blotting. We used overlapping polymerase chain reaction to map the whole genome of the strains. Phylogenetic analyses of the obtained sequences and related sequences available in GenBank generated four distinct groups (A–D). We built phylogenetic trees using predicted viral protein (VP) sequences. Unlike CAV VP2s and VP3s that were well conserved, the diversity of VP1s indicated that the new strains were virulent. Our epidemiological study provided new insights into the prevalence of CAV in clinical settings in recent years in China. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Introduction Funding: This research was supported by China Agriculture Research System (CARS-41-G15), http://www.zgrj.org/expert.asp?smallclassname=% B9%A6%C4%DC%D1%D0%BE%BF%CA% D2&SpecialName=%BC%B2%B2%A1%BF%D8% D6%C6%D1%D0%BE%BF%CA%D2, the funding institution was Chinese Ministry of Agriculture, and XMW was the recipient of the fundings. The funders had no role in study design, data collection The genus Gyrovirus, a diverse group of non-enveloped icosahedral viruses containing circular single-stranded DNA [1], infects a wide range of hosts. They also trigger several serious diseases in animals as causative agents. In particular, chicken anaemia virus (CAV), a member of family Anelloviridae genus Gyrovirus, is the etiological agent of chicken infectious anaemia [2]. CAV infects several bone marrow-derived cells which results in severe anaemia and immunosuppression in young chickens. In terms of older birds, CAV can jeopardize the immune responses [3, 4]. Since its first reported in 1979 [2], CAV infection has become epidemic among chicken populations on a global scale [5–7]. CAV also has dramatic financial impact in PLOS ONE | https://doi.org/10.1371/journal.pone.0210696 January 18, 2019 1 / 18 Molecular epidemiology of chicken anaemia virus and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. areas of intensive chicken farming. Vaccination is generally used to contain the spread of the virus [8]. In a recent study, a novel human gyrovirus was isolated from a skin swab and designated as human Gyrovirus (HGyV) [9]. Since Circovirus shares partial homology to CAV, the identification of HGyV signals possible threats for human pathogenesis, further investigation is yet required. The negative-sense CAV genome consists of 2,319 nucleotides and is replicated by a rolling-circle mechanism; but the packaging and egress of viral particles are poorly characterised [1, 10]. The CAV genome encodes multiple overlapping open reading frames (ORFs) [11] that are translated into three main distinct polypeptides: CAV viral protein 1 (VP1, 52 kDa), viral protein 2 (VP2, 24 kDa) and viral protein 3 (VP3, 16 kDa). VP1 is the major structural protein while the VP2 is a replicase with dual-specificity phosphatase activity [12]. VP3, also named apoptin, is also a non-structural protein that mainly implicats in the induction of apoptosis and viral cytotoxicity in host cells. In 1996, CAV was first reported from young broilers in China [13]. 42% of overall seroprevalence was shown in farms of five Chinese provinces in a domestic poultry survey [14]. In addition, a high prevalence of 87% resulted in studies of the virus on live bird markets in Southeast China [15]. In the present study, our group investigated the epidemiology of CAV in sick or dead chickens in 12 provinces throughout China from 2014 to 2015. Totally, we obtained 96 positive results for CAV infection in 722 clinical samples, 24 out of 149 in 2014, and 72 out of 573 in 2015. We analysed the infection type of CAV in association with other pathogens including Marek’s disease virus (MDV), reticuloendotheliosis virus (REV), avian leukosis virus (ALV), avian gyrovirus 2 (AGV2), and avian reovirus (ARV). We found that coinfection was the main infection type of CAV. In addition, we analysed the characteristics of the new CAV sequenced strains together with those available in GenBank. The analysis revealed that all the sequences could be clustered into four major groups. Furthermore, we compared the key amino acids in VP1 that determined the virulence of CAV, providing new insights into the epidemiology of CAV. Materials and methods Ethics statement All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The animal experiments were performed in strict compliance with the Guideline for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. The Committee of the Ethics of Animal Experiments at the Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Sciences (CAAS) approved the animal experiment protocols. A permission from China Agriculture Research System was issued f (...truncated)