Molecular epidemiology of chicken anemia virus in commercial farms in China

Virology Journal Molecular epidemiology of chicken anemia virus in commercial farms in China Yassir M Eltahir 0 1 Kun Qian 0 Wenjie Jin 0 Pingping Wang 0 Aijian Qin 0 0 Ministry of Education Key Lab for Avian Preventive Medicine, Yangzhou University , Yangzhou, 225009 , PR China 1 Department of Preventive Medicine and Veterinary Public Health, Faculty of Veterinary Science, University of Nyala , Nyala , Sudan Background: Chicken anemia virus (CAV) is the causative agent of chicken infectious anemia (CIA). A high prevalence of CAV has been reported in China. However, VP1 sequences of Chinese isolates show no clear genotype clustering or correlation with geographic origin. Therefore, the present study aimed to detect and characterize CAV isolates from China based on sequence and phylogenetic analysis of the VP1, VP2 and VP3 genes. Results: Of 460 spleen samples tested by PCR, 47 (10.22%) were found to be positive for CAV. A total of 25 CAV, approximately full genomes, from different commercial farms were characterized. Phylogenetic analysis of the Chinese CAV sequences together with strains from different countries resulted in four distinct groups (A-D) with significant high bootstrap values. The Chinese viral sequences were located as four different clusters within groups A and D. All the Chinese CAV genomes characterized in this study had glutamine (Q) at amino acid position 394, which indicated that all are highly pathogenic. Mutations associated with attenuation and weaker reactivity with monoclonal antibody 2A9 were absent in the Chinese sequences. Conclusions: We revealed that CAV prevalence was lower than that reported previously in commercial farms in China. We also showed four distinct sequence groups (A-D), and genetic variability in local CAV sequences that could be divided into four groups based on phylogenetic analysis. - Background Chicken anemia virus (CAV), a member of the family Circoviridae, is a non-enveloped, icosahedral virus with a negative-sense, single-stranded circular DNA genome [1]. The CAV genome consists of 2.3 kb, with three partially overlapping open reading frames (ORFs) for VP1, the major viral structural protein (51.6 kDa); VP2, a scaffolding protein (24 kDa); and VP3, a non-structural protein named apoptin (13.6 kDa) for its ability to induce apoptosis; VP1 and VP2 are the main targets of neutralizing antibodies [2]. The VP1 gene has the highest variability of the three overlapping ORFs, according to sequences that have been submitted to GenBank [3]. To date, all viruses seem to belong to the same worldwide serotypes. However, because there are currently only a few full genome sequences available for CAV strains from the USA, Asia, Australia and Europe, the emergence of new serotypes cannot be excluded, which would have important consequences for vaccine efficacy and serodiagnosis [4]. In China, CAV was first isolated in 1996 from 25-40day-old broilers [5]. A survey in domestic poultry in farms in 5 Chinese provinces (Beijing, Guangdong, Zhejiang, Shanghai, and Tianjin Shi) showed a 42% overall seroprevalence [6]. On the other hand, in Southeast China, studies undertaken on live bird markets also indicated a high prevalence (87%) of the virus [7]. Although considerable numbers of VP1 sequences from China are available in GenBank, to the best of our knowledge, no systematic full genome analysis of Chinese strains has been performed. Here, we report the detection and characterization of CAV genomes based on sequence and phylogenetic analysis of the entire coding regions (VP1, VP2 and VP3) of the genome from commercial broiler and layer breeder chickens in China. 36 weeks, during necropsy at veterinary hospitals in Anhui (n = 51),Fujian(n = 14), Hunan (n = 127) and Jiangsu (n = 158) provinces. In parallel, 110 spleen samples were collected from 1-7-day-old chickens from four different commercial farms from Jiangsu province. Chickens originated from 22 different flocks on commercial farms. Flocks comprised 900-30 000 chickens, and none of the farms were vaccinated against CAV. DNA extraction According to the manufacturers instructions, DNA was extracted from spleen samples using the commercially available Flexi Gene DNA Kit (Qiagen GmbH, Hilden, Germany). The DNA was then quantitated and stored at -20C until PCR was performed. Virus detection by PCR The extracted DNA was first screened by PCR for CAV DNA using specific primers, CAV1: 5-GCA GTA GGT ATA CGC AAG GC-3 and CAV2: 5-CTG AAC ACC GTT GAT GGT C-3, covering a 186-bp region on the highly conserved VP2 coding gene [2]. The PCR amplification was carried out in PCR buffer that contained 1.5 mM MgCl2, 200 M of each dNTP, 10 pmol each primer, and 1.0 U Taq DNA polymerase (Fermentas, Shenzhen, China) in a 25-l total reaction volume in an automated thermal cycler (Gene Amp PCR System 9700, Applied Biosystems, Foster City, CA, USA) using the following cycling profile: initial denaturation of 94C for 2 min, followed by 35 cycles of denaturation, annealing and extension at 94C for 30 s, 60C for 30 s and 72C for 1 min, respectively, and the final extension was carried out at 72C for 7 min. The PCR products were then analyzed by 1.5% agarose gel electrophoresis and imaged with the EpiChem system (UVP Bioimaging Systems, Garland, CA, USA). Amplification of the CAV genome Primers VP1F: 5-AGCCGACCCCGAACCGCAAGAA-3 and VP1R: 5-TCA GGG CTG CGT CCC CCA GTA CA-3 were used to amplify the VP1 region, and VP2F: 5-GCG CAC ATA CCG GTC GGC AGT-3 and VP2R: 5-GGG GTT CGG CAG CCT CAC ACT AT-3 were used to amplify the VP2 region from PCR-positive samples. These two primer sets covered the entire coding regions of CAV [8]. The PCR amplification was carried out in PCR buffer that contained 1.5 mM MgCl2, 200 M of each dNTP, 10 pmol each primer, and 1.0 U Takara LA Taq polymerase (TaKaRa Biotechnology Co., Ltd., Dalian, China) in a 25-l total reaction volume. The reaction was carried out in an automated thermal cycler (Gene Amp PCR System 9700, Applied Biosystems, Foster City, CA, USA). Amplification of 1390 bp of the VP1 region was carried out with initial denaturation of 94C for 4 min, followed by 34 cycles of denaturation, annealing and extension at 94C for 1 min, 60C for 1 min and 72C for 2 min, respectively, and the final extension was carried out at 72C for 15 min. Amplification of 713 bp of the VP2 region was carried out with initial denaturation of 94C for 4 min, followed by 34 cycles of denaturation, annealing, extension at 94C for 1 min, 63C for 1 min and 72C for 1 min, respectively, and final extension was carried out at 72C for 5 min. The PCR products were then analyzed by 1.5% agarose gel electrophoresis and imaged with the EpiChem system (UVP Bioimaging Systems). In all PCR reactions, a previously characterized CAV isolate maintained in our laboratory was uses as a positive control. The PCR mixture was used as a negative control. The VP1 and VP2 regions were purified using an agarose gel with (...truncated)