Avian gyrovirus 2 DNA in fowl from live poultry markets and in healthy humans, China.
So far, little is known about AGV2 in mainland China among chickens and humans. Because live poultry market (LPMs) play a critical role in the transmission of poultry pathogens to humans, we used PCR to investigate the presence of AGV2 in chickens (54 feather shaft samples) from 4 LPMs in Yangzhou and in 178 human blood samples from healthy persons living in Yangzhou. The DNA from the feather shafts and human blood were extracted as previously described (8). PCR was performed by using the following 2 primers: AGV2_F 5'-CGTGTCCGCCAGCAGAAACGAC-'3 and AGV2_R 5'-GGTAGAAGCCAAAGCGTCCACGA-'3. The PCR targets partial VP2 and VP3 genes that have an expected size of 346 bp. The parameters of the PCR were as follows: 1 cycle at 95[degrees]C for 5 min; then 30 cycles at 94[degrees]C for 30 s, 64[degrees]C for 30 s, and 72[degrees]C for 30 s; and 1 cycle at 72[degrees]C for 10 min. PCR showed that a band with the size of [approximately equal to]346 bp could be amplified in 10 of 54 chicken feather samples and in 2 of 178 human blood samples.
We confirmed the AGV2 specificity of these PCR-amplified bands by direct sequencing using the Sanger method. The sequence assay showed that the 12 sequences identified here had 98.3%-100% homology to each other and 92.2%-99.1% aa identity to AGV2 samples previously deposited in GenBank (see Figure legend for accession numbers). The positive rates for samples from the 4 LPMs tested were 25%, 12.5%, 15.8%, and 20%; the positive rate for the 178 human blood samples was 1.1%. The low positive frequency of AGV2 in human blood detected in this study is consistent with that found by investigation in other countries (3,4). Because the limit of detection of PCR in this study was estimated to be 2.7 copies of AGV2 DNA using dilutions of a plasmid with partial AGV2 sequence, we determined that the copy number of AGV2 in the 2 positive human blood samples was 2.7 x [10.sup.3] copies/mL plasma.
We also constructed a phylogenetic tree using the neighbor-joining method (1,000 bootstrap replications) with MEGA6 (9). The tree analysis revealed that the 12 AGV2 isolates we identified and 7 AGV2 isolates from GenBank clustered into 2 subgroups on the basis of the PCR amplified fragment (Figure). The 12 AGV2 sequences we identified clustered together with gyrovirus sequences detected in ferret and human samples in subgroup I, and the prototype sequence Ave3 was located in subgroup II. The 12 AGV2 showed -92.2%-93% aa identity to Ave3, and <99.1% homology with isolates CL33, G13, and 915F06007 detected in ferret and human samples. The 12 AGV2 sequences also showed [approximately equal to]93%-93.9% identities to ACV2 sequence that was previously identified in human fecal samples from mainland China (GenBank accession no. JQ690763). The China sequence also clustered with Ave3 in subgroup II. These findings indicate that [greater than or equal to]2 subgroups of AGV2 are circulating in mainland China.
Our results demonstrate the presence of AGV2 in LPMs and human blood in mainland China. The amplification and analysis of partial AGV2 sequences was the major limitation in our method. The high homology between sequences identified in LPMs and human blood indicates the LPMs are a potential source for AGV2 in humans. Unlike our 12 conserved AGV2, AGV2 identified by Santos et al. in southern Brazil varied <15.8%, and these variants of AGV2 were mainly detected in diseased chickens (8). However, little is known about the molecular epidemiology of these AGV2 variants in other countries. More recently, Varela et al. reported the detection of AGV2 in poultry vaccines, indicating the potential role of contaminated vaccines in the spread of AGV2 (10). Future studies should investigate the large geographic distribution of AGV2 and monitor the variants, the host range, and the associated diseases.
This work was supported by the National Natural Science Foundation of China (31402228), National College Student Innovation Training Project (201411117002), Key University Science Research Project of Jiangsu Province (14KJA230002), Jiangsu Province College Student Innovation training Projects (201411117002Z and 201411117056Y), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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Address for correspondence: Jianqiang Ye, Yangzhou University, 12 Wenhui East Rd, Yangzhou, Jiangsu, 225009, China; email: email@example.com, firstname.lastname@example.org; email@example.com
Jianqiang Ye,  Xiaoyan Tian,  Quan Xie, Yu Zhang, Yuanzhao Sheng, Zhenwen Zhang, Chengming Wang, Hong Zhu, Yumeng Wang, Hongxia Shao, Aijian Qin
Author affiliations: Ministry of Education Key Laboratory for Avian Preventive Medicine and Key Laboratory of Jiangsu Preventive Veterinary Medicine, Yangzhou University, Yangzhou, China (J. Ye, X. Tian, Q. Xie, Y. Zhang, Y. Sheng, H. Zhu, Y. Wang, H. Shao, A. Qin); Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou (J. Ye, Z. Zhang, C. Wang, H. Shao, A. Qin); College of Medicine, Yangzhou University, Yangzhou (Z. Zhang)
 These authors contributed equally to this article.
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|Author:||Ye, Jianqiang; Tian, Xiaoyan; Xie, Quan; Zhang, Yu; Sheng, Yuanzhao; Zhang, Zhenwen; Wang, Chengming|
|Publication:||Emerging Infectious Diseases|
|Date:||Aug 1, 2015|
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