| Duck virus hepatitis (DVH) is a highly fatal contagious disease of young ducklings.DVH is caused by three different viruses, including DHV-1(Picornaviridae, genus Avihepatovirus. Duck hepatitis A virus, DHAV), DHV-2(duck astrovirus), and DHV-3(another virus unrelated to DHV-1and DHV-2). DHAV can be classified as DHAV-1(formerly called DHV-1)ã€DHAV-2(formerly known as New DHV isolated in Taiwan) and DHAV-3(formerly called New DHV isolated in Korean)In order to have a better understanding of the prevalence of DHAV in the eastern China, we established a RT-PCR method for detection of DHAV, detected clinical samples collected from duck farm in Jiangsu, Shandong and Anhui from2009to2011, isolated the virus and performed a phylogenetic analysis. This study will be helpful for detection of DHAV and control of DHAV infection.1. Establishment of a RT-PCR method for detection of DHAV-1and DHAV-3According to VP1sequences of DHAV-1and DHAV-3deposited in GenBank, Two pairs of primers in a relatively conservative region were designed with the software of Primer Premier5.0. The target PCR productions for DHAV-1and DHAV-3were720bp and950bp, respectively. The RT-PCR methods for DHAV-1or DHAV-3could only amplify a corresponding band from DHAV-1strain YZ or DHAV-3strain JD respectively, but not from different serotype strain, avian influenza virus strain, duck plague virus strain, and Riemerella anatipestifer, and the minimum detectable limits for DHAV-1and DHAV-3RNA were5.2ng and6.8ng, respectively. The results of sensitivity and specificity of test demonstrated that the RT-PCR method provided an option assay for rapid detection of DHAV.2.Identification and phylogenetic analysis of DHAV isolated from the eastern China from2009to2011The RT-PCR methods for DHAV-1and DHAV-3were applied to detect15clinical samples. Fifteen out of15samples were DHAV-1positive and5out of15samples were DHAV-3positive. Therefore.66.6% of DVH cases were only DHAV-1infection, while33.3% fo DVH cases were co-infection with DHAV-1and DHAV-3. Fifteen strains were obtained by allantoic sac inoculation in duck embryo.720-bp of fragments were amplified and sequenced from all15isolates. Phylogenetic analysis showed that these sequences of VPl located in the same linage of DHAV-1reference strains.950-bp fragments were also obtained and sequenced from isolates DHV01and YN, Phylogenetic analysis showed that these sequences of VP１located in the same linage of DHAV-3reference strains and Korean isolates. The3D sequences of isolates DHV01and YN also showed that these two isolates belonged to DHAV-3.DHAV-1strain YZ was selected to immunized chicken for preparation of antiserum. Neutralization test results showed neutralization titer of the antiserum was328for DHAV-1isolate SY and zero for DHAV-1and DHAV-3mixture strain YN, indicating an obvious antigenic difference between SY and YN.In summary, there are two genotype strains, DHAV-1and DHAV-3, in duck farm in the eastern China. It is difficult for control of DVH when co-infection with DHAV-1and DHAV-3exists in duck farm. |
