| Duck virus hepatitis (DVH) is an acute and highly contagious disease affecting young ducklings, particularly those under 4 weeks of age. The disease is caused by at least three heterologous serotypes of viruses:Duck hepatitis A virus (DHAV), duck astrovirus type 1 and duck astrovirus type 2. The most widely distributed virus, DHAV belongs to a novel genus Avihepatovirus in the family Picornaviridae. Based on neutralization tests and phylogenetic analyses, DHAV have been divided into three different genotypes (DHAV-1, DHAV-2, DHAV-3). Among them, DHAV-1 is the most widely distributed, DHAV-2 has so far only been identified in Taiwan, and DHAV-3 recently has caused serious damage to the local duck industry in mainland of China and South Korea. There is no cross-neutralization between DHAV-1 and DHAV-2, and limited cross-neutralization between DHAV-1 and DHAV-3. The conventional diagnosis of DVH is based on the common clinical signs and gross pathological changes in ducklings, but it is not possible to distinguish between DHAV-1 and DHAV-3. To date, several neutralization tests and molecular biological methods for detection of DHAV isolates have been described. Neutralization tests including indirect hemagglutination test, agar gel diffusion precipitation, and direct fluorescence-antibody technique have been used in virus identification for years. Although the neutralization test is generally reliable for typing DHV, it seems time consuming and labour intensive. Molecular biological methods can quickly and specially detect DHAV directly from samples or from viral isolates, including RT-PCR assays and RT loop-mediated isothermal amplification assays. Therefore, it would be of great clinical value to develop a one-step duplex RT-PCR assay that can be used for routine evaluation and monitoring of DHAV-1 and DHAV-3 as an aid to improve the control of the circulation of DHAV.1. Development of a one-step duplex RT-PCR assay for detection of DHAV type 1 and 3For rapid diagnosis for DHAV-1 and DHAV-3, two pairs of primers were all designed in the conserved regions of DHAV-1 and DHAV-3 which were highly divergent from each other, based on the whole genome sequences alignments of DHAV retrieved from GenBank. The one-step duplex RT-PCR assay established in this study. The target PCR productions for DHAV-1 and DHAV-3 were 992bp and 1533bp, respectively. This assay was confirmed to be DHAV specific and don’t amplify healthy ducklings, Duck plague virus, Muscovy duck parvovirus, Duck circovirus, Avian influenza virus, Riemerella anatipestifer, Pasteurella multocida, Staphylococcus aureus, Escherichia coli, Salmonella anatis. The detection limit of the one-step duplex RT-PCR was estimated to be 10 pg total RNA templates DHAV-1 or DHAV-3 RNA.2. Utilizing the one-step duplex RT-PCR assay for detection of DHAV-1 and DHAV-3In this study, the assay was evaluated in experimentally infected ducklings and DHAV isolates. The experimental results were absolutely accorded with the theoretical results. Clinical samples collected from duck farm in Sichuan, Shandong, Hubei and Guangxi province from 2012 to 2014 are detected by the one-step duplex RT-PCR assay. A total of 52 clinical samples from different commercial farms were detected.41 out of 52 samples were DHAV-1 positive, and 11 out of 52 samples were DHAV-3 positive, we did not find any co-infections in these clinical samples. The result suggests that DHAV-1 and DHAV-3 both exist in China. In summary, the one-step duplex RT-PCR assay established in this study is feasible for rapid and diagnosis for infection of DHAV-1 and DHAV-3 in ducklings. It could be used for simultaneously detection of DHAV-1 and DHAV-3 infections. |
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