| Duck virus hepatitis (DVH) is an acute and fatal disease of young ducklings characterized primarily by hepatitis. The increasing morbidity and mortality in ducklings result in a severe loss. So far, three different viruses, duck hepatitis virus (DHV) types 1, 2 and 3, have been associated with these symptoms. DHV-1 and DHV-3 was classified as a picornavirus. However, DHV-2 was classified as an astrovirus. Among DHV types 1, 2 and 3, DHV-1 is now worldwide in distribution.According to DHV-1 conservative sequence, the genome sequence of DHV-1 strain 161/79/V was cloned by RT-PCR with specific primers. In this study, we reported and analyzed the complete nucleotide sequence and demonstrated clearly that the strain 161/79/V belong to DHV-1 which is a member of the family Picornaviridae. GenBank accession number is EU753359. The organization of the DHV-1 genome was comprised 7653 nt and contained a single, long open reading frame (ORF) encoding a polyprotein of 2249 aa. The long polyprotein was preceded by a 5'un-translated region (UTR) of 589nt, followed by a short 3'UTR of 314nt and a poly(A) tail. The polyprotein is processed auto-catalytically at the conserved inter-domain junctions by protease, which contains the following organization: VP0–VP3–VP1–2A1-2A2-2A3–2B– 2C–3A–3B–3C–3D. The genome organization and the molecular evolution and phylogeny were analysised by using a series of bioinformatics tools and software. The genome sequence of strain 161/79/V was closely related to strain JH2 and the pairwise percent of identity of the nucleotide and amino acid were 97.1% and 98.7%. Phylogenetic analysis of strain 161/79/V and other 22 strains DHV VP1sequence would be useful for studying the genetic relationships of DHV.The results of phylogenetic retionship show three groups of DHV by VP1 protein. The pairwise percent identity of the nucleotide and amino acid among three group were 68.8～72.8% and 69.3～77.3%. The results demonstrated that DHV may contains three genotypes. Screening Genetic and phylogenetic characteristic of DHV will provide more information on how DHV evolves and is transmitted in the world.The RT-PCR primers were designed based on the nucleotide sequence of DHV-1 strain 161/79/V to amplify VP1 gene. The PCR product was cloned into pET-32a(+) vector The recombinant plasmid pET-VP1 were transformed into E.coli Rosetta-gami(DE3)pLysS. The target protein was successfully expressed after induced 6 hours with 0.5 mmol/L IPTG. The recombination protein was unstably expressed with soluble formation under 16℃. But they were stably expressed with sedimentary formation under 37℃. The Trx-His-VP1 fusion protein was about 47ku and purified by His-Ni~+ affinity chromatography under denaturalized condition. The target protein was harvested with a concentration of 0.2mg/ml. The Western Blot and indirect ELISA analysis showed that the purified Trx-His-VP1 protein could react to positive serum of DHV-1, do not to negative serum. The result demonstrated hat the expressed protein possessed antigenicity. In conclusion, the results provide a technique support for further developing a fast and accurate diagnosis method. |
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