Complete Genome Sequencing Of New Type Duck Hepatitis And Cloning And Expression Of VP1 Gene

Duck Viral Hepatitis (DVH) caused by duck hepatitis virus (DHV) is a highly mortal disease and spreads rapidly in three-week-old ducklings characterized primarily by Hepatitis. Now, the traditional attenuated vaccines and the highⅠ-yolk antibody were the main methods for the prevention and treatment of DHV. However, many domestic areas frequently reported that immuned ducks outbreaks of duck hepatitis in recent years, which was restricted the development of ducks industry seriously.1. Isolataion and RT-PCR Identification of a Korea new type Duck Hepatitis VirusOne strain of duck hepatitis virus was isolated from Shandong province. The result of serum neutralization test showed that there was no cross-protection occured in strain JFX08 and typeⅠduck hepatitis virus (DHV-Ⅰ). Clinical symptom and pathological lesion could be reproduced in 3-day ducklings challenged by the isolated strain. A pair of primers was designed and synthesized according to the sequence of duck hepatitis virus and a reverse transcriptase polymerase chain reaction method was developed for strain JFX08.Nucleotide sequence identity between strain JFX08 and N-DHV from Korean(genotype C) was 93.2%~94.0%. However, it was lower between strain JFX08 and DHV-Ⅰor N-DHV from Taiwan than N-DHV from Korean. Phylogenetic and evolutionary analysis showed that our isolated strain JFX08 belonged to genotype C, which was on different branches from genotype A and B. 2. Sequencing and analysis of the Genome of New Duck Hepatitis Virus JFX-08The genome of duck hepatitis virus strain JFX08 isolated from China has been sequenced and analyzed. The results showed that the full length of the genome was 7793nt; including 652nt of 5'untranslated region (UTR), an open reading frame of 6753nt encoded a polypeptide of 2251 amino acids, 366nt of 3'untranslated region. The pairwise percent identity of the amino acid sequences at ORF region between strain JFX08 and Korea new type DHV were higher than between strain JFX08 and I type DHV or Taiwanese new type DHV. There were 2 amino acids insertion and many variant sites and the hypervariable regions were found in 180-194aa and 213-219aa of VP1 gene. A lot of variant sites and insertion/ deletion were found in untranslated region. Phylogenetic and evolutionary analysis based on polyprotein, VP0, VP3, VP1, 2C and 3D indicated that strain JFX08 had a very close relationship with Korea new type DHV, which affiliated with genotype C.3. Cloning and Expression of VP1 Gene of Duck Hepatitis Virus Genotype C and Preparation of Its Polyclonal AntibodyThe VP1 gene of strain JFX08 of duck hepatitis virus genotype C was amplified by reverse transcription-polymerase chain reaction (RT-PCR). To obtain expression plasmid pET-32a -VP1, the VP1 gene was cloned into pMD18-T and pET-32a(+)vectors. SDS-PAGE analysis revealed that the recombinant VP1 protein of Genotype C of duck hepatitis virus was expressed in Escherichia coli BL21 (DE3) at a high level after being induced with Isopropylthio-β-D-galactoside (IPTG) stably. Western-blot revealed that the recombinant protein was recognized specifically by antisera against the Genotype C of duck hepatitis virus. 4-week-old SPF chickens were immunized with the purified recombinant protein. ELISA established by the purified recombinant protein and DHV Genotype C virus revealed that the titer of antiserum was 1:25 600 and 1:51 200 respectively, which indicated that the recombinant protein had good immunogenicity.