Sequence Analysis Of The Full-length Genome Of Attenuated Strain A66 And Establishment Of RT-nested-PCR Method For Duck Hepatitis Virus Type Ⅰ

Duck hepatitis virus(DHV) is the causative agent of duck viral hepatitis(DVH),a fatal and rapidly spreading viral infection of young ducklings.According to the complete genome sequences of DHV-Ⅰ03D strain and C80 strain which published on GenBank,six pairs of primers covering the complete genome of DHV-Ⅰ,were designed and synthesized by Primer Premier 5.0.A total of six fragments of attenuated strain A66 were amplified with RT-PCR.The cDNA fragment from the 3'end of the viral genome was amplified by the "Rapid Amplification of cDNA Ends" (RACE) method.The purified PCR products were sequenced after ligated to the pMD-19T. The genome sequence of A66 strain was analyzed by DNAStar soft ware.The results were as following:The complete sequence of Duck hepatitis virus typeⅠ(DHV-Ⅰ) attenuated strain A66 was determined(GenBank accession number DQ886455).The results showed that the full genomic length of A66 was 7704nt without poly(A) tail,with a 626nt of 5'UTR, followed by a long open reading frame(ORF) of 6750nt,and a 314nt of 3'UTR.Results showed that except two N-DHV-Ⅰstrains 90D and 04G,the identities of the complete nucleotide sequences of A66 were all between 94.5%～99.7%compared with the other reference DHV-Ⅰstrains published on GenBank;the identities of the nucleotide and amino acid sequences of ORF of the genome of A66 were all between 94.3%～99.7%and 97.3%～99.6%respectively compared with the other reference DHV-Ⅰstrains published on GenBank.The primary structure of the genome of DHV-Ⅰwas high conservative.Phylogenetic analysis based on the nucleotide and amino acid sequences of complete genome,ORF and VP1 of A66 strain and 31 reference DHV-Ⅰstrains indicated that the 32 DHV-Ⅰsequences were clustered into two major genotypes(A and B). Genotype A contained 30 DHV strains determined in this study and viruses from China the USA,the UK and South Korea.Genotype B contained two N-DHV-Ⅰstrains(90D and 04G) isolated in Taiwan in 1990 and 2004,respectively.The attenuated strain A66 was grouped into one lineage with attenuated strain C80 and virulence strain JX,which these evolution were closer than the other strains.According to the sequence of RNA polymerase encoding region of A66 strain,two pairs of primers were designed and synthesized.A RT-nested-PCR assay for detecting DHV-Ⅰwas established.A specific 304 bp fragment was amplified from RNA templates of DHV-Ⅰattenuated strain A66 and virulence strain R85952,but no bands were amplified with templates extracted respectively from normal chicken embryo,health duck liver tissue, duck plague virus(DPV),gosling parvovirus(GPV),avian influence virus(AIV H9 subtype),newcastle disease virus(NDV),infectious bursal disease virus(IBDV),egg drop synedrome virus(EDSV),Duck origin Escherichia coli(E.coli),Riemerella anatipestifer(RA),Duck origin Pasteurella multocida(PM).Sensitivity of the first and second amplifications by the RT-nested-PCR assay was 100 pg and 1 fg,respectively.The sensitivity of the second amplifications was increased by 100000 times.Clinical cases were detected initially by the RT-nested-PCR method and the results were identical with the virus isolation.Results showed the RT-nested-PCR assay improved the detection sensitivity when it was used for the detection of DHV-Ⅰ.The RT-nested-PCR technique could be used as a method to diagnosis and detect samples of clinical cases and investigate molecular epidemiology of DVH,which has application value.