Sequencing And Analysis Of The Full-length Genome Of ZJ-V Strain And Establishment Of RT-PCR For Detecting DHV-Ⅰ

A total of five fragments,covering the complete genome of DHV-IZJ-V isolate,were amplified with RT-PCR,using specific primers at several positions along the template RNA based on the published DHV-â… sequence.The cDNA fragment from the 3'end of the viral genome was amplified by the "Rapid Amplification of eDNA Ends"(RACE) method.The purified PCR products were sequenced after ligation to the pMD-18T.With the help of molercuLar biology soft ware,the genome of ZJ-V isolate was analyzed. The results were as following:The ZJ-V DHV-â… genome consisted of 7691 nucleotides(excluding the poly(A)tail) and contained a single open reading frame(ORF)containing 6747 nucleotides that terminates in a UGA codon.No additions or deletions were observed in the genomic sequence of ZJ-V when compared with that of 03D.The 5'UTR of the ZJ-V strain consisted of 626 nucleotides and the 3'UTR consisted of 318 nucleotides.Sequence analyses revealed that the VP1 genes of these DHV-â… isolates all had the same size of 714 bp as those of most other reported DHV-â… isolates.Among all the cited DHV-â… isolates,they shared 93.3%～95.9%nucleotide and 92.9～95.0%amino acid sequence identities in the VP1 gene.Most of the amino acid substitutions located in the carboxyl terminus of VP1 gene,and three Chinese attenuated DHV-â… isolates showed four coincident mutations(S₁₈₁→L₁₈₁,E₂₀₅→K₂₀₅,R₂₁₇→K₂₁₇,N₂₃₅→D₂₃₅),suggesting that VP1 might contain some virulence determined sites.Phylogenetic analysis confirmed the existence of two distinct genotypes of DHV-â… Phylogenetic analysis confirmed the existence of two distinct genotypes of DHV-â… (Groupâ… and Groupâ…¡).Interestingly,the DHV-â… isolates from Groupâ… were all tissue-adapted viruses;on the other hand,ten of eleven field DHV-â… isolates were clustered within Gâ…¡, which meaned that the genome of DHV-â… had mutated after passaged in chicken/or duck embryo embryo and these mutations might result in the attenuation of field DHV-â… isolates.The secondary structure of NCR of DHV was predicted and analyzed by MFOLD software online.The results showed that there were four stem-loops(SL)in 3'NCR, named as SL1,SL2,SL3 and SL4,respectively.Apart from SL1,the other structure domains show high stability,which may be related to the replication,host specificity of DHV.The secondary structure of 5'NCR has one conservative loop-stem structure domin,which was similar to that of RDL-62 isolate.The secondary structure and B lymphocyte antigen epitopes of structural protein of DHV-â… were predicted.The result showed that the secondary structure of VP0,VP1 and VP3 all were complicate and two dominant antigen epitopes located in the N-terminal of protein VP1 and VP3,respectively.A RT-PCR technology for diagnosing DHV-â… has been constructed,and the results showed that the method was quick,high specificity and sensitivity over traditionary detecting methods.In a world,the method can be wildly applied in many fields,such as epidemiologic survey or clinical diagnosis et al.