Sequenceing And Analysis Of Zhejiang Strain And The Establishment Of A RT-PCR Mothod To Detect DHV-I

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 virus, duck hepatitis virus (DHV) type I, II and III, have been associated with these symptoms. DHV-I and DHV-III was classified as a picornavirus. However, DHV- II was classified as an astrovirus. Among DHV type I, II and III, DHV-I is now worldwide in distribution.The authentic 3′end sequences of genomes of duck hepatitis virus (DHV) of four strains (ZYM strain,Z05 strain,Z07 strain and Z10 strain ) was obtained by 3′RACE.The analysis showed that 3′end sequences in the genomes of the four DHV strains including 3D region, 3′untranslated region (3′UTR) and Poly(A) tail. The result shows that the amplified sequences is 597 nt in length (unincluding polyA tail), the 3′end sequences of genomes of DHV share high nucleotide acid identity among the four DHV strains range from 96.3 % to 100 %, Compared with five other DHV strains respectively,The nucleotide acid sequence identities of 3′end sequences range from 93.5 % to 99.7 %, Phylogenetic analysis of 3′end sequences indicated that share high nucleotide acid identity.A total of eight fragments,covering the complete genome of DHV-I Z10 isolate,were amplified with RT-PCR,using specific primers at several positions a long the template RNA based on the published DHV-I sequence.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 weres equenced after ligation to the pMD-18T. Sequencing after splicing, the online BLAST homology search, identified as duck hepatitis virus type I genome sequence, With the help of molercular biology soft ware, the genome organization and the molecular evolution and phylogeny of Z10 isolate were analyzed. The results were as following: The Z10 DHV-I genome consisted of 7688 nucleotides(excluding the poly(A) tail) and contained a single open reading frame (ORF) encoding a polyprotein of 2249 aa. 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. Compared with the genome sequences of six DHV-I isolates published in GenBank。The results demonstrated that Z10 isolate had the most recently genetic relationship with CL isolate and they had a nucleotide sequences homology by 98.4 %; It had the lowest nucleotide sequence homology with the H strain by 95.3 %.No additions or deletions were observed in the genomic sequence of Z10.when compared with that of 03D.The 5'UTR of the Z10 strain consisted of 626 nucleotides and the 3'UTR consisted of 314 nucleotides.According to the sequences of duck hepatitis virus type I (DHV-I) published in GenBank a pair of primers were designed and synthesized. VP1 gene cDNA fragments of flour strains of DHV typeⅠ(named Z05,Z06,Z07,Z10)were amplified by RT-PCR respectively. PCR products were cloned and sequenced. The results suggested that the full-length of DHV serotype I VP1 gene all comprised 714 bp , coding for 238 amino acids. The nucleotide sequence homologies among 4 strains were between 93.0 %～99.7 % , and those of the amino acid sequence were between 95 %～100 %. There was a 93.0 %～99.7 % identity of the VP1 nucleotides sequence among the four isolates and 91.2 %～99.7 % identity of the amino acid sequence with the DHV-I isolates from GenBank. Phylogenetic analysis confirmed that three isolates from zhejiang province were clustered within the same gene group, Most of the amino acid substitutions located in the carboxy terminus of VP1 gene, amino acid replacement appears, suggesting that VP1 might contain some virulence determined sites of DHV-I. The results demonstrated that DHV may cintains there genotypes. Screening Genetic and phylogenetic characteristic of DHV will provide more information on how DHV evolves and is transmitted in the world.A RT-PCR technology for diagnosing DHV-I had 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.