Development And Characterizations Of An Infectious Molecular Clone And Its Derivatives Of Duck Hepatitis Virus Type Ⅰ

Duck hepatitis virus (DHV) causes a highly contagious disease in young ducks often associated with liver necrosis, hemorrhages and high mortality. We inoculated BHK-21 cells with supernatant of liver tissue grinding fluid. After blind passages, Obvious cytopathic effect (CPE) was observed.The morphology of the virus was spherical in shape, non-envelope with diameter of approximately 30 nm , which was examined by electron microscope via negative staining in supernatant fluid of the infected cell culture lysis products. Results of indirect immunofluorescence assay showed that the virus was specifically reacted with anti-DHV-Ⅰserum. A sequence with the size of 238bp was amplified by reverse transcription PCR (RT-PCR) with specific primers. Sequence analysis showed that similarity of the isolated virus and DHV-Ⅰproved to be 97.1% .These data showed that the virus isolated from BHK-21 cells was DHV-Ⅰand designated as ZJ-V/2006.It has been reported that reverse genetics system (RGS) is an important tool for studying on the pathogenesis mechanism, genome structure of RNA virus. We developed for the first time the reverse genetics of DHV-Ⅰ.First,five pairs of oligonucleotides were designed based on the full length genomic sequence of DHV ZJ-V strain. Using RT-PCR technique, five overlapping cDNA fragments, designated as A, B, C, D and E were amplified, respectively. And D and E fragments were fused by PCR, designated as ED. Using pBluescriptⅡKS ( + ) as a plasmid vector , the full-length cDNA clone pBLDHV of DHV ZJ-V strain was obtained by connecting the four cDNA fragments utilizing single restriction endonuclase site. A ApaⅠsite and a SP6 promoter were introduced immediately upstream of 5′end, while a NruⅠSite was engineered down stream of 3′end of DHV poly (A) tail ( containing 20 As). The results of sequencing and analysis showed that there were 99.9% identical between the construction of the full-length cDNA sequences and cDNA sequences of DHV-ⅠZJ-V strain, the only differences in sequence being at 6 positions, none of which affected the amino acid sequence. Second, the plasmid pBLDHV was linearized with NruⅠ, the DNA was used for in vitro transcription with the SP6 RiboMAX transcription kit.and acquired viral RNA by purified method. In vitro-transcribed RNA was mixed with DMRIE-C, and transfected BHK-21 cell, observed cytopathic effect (CPE). Apparent CPE were observed after 24h transfection.At 48h, CPE became more evident, and there was the phenomenon of cell detachment. At 72h, the detached cells were to 70%. Then, the rescued virus was identified by RT-PCR, IFA and electron microscope, respectively. The results showed that CPE could be observed clearly, 24h post-transfection, and viral antigen could also be detected by IFA. The results showed that viral protein was expressed in the transfected cells. Immune electron microscopic observation of the virus particles also revealed that the particles were rotundity with a diameter of 30nm. To exclude the possibility that the virus recovered from the transfected cells was due to contamination by the parental virus, the RT-PCR product from parental or recombinant viral RNA digested with EcoRⅠ. The RT-PCR product generated two fragments of 124 and 285 bp. In contrast, the PCR fragment derived from the parental isolated was not cleaved, the result clearly showed that viruses recovered from the transfected cells were derived from the infectious full-length RNA transcripts and not from contaminating parental viruses.Based on the foregoing research, we also studied on the biological characterictics of the recombinant virus. The results of TCID50 showed that the titre of the recovered virus can reach the peak (104.6/mL) at 48h post-transfection. Wild-type and the recombinant virus shared very similar overall patterns of replication, these data confirm that the recombinant virus shares phenotypic properties with the wild type ZJ-V strain in cell culture. The results of qRT-PCR showed that the genomic copies of the recombinant virus were related with the culture time of virus. The levels of DHV-1 RNA were quantified by real-time RT-PCR in BHK-21 cells 6-72 h after transfection with RNA transcribed from the full length cDNA clone. Virus genome copies determined by qRT-PCR, the genomic copies of the recombinant virus increased gradually, and the numerous reached the peak of replication (3.27×109 copies/mL) at about 48h, and then the genomic copies of the rescued virus gradually decreased. Furthermore, To determine whether the rescued virus, as well as the parental virus (ZJ-V strain), displayed the same phenotype in vivo, their virulence was assessed in duckling by inoculating i.p. with either rescued virus or the parental virus.The results showed that there were no obviservable differences in severity or quality of the clinical sign, and the survival curves were very similar.A real-time PCR assay (rPCR)was developed for efficient detection of Duck hepatitis virus typeⅠ(DHV-Ⅰ). A pair of specific primers was designed against the conserved region in the 3D gene that encodes the RNA dependent RNA polymerase. A real-time PCR assay based on SYBR GreenⅠwas developed for detection of the DHV-Ⅰ.The standard curve was constructed by using pMD-3D plasmid. The specifictiy, sensitivity and reproducibility of the assay were evaluated. No cross-reactions were found in specimens containing DEDSV,DHBV,DPMV,DPV,DRV and RA.The detection limit of this assay was 1 TCID50 /0.1 mL and it was highly specific to DHV-Ⅰ.The coincidence rates between the real-time PCR and the virus isolation were 100%. All results showed that the real-time PCR has high sensitivity and specificity to detect DHV-Ⅰusing SYBR Green I PCR assay. The rapid, sensitive and specific rPCR assay will be a powerful tool for detection of suspected cases of DHV-Ⅰ, distribution pattern of DHV-Ⅰin vivo and molecular epidemiological screening.In conclusion, we have successfully isolated to DHV-Ⅰfrom BHK-21 cell, designated as ZJ-V/2006. And constructed a genetically stable infectious clone of DHV-Ⅰ, and systematic studied on the bionomics of rescued virus, the recombinant virus and the parental virus showed similar biological properties in terms of growth kinetics, replication and virulence characteristics. The revers genetics system would allow for the development of DHV-Ⅰas a vaccine vector in domesticated animals. Furthermore, The model offers a novel approach to elucidate the mechanisms involved in viral pathogenesis, and the molecular basis attenuated after passage in tissue culture.