Molecular Characterization Of Duck Hepatitis B Virus Isolates From Hubei Ducks And Development Of A Standard DHBV Animal Model

Hepatitis B virus (HBV) is a human hepadnavirus that causes acute and chronic hepatitis and hepatocellular carcinoma. The long-term objectives of our research are to design therapeutic strategies and to improve vaccines for human HBV infection. Our understanding of the immune response to HBV is incomplete, largely due to the narrow host restriction of this pathogen and the limitations of existing experimental models. Much of our current understanding of the viral life cycle of HBV infection is derived from studies of duck HBV (DHBV) and woodchuck HBV (WHV) infection in their natural hosts, HBV-infected chimpanzees, and HBV transgenic mice. For various reasons, however, none of these models is ideal. DHBV and WHV are genetically divergent from HBV, and immunological studies in genetically outbreed and immunologically uncharacterized ducks and woodchucks are difficult. Chimpanzee experiments are limited by cost, availability, and ethical considerations, whereas transgenic mice are immunologically tolerant to the virus. In summary, whilst the duck model has recognized limitations, it has proven to be an exceptionally useful system for the elucidation of hepadnaviral replication strategy and antiviral drug screening.One of the major advantages of the DHBV model is that its natural host, domestic duck, is inexpensive, easy to handle, and available from commercial breeders. To use the DHBV model as an in vivo experimental system, it is essential to have an efficient and reproductive duck infection method leading to the development of persistent infection in all animals. We currently use one method of DHBV transmission, which can experimentally infect ducklings with a pool of DHBV-positive serum, as it allows a rapid generation of a large flock of chronically infected ducklings of the same age. However, strain-related differences in the histological and virological progression of infection have not been documented. Although intralaboratory strain use can be assumed to be standardized, since individual groups tend to be consistent in their choice of animal supply and viral inocula, there are no established interlaboratory standards. So, it is necessary to develop a standard DHBV animal model to enhance our understanding of the replication strategy, natural history, and pathogenesis of HBV infection.Objective1. To clone and analyze the genome of a duck hepatitis B virus (DHBV) isolated from a Hubei brown duck.2. To construct the recombinant plasmid containing 1.5-fold-overlength genome of DHBV and obtain the DHBV inocula collected from culture supernatant of DHBV transfected the avain LMH line.3. To develop a standard DHBV animal model and use it as an in vivo experimental system to study antiviral strategies.Methods1. DHBV DNA-positive serum were collected from the investigation of the natural carrying rate of DHBV in adult ducks in Hubei area. The complete genome of a DHBV strain was amplified by polymerase chain reaction (PCR) prior to cloning into T vector and sequencing.2. 4.4 kb fragment of DHBV genome, derived from pMD-DHBV and DHBV positive serum, was cloned into Sac I and Not I site of the vector PCR2.1 to construct the recombinant plasmid PRC2.1-DHBV1.5. And to identify its inserted direction by endonucleases digestion.3. The avain LMH hepatoma cells were transfected with plasmid PCR2.1-DHBV1.5 by using LipofectineTM2000 transfection reagent. After 48 hours, the lever of intracellular viral DNA in the supernatant of LMH cells was analyzed by Southern blot hybridization. And the DHBV could be observed by electron microscope.4. 2-day-old ducklings can be experimentally infected with the supergenomic DNA via the intraperitoneal. Blood samples were taken twice or thrice a week during post-inoculation. Viremia was quantified by serum Real-Time PCR to show the viremia peak.5. Antiviral treatment to the DHBV-infected ducklings was started 3 d post-inoculation. Animals received oral administration of 3TC at a dose of 25mg/kg/d for 5 d, followed by a maintenance therapy thrice weekly for 3 more weeks. Viremia was quantified by serum Real-Time PCR to show the viremia peak. Southern Blot analysis of liver biopsies at the end of antiviral drug treatment.Results1. The natural carrying rate of DHBV in Hubei adult ducks was 10%. The strain had DHBV genomes of 3,024 nucleotides with three characteristic overlapping reading frmes encoding the polymerase, surface and core gene products. Comparison of the strain with 17 DHBV strains in GenBank revealed a homology of 89.3% to 93.5% at the nucleotide level. On translation of the open reading frames, the S protein,core protein,and functional domain of the Pol protein were high conserved in all of the DHBV strains. The Hubei strain was found to share signature amino acids in the polymerase genes with the"Chinese"DHBV isolates as opposed to those of the"Western"country isolates.2. We got successfully the recombinant plasmid PRC2.1-DHBV1.5 containing 1.5-fold-overlength genome of DHBV and identified its sense direction by endonucleases digestion.3. All expected DHBV replicative intermediates were verified by Southern blot analysis in the supernatant of LMH cells. We can use the transfected supernatant as experimental inoculum.4. The experimental infecting rate of 2-day-old ducklings was 87.5%. They were inoculated intravenously with transfected supernatant (8×107 virus genome equivalents/animal). Viremia started to be detectable at day 7 and reached a peak at day 11 post-inoculation, followed by a decrease and fluctuations.5. Four weeks of oral administration of 3TC leaded to a significant decrease in viremia peak during drug treatment. This effect is not sustained, as a rebound in viremia is observed after drug withdrawal. Similarly, the analysis of liver biopsies at the end of 3TC treatment showed a marked decrease in DHBV DNA. However, after drug withdrawal a rebound of intrahepatic DHBV DNA is observed in duck livers.Conclusions1. The present investigation shows Hubei ducks are one of the best species for experimental DHBV infection. The HB DHBV isolate might belong to a subtype of the Chinese DHBV isolates. The different folding of the encapsidsation signal (ε) reflect the lower intra-gron divergence fownd in the case of"Western"isolates, whereas the Chinese isolates are more divergent as a group.2. The recombinant plasmid PRC2.1-DHBV1.5 containing a replication-competent, which initiates viral replication efficiently in infected avian hepatoma cells, is expected as a useful source of DHBV inoculum in vivo.3. The Hubei duck model with experimental DHBV infection of transfected supernatant is more suitable for the hepadnavirus biologic research.