Background & Aims: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a

Resistance of hepatitis B virus (HBV) to antivirals has become a major clinical problem. Our objective was to develop a new method for the cloning of naturally occurring HBV genomes and a phenotypic assay capable of assessing HBV drug susceptibility and DNA synthesis capacity in vitro. Viral DNA was extracted from sera and was amplified by polymerase chain reaction, and amplicons were cloned into vectors that enable, after cell transfection, the initiation of the intracellular HBV replication cycle. Single or multiple clones were used to transfect Huh7 cells. The viral DNA synthesis capacity and drug susceptibility were determined by measuring the level of intracellular DNA intermediate, synthesized in absence or presence of antiviral, using Southern blot analysis. We have developed, calibrated, then used this phenotypic assay to determine the drug susceptibility of HBV quasispecies isolated throughout the course of therapy from patients selected according to their mutation profile. A multiclonal and longitudinal analysis enabled us to measure the variation of drug susceptibility of different viral quasispecies by comparison of IC(50)/IC(90)s with standards. The presence of famciclovir- or lamivudine-induced mutations in the viral population caused a change in viral DNA synthesis capacity and drug susceptibility in vitro, demonstrating the clinical relevance of the assay. In conclusion, our phenotypic assay enables the in vitro characterization of DNA synthesis capacity and drug susceptibility of HBV quasispecies isolated from patients. This assay should allow a better monitoring of patients undergoing antiviral therapy, as well as the screening of novel drugs on emerging resistant strains.

The immunisation properties of an inactivated bluetongue virus serotype 8 (BTV-8) vaccine were evaluated in sheep and cattle. Five sheep were vaccinated with one dose of vaccine and five cattle were vaccinated with two doses 28 days apart. Six sheep and five cattle served as unvaccinated controls. All animals were subjected to a virulent BTV-8 challenge, and safety and antibody responses were monitored. All control animals developed disease and viraemia, while vaccinated animals were clinically protected and viraemia was completely prevented.

Resistance of hepatitis B virus (HBV) to antivirals has become a major clinical problem. Our objective was to develop a new method for the cloning of naturally occurring HBV genomes and a phenotypic assay capable of assessing HBV drug susceptibility and DNA synthesis capacity in vitro. Viral DNA was extracted from sera and was amplified by polymerase chain reaction, and amplicons were cloned into vectors that enable, after cell transfection, the initiation of the intracellular HBV replication cycle. Single or multiple clones were used to transfect Huh7 cells. The viral DNA synthesis capacity and drug susceptibility were determined by measuring the level of intracellular DNA intermediate, synthesized in absence or presence of antiviral, using Southern blot analysis. We have developed, calibrated, then used this phenotypic assay to determine the drug susceptibility of HBV quasispecies isolated throughout the course of therapy from patients selected according to their mutation profile. A multiclonal and longitudinal analysis enabled us to measure the variation of drug susceptibility of different viral quasispecies by comparison of IC50/IC90s with standards. The presence of famciclovir‐ or lamivudine‐induced mutations in the viral population caused a change in viral DNA synthesis capacity and drug susceptibility in vitro, demonstrating the clinical relevance of the assay. In conclusion, our phenotypic assay enables the in vitro characterization of DNA synthesis capacity and drug susceptibility of HBV quasispecies isolated from patients. This assay should allow a better monitoring of patients undergoing antiviral therapy, as well as the screening of novel drugs on emerging resistant strains. (Hepatology 2004;40:855–864).

This study used a duck hepatitis B virus (DHBV) model to evaluate whether a novel DNA vaccination protocol alone or associated with antiviral (lamivudine) treatment was able to clear the intrahepatic covalently closed, circular viral DNA (cccDNA) pool responsible for persistence of infection. DHBV carriers received DNA vaccine (on weeks 6, 10, 13, 14, 28 and 35) targeting the large envelope and/or core proteins alone or combined with lamivudine treatment (on weeks 1-8) or lamivudine monotherapy. After 10 months of follow-up, a dramatic decrease in viraemia and liver DHBV cccDNA (below 0.08 cccDNA copies per cell) was observed in 9/30 ducks (30 %) receiving DNA mono-or combination therapy, compared with 0/12 (0 %) from lamivudine monotherapy or the control groups, suggesting a significant antiviral effect of DNA immunization. However, association with the drug did not significantly improve DHBV DNA vaccine efficacy (33 % cccDNA clearance for the combination vs 27 % for DNA monotherapy), probably due to the low antiviral potency of lamivudine in the duck model. Seroconversion to anti-preS was observed in 6/9 (67 %) ducks showing cccDNA clearance, compared with 1/28 (3.6 %) without clearance, suggesting a significant correlation (P,0.001) between humoral response restoration and cccDNA elimination. Importantly, an early (weeks 10-12) drop in viraemia was observed in seroconverted animals, and virus replication did not rebound following the cessation of immunotherapy, indicating a sustained effect. This study provides the first evidence that therapeutic DNA vaccination is able to enhance hepadnaviral cccDNA clearance, which is tightly associated with a break in humoral immune tolerance. These results also highlight the importance of antiviral drug potency and an effective DNA immunization protocol for the design of therapeutic vaccines against chronic hepatitis B.