A new strategy for studying in vitro the drug susceptibility of clinical isolates of human hepatitis B virus
Résumé
Background/Aim : Hepatitis B virus resistance 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. Methods : Viral DNA was extracted from sera, PCR amplified with newly designed primers, and 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. Results : We have developed, calibrated, then used this phenotypic assay to determine the drug susceptibility of HBV quasi-species isolated throughout the course of therapy from patients selected according to their mutation profile. A multiclonal and longitudinal analysis enabled to measure variation of drugs susceptibility of different viral quasi-species 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. Conclusion : Our phenotypic assay enables the in vitro characterization of the DNA synthesis capacity and drug susceptibility of HBV quasi-species 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.
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