Analysis of the replication and drug resistance of patient serum hepatitis B virus (HBV) populations can contribute to the therapeutic management of chronic hepatitis B. We developed a procedure for cloning serum HBV quasispecies populations and for phenotypic analysis of the cloned populations for in vitro drug susceptibility. Equivalent sequences were compared to the respective serum HBV DNAs of the cloned quasispecies by population sequencing. Analysis of individual clones revealed that each population contained a diversity of HBV quasispecies. Furthermore, secreted HBV in the supernatant following transfection of the quasispecies populations remained mostly unchanged from the respective input populations. HBV obtained from patients who had developed resistance to adefovir or lamivudine, as demonstrated by development of the rtA181V or rtL180M/M204V mutations in HBV polymerase, respectively, were tested. Phenotypic analysis demonstrated that a population containing the HBV rtA181V mutation showed a 2.9-fold increase in the 50% effective concentration (EC50) for adefovir compared to the wild-type baseline isolate, while the lamivudine-resistant HBV quasispecies population showed a >1,000-fold increase in the lamivudine EC50. In summary, a strategy of cloning full genome HBV quasispecies populations from patient sera was developed, which could provide a useful tool in clinical HBV drug resistance phenotyping and studies of the evolution of clinical viral species.
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