In Vitro Characterization of Viral Fitness of Therapy-Resistant Hepatitis B Variants

Abstract

Because of the overlapping of polymerase and envelope genes in the hepatitis B virus (HBV) genome, nucleoside analog therapy can lead to the emergence of complex HBV variants that harbor mutations in both the reverse transcriptase and the envelope proteins. To understand the selection process of HBV variants during antiviral therapy, we analyzed the in vitro fitness (the ability to produce infectious progeny) of 4 mutant viral genomes isolated from one patient who developed resistance to a triple therapy (lamivudine, adefovir, and anti-HBV immunoglobulins). The 4 mutant and the wild-type forms of HBV were expressed from vectors in hepatoma cell lines; replication and viral particle secretion capacities then were analyzed. The impact of envelope gene mutations on infectivity was tested in HepaRG cells using the hepatitis delta virus (HDV) model as a reporter for infection. The dominant HBV variant characterized from the therapy-resistant patient was found to have the best replicative capacity in vitro in the presence of high concentrations of lamivudine and adefovir. The expression of envelope proteins and secretion of subviral and Dane particles by this mutant was comparable with that of wild-type HBV. HDV particles enveloped by surface proteins from the selected mutant had the highest rates of infection in HepaRG cells compared with other mutants. These results illustrate the importance of viral fitness and infectivity as a major determinant of antiviral therapy resistance in patients. Understanding HBV mutant selection in vivo will help to optimize new anti-HBV therapeutic strategies.