Stepwise process for the development of entecavir resistance in a chronic hepatitis B virus infected patient

. Background/Aims Complex mutants may be selected under sequential anti-VHB pressures. We analyzed the genotypic and phenotypic evolution of the viral quasi-species of a patient who developed resistance to entecavir following lamivudine breakthrough. Methods The polymerase gene was amplified, cloned and sequenced at different time points. Hepatoma cell lines were transfected to compare the replication capacity of HBV mutants and their drug susceptibility. Results A mixture of lamivudine-resistant HBV strains coexisted following viral breakthrough to lamivudine, all harboring the rtM204V mutation. The rtV173L+L180M+M204V dominant mutant displayed strong lamivudine-resistance and the highest replication capacity. Following the switch to entecavir, the viral load dropped but the lamivudine-resistant strains continued to be selected. Three years later, the viral load rose again, and a complex mixture of entecavir-resistant strains, all harboring the lamivudine-resistance signature rtL180M+M204V and the rtS202G mutation were observed. Although the rtL180M+S202G+M204V variant, that prevailed at the end of entecavir therapy, did not show the highest viral genome replication capacity, it conferred one of the strongest resistance levels to entecavir. Conclusions We report the selection of complex HBV mutants that escaped lamivudine and entecavir antiviral pressures. Genotypic and phenotypic analysis provided additional information to understand the process of HBV variant selection.