Genotypic succession of mutations of the hepatitis B virus polymerase associated with lamivudine resistance

Background/Aims: Hepatitis B mutant strains of virus emerging during treatment with the nucleoside analog lamivudine are being increasingly recognized. In the majority of lamivudine-resistant isolates the mutations have been reported to occur within the YMDD motif of the viral polymerase, either as a single mutation M552I or as M552V concomitant with L528M. We analyzed the time course and genetic succession pattern during the emergence of lamivudine resistance. Methods: Seven patients with breakthrough viremia in the setting of chronic hepatitis (n=5) or recurrent HBV after liver transplantation (n=2) were investigated, Pre- and post-breakthrough serum samples were evaluated by single- or second-round PCR amplification and sequencing analysis, Results: Genotypic succession of the virus populations was observed to occur from M552I to M552I/L528M (n=2) and from L528M to M552V/L528M (n=1). The double mutations M552I/L528M (n=4) or M552V/L528M (n=2) were found in six out of seven patients, and represented the stable virus populations throughout the follow-up period. Breakthrough viremia was not associated with the single L528M mutation. The mean duration of uninterrupted treatment with lamivudine until breakthrough was 422 days (range 182-642) and was longer in the setting of chronic hepatitis B than in recurrent hepatitis B after liver transplantation. HBV DNA levels after breakthrough were lower than pretreatment levels in the majority of patients with chronic hepatitis but higher after liver transplantation. Conclusion: Our observations show that the virus populations conferring resistance to lamivudine can evolve from single to double mutations at amino acid 552 and 528 of the HBV polymerase, and that M552I/L528M or M552V/L528M seem to be the predominant mutations arising during long-term antiviral therapy with lamivudine.