Characterization of the tenofovir resistance-associated mutations in the hepatitis B virus isolates across genotypes A to D

Tenofovir is wildly used to treat chronic hepatitis B (CHB) infection due to good potency and high genetic barrier to drug resistance. To date, it remains controversial whether hepatitis B virus (HBV) could be resistant to tenofovir. This study used multiple HBV isolates across genotypes A to D to characterize the reported tenofovir resistance-associated mutations identified from a few clinical case reports. Our data demonstrated that all the rtS78T, rtA194T, and CYEI (S106C, H126Y, D134E, and L269I) mutants exhibited no resistance to tenofovir in vitro. In contrast, the quadruple mutation MLVV (rtL180M, rtT184L, rtA200V, and rtM204V) decreased tenofovir susceptibility, with increased half maximal efficient concentration ranging from 3.28-fold to 5.34-fold, but it severely impaired viral fitness by reducing both replication capacity and infectivity of the mutants. Interestingly, basal core promoter (BCP) mutations A1762T/G1764A and precore (PC) mutation G1896A restored the replication capacity of the MLVV mutant to a level even higher than the wild-type virus without BCP and PC mutations. In conclusion, our data support the role of tenofovir as a first-line agent to treat CHB infection but also indicate tenofovir-resistant mutants could emerge due to multiple mutations accumulated during long-term therapy, particularly in hepatitis B e antigen-negative patients.