Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients

[Display omitted] •Among oral antivirals for HBV infection, only tenofovir has revealed no genotypically resistant HBV.•However, there are patients with incomplete viral response during tenofovir-containing treatment.•We consistently identified 7 common mutations including rtS106C (C), rtH126Y (Y), rtD134E (E), and rtL269I (I).•The mutations C, Y, and E were novel mutations associated with drug resistance.•The quadruple CYEI mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC50 and 26.3-fold in IC90.•All tenofovir-resistant mutants with/without entecavir resistance were susceptible to a novel capsid assembly modulator. Tenofovir disoproxil fumarate (TDF) is one the most potent nucleot(s)ide analogues for treating chronic hepatitis B virus (HBV) infection. Phenotypic resistance caused by genotypic resistance to TDF has not been reported. This study aimed to characterize HBV mutations that confer tenofovir resistance. Two patients with viral breakthrough during treatment with TDF-containing regimens were prospectively enrolled. The gene encoding HBV reverse transcriptase was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The relative frequency of mutants was evaluated by ultra-deep sequencing and clonal analysis. Five mutations (rtS106C [C], rtH126Y [Y], rtD134E [E], rtM204I/V, and rtL269I [I]) were commonly found in viral isolates from 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC50 value for wild-type HBV was 3.8 ± 0.6 µM, whereas the IC50 values for CYE and CYEI mutants were 14.1 ± 1.8 and 58.1 ± 0.9 µM, respectively. The IC90 value for wild-type HBV was 30 ± 0.5 µM, whereas the IC90 values for CYE and CYEI mutants were 185 ± 0.5 and 790 ± 0.2 µM, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3–778 (IC50