This study aimed to clarify whether rtA186T and rtI163V substitutions of hepatitis B virus (HBV) contributed to entecavir (ETV) resistance. A total of 22,009 Chinese patients with chronic HBV infection who received resistance testing at Beijing 302 Hospital from 2007 to 2016 were enrolled. Among them, 6170 patients had been treated with ETV. The HBV reverse transcriptase gene was screened by direct sequencing and verified by clonal sequencing. Phenotypic analysis was performed for evaluating replication capacity and drug susceptibility. Classical ETV-resistance mutations rtT184/S202/M250substitution+rtM204V/I±L180M (LAM-r), rtA186T, and rtI163V were detected in 1252 (5.69%), 14 (0.06%), and 230 (1.05%) of the 22,009 patients, respectively. The rtA186T mutation always coexisted with LAM-r, but not with rtI163V. The 14 rtA186T-positive patients were all treated with LAM and ETV, and the emergence of the rtA186T+LAM-r was closely associated with virological breakthrough or inadequate virological response to ETV. By contrast, the emergence of rtI163V was not related to ETV treatment. Six rtA186T-positive patients were followed up longitudinally, showing that these patients all had received sequential adefovir and LAM monotherapies prior to ETV treatment. Compared to wild-type strain, two patient-derived mutants' rtL180M+A186T+M204V and rtL180M+T184S+A186T+M204V had 86.7% and 89.2% decreased replication capacity, 210- and 555-fold increased ETV resistance, respectively; and artificial elimination of rtA186T largely restored their ETV sensitivity. The rtA186T mutants remained sensitive to tenofovir. In conclusion, our study confirmed that rtA186T plus LAM-r is a novel ETV-resistance mutation pattern which conferred ETV resistance in multiple Chinese patients.
Keywords: Entecavir; Hepatitis B virus; Mutation; Resistance; rtA186T; rtI163V.
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