Analysis Of Evolution And Virological Characteristics Of RtI233V Mutations In The Hepatitis B Virus Reverse Transcriptase Domain

Objective: To analyze the evolution of rt I233 V mutation within the reverse transcriptase domain of hepatitis B virus(HBV) and its association with adefovir dipivoxil(ADV) resistance. Methods: Detection frequency of rt I233 V mutation in 9830 patients with chronic HBV infection was analyzed. HBV RT genes isolated from two patients’ serial serum samples were amplified by nested PCR, and clonal sequencing(>20 clones/sample) was performed to analyze the evolution of rt I233 V mutations. The replicative competence p Tri Ex-HBV1.1 vectors harboring wild-type(WT) and three mutation(rt I233 V, rt N236 T, rt I233V+rt N236T) strains and site-directed mutagenesis obtained rt N236 T strains were respectively constructed and transient transfected into Hep G2 cells. Then serially diluted or the highest concentrated solution of LAM, ADV, ETV and TDF were added to cells. Then the supernatant HBV DNA production was quantitatively detected by real-time PCR and HBV mutants’ replication competence and phenotypic characteristics under the drug pressures was analyzed. Results: The detection rate of rt I223 V mutation in 9830 nucleos(t)ide analogues-treated patients was 0.28%(28/9830), including 0.19%(19 patients) with rt I223 V individual mutation and 0.09%(9 patients) with rt I233 V mutation combining with rt N236 T or other mutations. All of the patients detected with rt I233 V had ADV exposure history: 16(57.1%) of them received ADV monotherapy for over six months; 12(42.9%) of them received ADV combined sequential therapy for over 12 months. The relative viral replication capacity and phenotypic resistance analysis of patient one showed rt I233 V mutant and wild-type strains had similar viral replication competence(98.2%), while rt N236 T exhibited significantly lower replication competence compared with wild-type strains(55.4%); rt I233V+rt N236 T mutant was 100.4% of the wild-type strain, showed rt I233 V have the ability to restore the defected replication capacity of rt N236 T strains. Susceptibility of mutant harboring rt N236 T, rt I233V+rt N236 T and rt I233 V was 1/6.82, 1/5.28 and 1/1.57 respectively to ADV compared to wild-type virus; rt N236 T and rt I233V+rt N236 T mutants confer resistance but rt I233 V mutant remained susceptible to adefovir. Rt I233 V had little impact on drug resistance when occurred in combination with rt N236 T. Patient two: The relative viral replication capacity of rt I233 V mutant was 102.5% of the wild-type strain. Susceptibility of rt I233 V mutant was 1/0.76 to ADV compared to wild-type virus. Rt I233 V mutant was susceptible to adefovir, lamivdine, entecavir, and tenofovir. ADV showed similar inhition rate to rt I233V+rt N236 T strains identified in clinic and rt N236 T obtained by site-directed mutagenesis in laboratory, and further confirmed rt I233 V strains had little impact on ADV resistance. Conclusion: The rt I233 V mutation is associated with sub-optimal response to ADV. Though it does not directly reduce virus sensitivity to ADV, rt I233 V mutation enhances ADV resistant strains replication competence, and serves as a complementary mutation.