Analysis For Phenotypic Resistant Characteristics Of A Novel Mutation RtN236V In The Reverse-transcriptase Domain Of Hepatitis B Virus Isolated From Two Adefovir Dipivoxil-refractory Patients With Chronic Hepatitis B

Objective:Adefovir dipivoxil (ADV) which belongs to nucleotide analogue is commonly used in clinical for anti-hepatitis B virus (HBV) therapy, but long-term use can lead to the occurrence of drug-resistance. Until now rtN236T and rtA181V are the classic primary resistant mutations reported for ADV, However other substitutions at the residue rtN236has not been reported. This study aimed to identify a novel mutation rtN236V in the HBV reverse transcriptase (RT) region of two ADV-refractory patients with chronic hepatitis B and analyze its phenotypic resistant characteristics. Methods:HBV DNA was extracted and the full-length RT region was amplified by nested PCR, then the PCR product was cloned into the pGEM-Teasy vector for sequence analysis. Sixteen clones from patient one’s sample and thirty-four clones from patient two’s sample were randomly selected for DNA sequencing and drug-resistance-associated mutations were analyzed. After restriction double enzyme digestion and ligation procedure, the1.1-ploid genome length HBV recombinant plasmids harboring wild type and the different mutants in RT region were constructed. Then the replication-competent constructs were transiently transfected into the HepG2cells and cultured in the presence or absence of serially-diluted nucleos(t)ide analogs. Four hours post-transfection, new medium containing different concentrations of lamivudine, adefovir dipivoxil, entecavir, and tenofovir was supplemented every other day for four days. Viral replication capacity was determined by measuring supernatant HBV DNA using real-time quantitative PCR. To analyze the phenotypic characteristics of the HBV mutants under the drug pression, the supernatant was collected and HBV DNA production was quantitated using real-time quantitative PCR. Results:Sequence analysis of16clones from patient one showed that there were13(81.25%) for rtN236V,2(12.5%) for rtN236T,1(6.25%) for wild type. Sequence analysis of34clones from patient two showed that there were20(59%) for rtN236V,11(32%) for rtN236T,2(6%) for wild type, and1(3%) for rtA181V+N236V. The1.1-ploid genome length HBV recombinant plasmids harboring wild type and different mutants in RT region were constructed successfully. Patient one:The relative viral replication capacity of rtN236T, rtN236V mutants was84.76%and80.02%of the wild-type strain respectively. Patient two:The relative viral replication capacity of rtN236T, rtN236V and rtA181V+N236V mutants was89.43%,83.60%and66.44%of the wild-type strain respectively. Phenotypic resistance analysis showed that compared with wild-type strain, rtN236V mutants from patient1and patient2decreased3.90-fold and3.10-fold, and rtN236T mutant decreased4.50-fold and4.75-fold susceptibility to ADV respectively in vitro. The rtA181V+N236V mutant from patient2decreased5.10-fold susceptibility to ADV. The three mutants were still susceptible to lamivdine, entecavir and tenofovir. Conclusion: A novel mutation rtN236V concomitant with rtN236T and rtA181V mutations was first identified in viral pool of two ADV-refractory patients with virologic breakthrough. The mutant rtN236V had significantly decreased the viral replication capacity and led to less susceptibility to ADV, the evidence conferred rtN236V as a novel ADV-resistance-associated mutation.