ABSTRACT
Objective To establish a novel and convenient method to study the phenotype of drug resistant hepatitis B virus (HBV) isolates,and to analyze the drug susceptibility by replacing the reverse transcriptase (RT) domain of wild-type HBV with that of the drug resistant HBV isolates.Methods Full length of HBV isolates was amplified and cloned from the sera of patients prior to nucleoside/nucleotide analogues (NA) treatment.Wild-type full-length HBV genomes was used to construct the recombinant expression plasmids PHY536207 (genotype B) and PHY97 (genotype C).The restriction enzyme sites were introduced in the upstream and downstream region of reverse transeription (RT) domain to construct plasmid,which were named as mPHY536207 and mPHY97,respectively.Lamivudine (LAM) resistant mutant and adefovir (ADV) resistant mutant were isolated and cloned to construct recombinant expression plasmids PHY634 and PHY6923,respectively.Subsequently,the RT domain of mPHY536207 was replaced by that of drug resistant mutant to construct the plasmids RT634 (LAM-resistant) and RT6923 (ADVresistant).The HBV constructs were transfected into Huh7 cells.The HBsAg levels in supernatant were determined by enzyme-linked immunosobent assay (ELISA),and the amount of intracellular HBV DNA was assayed by real-time polymerase chain reaction and Southern blot analysis.Results The plasmids PHY536207 and PHY97 containing genotype B and genotype C wild-type fulllength HBV genomes were constructed successfully,both of which could replicate in Huh7 cells.Intracellular HBV DNA extracted from cells in each of six-well culture plates was more than 1 × 107 copy/ mL,and the introduction of Pst Ⅰ restriction enzyme site did not affect the viral replication and HBsAg secretion.PHY634 and RT634,in which mutant RT domain was replaced into a wild type HBV expressing vector,exhibited the same HBV DNA replication under the treatment with different doses of LAM,the value of 50％ inhibitory concentration (IC50) was ＞100 μmol/L,while the IC50 of mPHY536207 was 0.18μmol/L.Moreover,wild-type isolate was sensitive to ADV (IC50 =1.2 μmol/L),while PHY6923 and RT6923 were resistant to ADV treatment (IC50 ＞100 μmol/L).Conclusion The phenotypic assay is successfully developed in this study based on replacing RT domain of wild-type HBV strains with that of clinical isolated drug resistant strain.
ABSTRACT
Objective To investigate the effects of adefovir resistance related mutations in hepatitis B virus (HBV) reverse transcription (RT) region on the viral replication and hepatitis B surface antigen (HBsAg) secretion. Methods Twelve adefovir treated chronic hepatitis B (CHB) patients who experienced a viral breakthrough were enrolled in this study. The RT region was amplified by polymerase chain reaction (PCR) using HBV DNA extracted from sera as the template. PCR products were then sequenced and analyzed to find out mutation patterns. Full-length HBV genome was amplified from 4 representative serum samples followed by direct sequencing. The dominant strain was cloned into vector PHY106 to construct a recombinant plasmid containing the 1.1 unit of HBV genome Which was transfected into Huh7 cells. HBsAg and hepatitis B e antigen (HBeAg) expression were determined by enzyme-linked immunosorbent assay (ELISA), meanwhile intracellular HBV DNA level was determined by quantitative real-time PCR. Furthermore strain harboring rtA181T/sW172 · mutation and strain without rtA181 mutation were cotransfected into Huh7 cells. HBsAg and intracellular HBV DNA were also determined after transfection. Results Ten out the 12 patients enrolled in this study exhibited mutations conferring resistance to adefovir. The rtA181T mutation was detected in 5 cases, and the rtA181T/S+rtN236T mutation was observed in 4 cases. Different mutants showed variable HBsAg secretion competency in vitro. Despite the defect of HBsAg secretion of the rtA181T/sW172 · mutant, the replication efficiency was almost the same in different mutants. When the strains with and without rtA181 mutation were cotranfected into cells, the HBsAg level increased in accordance with the amount of stains without rtA181 mutation. However, the intracellular HBV DNA level was not changed significantly. Conclusions The rtA181 mutation is common in patients with adefovir resistance, of which the rtA181T mutation is the major pattern. In vitro analysis reveals that the rtA181T/sW172 · mutant is defective in HBsAg secretion which could be rescued by coexistence of wild-type strains. The replication efficiency in various mutants shows no obvious differences.