Identification and Characterization of Clevudine-Resistant Mutants of Hepatitis B Virus Isolated from Chronic Hepatitis B Patients

Abstract

Clevudine (CLV) is a nucleoside analog with potent antiviral activity against chronic hepatitis B virus (HBV)infection. Viral resistance to CLV in patients receiving CLV therapy has not been reported. The aim of this study was to characterize CLV-resistant HBV in patients with viral breakthrough (BT) during long-term CLV therapy. The gene encoding HBV reverse transcriptase (RT) was analyzed from chronic hepatitis B patients with viral BT during CLV therapy. Sera collected from the patients at baseline and at the time of viral BT were studied. To characterize the mutations of HBV isolated from the patients, we subjected the HBV mutants to in vitro drug susceptibility assays. Several conserved mutations were identified in the RT domain during viral BT, with M204I being the most common. In vitro phenotypic analysis showed that the mutation M204I was predominantly associated with CLV resistance, whereas L229V was a compensatory mutation for the impaired replication of the M204I mutant. A quadruple mutant (L129M, V173L, M204I, and H337N) was identified that conferred greater replicative ability and strong resistance to both CLV and lamivudine. All of the CLV-resistant clones were lamivudine resistant. They were susceptible to adefovir, entecavir, and tenofovir, except for one mutant clone. In conclusion, the mutation M204I in HBV RT plays a major role in CLV resistance and leads to viral BT during long-term CLV treatment. Several conserved mutations may have a compensatory role in replication. Drug susceptibility assays reveal that adefovir and tenofovir are the most effective compounds against CLV-resistant mutants. These data may provide additional therapeutic options for CLV-resistant patients.Chronic hepatitis B virus (HBV) infection is a major health problem worldwide and leads to chronic hepatitis, cirrhosis, and hepatocellular carcinoma (13). Antiviral treatment for chronic hepatitis B improves the outcome of the disease and prevents the development of hepatocellular carcinoma (14). Currently, several oral antiviral agents, including lamivudine (LMV), adefovir (ADV), and entecavir (ETV), have been approved for the treatment of chronic HBV infections (8). However, oral antiviral treatment does not provide a cure or durable remission and it has limited long-term efficacy due to the emergence of resistance (12). Long-term treatment with nucleos(t)ide analogs is associated with an increased risk of drug resistance. Antiviral drug resistance in patients infected with HBV is associated with subsequent virologic breakthrough (BT), viral rebound, and biochemical BT.Clevudine [1-(2-deoxy-2-fluoro-␤-arabinofuranosyl)thymine, L-FMAU] (CLV) is a pyrimidine analog with potent antiviral activity against HBV (4). CLV inhibits the DNA-dependent DNA activity of HBV polymerase, as well as reverse transcription and priming (1, 16). Phase III clinical trial results have shown that CLV therapy for 24 weeks has a potent and sustained antiviral effect in both HBeAg-positive and -negative chronic hepatitis B patients (23,24). Clinica...

Resistance mutations of hepatitis B virus in entecavir‐refractory patients

“…The rtL269I mutation was also detected in NA treatment‐failure patients. This mutation has been reported to enhance the replication efficiency and to be associated with enhanced replication efficiency when combined with the rtM204I mutation, although rtL269I alone did not alter susceptibilities to NAs 25, 27. In our experiment, rtL269I but not rtN238H could enhance ETV resistance when combined with rtL180Q/M204V, which is consistent with previous data 27, 28…”

“…By the addition of rtL269I to clone B1+QV, ETV resistance was enhanced and EC 50 was increased to a level similar to clone B2, although the addition of the rtN238H mutation had minimal effects on ETV resistance. The rtN238H mutation was sometimes detected in NA‐treated patients and has been reported not to influence the susceptibilities to LAM, ADV, or other NAs 24, 25, 26. The rtL269I mutation was also detected in NA treatment‐failure patients.…”

The Impact of the Hepatitis B Virus Polymerase rtA181T Mutation on Replication and Drug Resistance Is Potentially Affected by Overlapping Changes in Surface Gene

“…1), by using the QIAamp MinElute virus spin kit (Qiagen) according to the manufacturer's manual. After the RT gene PCR, HBV1.2mer replicons harboring the patient-derived RT gene were constructed using the pGEM-4z vector (Promega) and sequenced as previously described (24). All artificial HBV1.2mer clones, including rtI233V, rtA181T/sW172S, rtA181T/sW172*, rtN236T, rtA181T/sW172*ϩrtN236T, rtA181T/sW172SϩrtI233V, and rtA181T/sW172*ϩrtI233V, were generated by site-directed mutagenesis using WT HBV1.2mer.…”

“…Then, 2-g portions of WT and mutant HBV replicons were transfected into Huh7 cells using Lipofectamine 2000 (Invitrogen). At 4 h after transfection, the medium was replaced with a fresh medium containing the indicated concentrations of LMV, ADV, TDF, and ETV, as previously described (24). A fresh drug-containing medium was replaced every day for 4 days, and the cells were harvested.…”

“…In vitro drug susceptibility and replication assays were performed using Southern blotting as described previously (24,25). Briefly, cell pellets were lysed with 100 l of HEPES buffer containing 1% NP-40.…”