Identification and Characterization of Besifovir-Resistant Hepatitis B Virus Isolated from a Chronic Hepatitis B Patient

Abstract

Hepatitis B virus (HBV) is known to cause severe liver diseases such as acute or chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Chronic hepatitis B (CHB) infection is a major health problem with nearly 300 million individuals infected worldwide. Currently, nucleos(t)ide analogs (NAs) and interferon alpha are clinically approved treatments for HBV infection. NAs are potent antiviral agents that bind to HBV polymerase and block viral reverse transcription and replication. Besifovir dipivoxil maleate (BSV) is a newly developed NA against HBV in the form of acyclic nucleotide phosphonate that is available for oral administration similar to adefovir and tenofovir. Until now, resistance to BSV treatment has not been reported. In this study, we found a CHB patient who showed viral breakthrough after long-term treatment with BSV. The isolated HBV DNA from patient's serum were cloned into the replication-competent HBV 1.2 mer and the sequence of reverse transcriptase (RT) domain of HBV polymerase were analyzed. We also examined the drug susceptibility of generated clones in vitro. Several mutations were identified in HBV RT domain. A particular mutant harboring ten RT mutations showed resistance to BSV treatment in vitro. The ten mutations include rtV23I (I), rtH55R (R), rtY124H (H), rtD134E (E), rtN139K (K), rtL180M (M), rtM204V (V), rtQ267L (L), rtL269I (I) and rtL336M (M). To further identify the responsible mutations for BSV resistance, we performed in vitro drug susceptibility assay on several artificial clones. As a result, our study revealed that rtL180M (M) and rtM204V (V) mutations, already known as lamivudine-resistant mutations, confer resistance to BSV in the CHB patient.

Keywords: besifovir dipivoxil maleate (BSV); drug resistance; hepatitis B virus; nucleos(t)ide analog; reverse transcription.

Figure 1

Clinical course and liver ultrasonography of the CHB patient with incomplete virological response during BSV treatment. (a) Clinical course during treatment of BSV. The patient first was treated with BSV in December 2018. In May 2020, drug regimen was changed from BSV to TAF. Serum samples were analyzed for HBV-DNA and ALT measurement. The time points of serum sampling are indicated by arrows. ETV, entecavir; BSV, besifovir dipivoxil maleate; TAF, tenofovir alafenamide fumarate. (b) The abdominal ultrasound showed evidence of early liver cirrhosis.

Figure 2

BFV susceptibility of the HBV RT mutants isolated from the CHB patient. (a) The amino acid sequences of HBV RT domains isolated from the CHB patient were analyzed by cloning and sequencing. (b) The HBV 1.2 mers cloned using the patient-derived RT domain were transfected into the Huh7 cells. HBV DNA and antigen levels were measured by Southern blot and ELISA, respectively. The sensitivity of Huh7 to BFV was determined by southern blot using an HBV-specific digoxigenin (DIG) labeled probe. The 3.2 kb marker is shown in lane 1 (c) IC₅₀ values were determined by quantitative real-time PCR. ** p < 0.01; *** p < 0.001. Data were obtained from at least three independent experiments (mean ± SD). RT, reverse transcriptase; WT, wild type; BFV, besifovir.

Figure 3

Identification of BFV-resistant mutation in patient-derived HBV mutant. (a) The sequences of patient-derived and artificial replicons constructed in this study are compared with that of WT. (b,c) The constructed HBV 1.2mer replicons were transfected into Huh7 cells, and BFV was treated every day in a dose-dependent manner as indicated. At 5 h post-transfection, BFV was treated for 3 days. The level of HBV DNA and secreted antigen were determined Southern blot and ELISA, respectively. ELISA was performed to confirm the transfection yield. (d) IC₅₀ values were measured by quantitative real-time PCR. ** p < 0.01; *** p < 0.001. The level of HBV replication without drug treatment was set to 100 %. (e) Replication ability and BFV resistance of each clone were compared with WT through real-time PCR. All data were obtained from at least 3 independent experiments (mean ± SD).

Figure 4

Susceptibility of BFV-resistant mutants to other antiviral drugs. (a,b) The in vitro drug susceptibility assay of patient-derived (1-1 and 1-2) and artificial MV clones was performed after treatment with ETV or TFV. After transfection with 2 µg of the indicated HBV 1.2mer mutants into Huh7 cells were treated with indicated antiviral agents (ETV or TFV) for 4 days. HBV replicative capacity was analyzed using Southern blot and detected with a DIG-labeled probe. The secreted HBV antigen levels (HBeAg, HBsAg) were analyzed by ELISA. (c) The drug susceptibility assay of patient-derived and artificial MV clones was performed after treatment with capsid assembly inhibitor, NVR 3-778. After transfection with indicated HBV 1.2 mer mutants into Huh7, cells were treated with NVR 3-778 for 4 days. HBV replicative capacity was analyzed using Southern blot.