Resistance mutations of hepatitis B virus in entecavir‐refractory patients

Abstract

The emergence of resistance mutations in the reverse transcriptase gene of hepatitis B virus (HBV) is associated with treatment failure. Entecavir (ETV) is one of the most potent anti‐HBV reagents; it has a very low resistance rate and is used as the first‐line treatment for chronic hepatitis B. In this study, we isolated HBVs in 4 ETV‐refractory patients (2 with viral breakthrough, 1 with partial virological response, and 1 with flare‐up) and assessed ETV resistance using replication‐competent 1.38‐fold HBV genome‐length molecular clones. The full genome sequences of infected HBVs in ETV‐refractory patients were determined. The HBV molecular clones were generated with the patient‐derived sequences. After transfection of these molecular clones into HepG2 cells, viral replications and ETV susceptibilities were evaluated by measuring the amount of intracellular core‐particle‐associated HBV DNA using Southern blotting and real‐time polymerase chain reaction. Among these cases, ETV‐resistant variants were detected in 2 patients with viral breakthrough and responsible amino acid mutations in reverse transcriptase were successfully identified in these variants. No ETV‐resistant mutation was detected in the other cases. The identified ETV‐resistant mutations did not confer resistance to tenofovir disoproxil fumarate. Conclusion: The HBV replication model with patient‐derived sequences is useful for assessing replication efficiency, susceptibility to anti‐HBV reagents, and responsible resistance mutations and can aid in choosing the appropriate treatment strategy for treatment‐failure cases of chronic hepatitis B. (Hepatology Communications 2017;1:110‐121)

Anti‐viral effects of interferon‐λ3 on hepatitis B virus infection in cell culture

“…The replication‐competent molecular clone of HBV with a 1.38‐fold genome length was constructed by using the viral genome sequence of a patient infected with HBV. The detailed methods and the structure of the construct were described previously . The HBV clone of genotype C (GT‐C; clone C_JPN22) with the introduction of the precore stop mutation (G1896A) was used .…”

“…The detailed methods and the structure of the construct were described previously. 11 The HBV clone of genotype C (GT-C; clone C_JPN22) with the introduction of the precore stop mutation (G1896A) was used. 12 Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used for the transfection of the HBV molecular clone.…”

“…The HBV DNA titer was measured by real-time polymerase chain reaction (PCR) with a primerprobe set targeting the HBs region after treatment with DNase (RQ1 RNase-Free DNase, Promega, Madison, WI) before DNA extraction. 11,16 HBV was inoculated into HepG2/NTCP-C4 cells at 500 genome equivalents (GEq) per cell. The virus was washed out after 16 h, and the culture media were replaced with media containing the reagents of interest.…”

“…19 Detection of core-associated HBV DNA The intracellular core-particle-associated HBV DNA was isolated as described previously. 11 The HBV DNA titer was measured by real-time PCR with primers and probe as described previously. 11,16 Gene expression analysis mRNA expression levels of ISGs were quantified using TaqMan Gene Expression Assays (Thermo Fisher Scientific).…”

“…11 The HBV DNA titer was measured by real-time PCR with primers and probe as described previously. 11,16 Gene expression analysis mRNA expression levels of ISGs were quantified using TaqMan Gene Expression Assays (Thermo Fisher Scientific). Assay IDs of the examined ISGs are shown in Supplementary Table 1.…”