Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients

Eun Sook Park, Ah Ram Lee, Doo Hyun Kim, Jeong Hoon Lee, Jeong Ju Yoo, Sung Hyun Ahn, Heewoo Sim, Soree Park, Hong Seok Kang, Juhee Won, Yea Na Ha, Gu Choul Shin, So Young Kwon, Yong Kwang Park, Byeong Sun Choi, Yun Bin Lee, Nakcheol Jeong, Yohan An, Young Seok Ju, Su Jong YuHee Bok Chae, Kyung Sang Yu, Yoon Jun Kim, Jung Hwan Yoon, Fabien Zoulim, Kyun Hwan Kim

Research output: Contribution to journalArticlepeer-review

101 Citations (Scopus)


Background & Aims: Tenofovir disoproxil fumarate (TDF) is one the most potent nucleot(s)ide analogues for treating chronic hepatitis B virus (HBV) infection. Phenotypic resistance caused by genotypic resistance to TDF has not been reported. This study aimed to characterize HBV mutations that confer tenofovir resistance. Methods: Two patients with viral breakthrough during treatment with TDF-containing regimens were prospectively enrolled. The gene encoding HBV reverse transcriptase was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The relative frequency of mutants was evaluated by ultra-deep sequencing and clonal analysis. Results: Five mutations (rtS106C [C], rtH126Y [Y], rtD134E [E], rtM204I/V, and rtL269I [I]) were commonly found in viral isolates from 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC50 value for wild-type HBV was 3.8 ± 0.6 µM, whereas the IC50 values for CYE and CYEI mutants were 14.1 ± 1.8 and 58.1 ± 0.9 µM, respectively. The IC90 value for wild-type HBV was 30 ± 0.5 µM, whereas the IC90 values for CYE and CYEI mutants were 185 ± 0.5 and 790 ± 0.2 µM, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3–778 (IC50 <0.4 µM vs. IC50 = 0.4 µM, respectively). Conclusions: Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC50 and 26.3-fold in IC90. These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high. Lay summary: Tenofovir is the most potent nucleotide analogue for the treatment of chronic hepatitis B virus infection and there has been no hepatitis B virus mutation that confers >10-fold resistance to tenofovir up to 8 years. Herein, we identified, for the first time, a quadruple mutation that conferred 15.3-fold (IC50) and 26.3-fold (IC90) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.

Original languageEnglish
Pages (from-to)1093-1102
Number of pages10
JournalJournal of Hepatology
Issue number6
Publication statusPublished - 2019 Jun

Bibliographical note

Funding Information:
Park ES was supported by the National Research Foundation of Korea (NRF) grant funded by Korea Government (MSIP) (No. NRF-2016R1A2B4007531) and by extramural grants from the Korea National Institute of Health (No. 2016-ER5101-00). Lee JH was supported by the Seoul National University Hospital Research Fund (No. 03-2016-0380). Ju YS was supported by Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), which was funded by the Ministry of Health & Welfare (No. HI16C2387). Kim KH was supported by the NRF grants funded by MSIP (No. NRF-2016R1A5A2012284 and NRF-2017R1A2B3006335).

Publisher Copyright:
© 2019


  • Antivirals
  • CYEI
  • Capsid assembly modulator
  • Entecavir
  • HBV
  • Nucleotide analogue

ASJC Scopus subject areas

  • Hepatology


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