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Antivir Ther. 2019 Mar 18. doi: 10.3851/IMP3305. [Epub ahead of print]

Molecular cloning and phenotypic analysis of drug-resistance mutants with relevant S-region variants of HBV for a patient during 189-month anti-HBV treatment.

Author information

Institute of Infectious Diseases, Beijing 302 Hospital, Beijing, China.
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
Department of Infectious Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Institute of Virology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany.



A unique chronic hepatitis B patient was followed over 189 months of nucleoside/nucleotide analogue (NA) therapies with the analysis of multiple drug-resistance HBV mutants.


Clonal sequencing (≥20 clones/sample) was performed on sera sampled at 41 time points, and the phenotypic features of eight representative mutants were analysed.


Lamivudine (LAM)-, adefovir dipivoxil (ADV)-, entecavir (ETV)- and repeat ADV-resistance mutants emerged upon individual sequential NA monotherapy. The efficacy of NA combination rescue therapies ranked as LAM+ADV < ETV+ADV < ETV+ tenofovir disoproxil fumarate (TDF). Specifically, LAM+ADV and ETV+ADV suppressed viral loads to <100 IU/ml for a long period of time, either with or without late stage HBV DNA fluctuations. Furthermore, ETV+TDF suppressed the viral load to <10 IU/ml. During the LAM+ADV and ETV+ADV combination therapies, ETV-resistance mutants dominated at most time points, and multidrug-resistance (MDR) mutants that harboured LAM-, ETV- and ADV-resistance mutations were intermittently detected. Interestingly, the rtA181T-causative sW172stop to sW172non-stop mutation transition was observed at HBV DNA fluctuations. In a phenotypic analysis, two MDR strains had cross-resistance to LAM, ETV and ADV, and a lower susceptibility to TDF (<10-fold decrease compared with the wild-type strain). In contrast, the natural replication capacity was inversely associated with the number of primary resistant mutations which would limit MDR mutant development.


Taken together, viral drug susceptibility, replication capacity, and perhaps immunological adaptation may play coordinated roles in the fitness of drug-resistance mutants. ETV+TDF therapy is the preferred option for treating chronic hepatitis B patients with multiple drug failure.


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