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Virological breakthrough is a clinical manifestation in patients infected with chronic hepatitis B (CHB), who undergo treatment with nucleoside/nucleotide analogs (NUCs). The current understanding of the underlying mechanism of virological breakthrough is limited. Ultra‑deep pyrosequencing (UDPS) is a novel and powerful tool used to investigate minor viral variants and viral evolution. The present study used UDPS to investigate the viral evolution pattern during virological breakthrough in patients with CHB treated with NUCs. A total of 12 patients who experienced virological breakthrough were recruited in the present study. During the treatment with lamivudine, adefovir was added as a rescue therapy when virological breakthrough emerged, and the therapy was continued until week 96. Serum samples from each patient were collected at different time points for UDPS analysis. Treatment with lamivudine resulted in an increased rate of the viral mutations, rtM204V/I, rtL180M and rtL80I. Virological breakthrough was accompanied by significant rtM204I/V substitutions in eight of the patients. A total of three types of rt204 mutation, associated with virological breakthrough, were observed, including YIDD variant‑dominated, YVDD variant‑dominated and YMDD wild‑type‑dominated virological breakthrough. YVDD variants reverted to the wild‑type following the adefovir add‑on rescue therapy, although the YIDD variants remained dominant following the combination therapy. The mechanism underlying virological breakthrough was revealed to be complex and associated with the rapid replication of mutated variants. UDPS analysis, therefore, provided a useful tool to investigate the dynamic evolution pattern of hepatitis B virus.

molecular medicine reports

Evaluation of the dynamic pattern of viral evolution in patients with virological breakthrough during treatment with nucleoside/nucleotide analogs by ultra-deep pyrosequencing