Screening and identification of a novel adefovir dipivoxil resistance associated mutation, rtN236V, of HBV from a large cohort of HBV-infected patients.

BACKGROUND The study aimed to clarify whether rtN236V mutation of HBV derived from adefovir dipivoxil (ADV)-refractory patients was associated with drug resistance. METHODS A total of 18,419 patients from Beijing 302 Hospital were investigated. HBV complete reverse transcriptase region of polymerase was screened by direct sequencing and verified by clonal sequencing if necessary. Replication-competent wild-type and mutant HBV genomic amplicons were constructed, transfected into HepG2 cells and cultured in the presence or absence of serially diluted nucleoside/nucleotide analogues. Intracellular HBV replicative intermediates were quantitated for calculating the 50% effective concentration of drug. RESULTS rtN236V was detected in six ADV-refractory patients; signature ADV-resistant mutations rtA181V and rtN236T were detected in 1,311 patients. rtN236V mutants emerged predominantly with virological breakthrough in the clinical course of the six patients. Phenotypic analysis of the mutants from two patients was performed. rtN236V mutants from patient 1 and patient 2 exhibited 3.90-fold and 3.10-fold decreased susceptibility to ADV, respectively, compared to the wild-type virus; by contrast, rtN236T mutants from the patients had 4.50-fold and 4.75-fold decreased susceptibility, respectively. Both mutants had a relatively lower viral replication capacity compared to wild-type virus in the absence of antivirals and remained susceptible to lamivudine, entecavir and tenofovir disoproxil fumarate. In clinical practice, switching to entecavir rescue therapy suppressed HBV DNA to an undetectable level and normalized alanine aminotransferase level for both patients. CONCLUSIONS rtN236V was a novel infrequently occurring ADV-resistance-associated mutation. It conferred a moderate resistance to ADV with relatively lower natural replication capacity.