Treatment of chronic HBV-infection is limited by selection of resistance. The rtM204I/V mutations in the YMDD motif of HBV reverse transcriptase are well documented resistance determinants against lamivudine and entecavir, but not against adefovir or tenofovir. Limited systematic phenotypic data are available for the latter two drugs. Methods: rtM204 mutations (rtM204A/I/K/L/Q/S/T/V) were systematically introduced into replication-competent 1.1-fold HBV-overlength constructs under control of a

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... MV promoter. Viral replication fitness was determined by selective qPCR after normalized transient transfection. In vitro drug susceptibilities were evaluated by determining IC50 values of lamividune, entecavir, adefovir, and tenofovir using standardized high-throughput phenotypic assays. Infectivity was analyzed by infection of HepaRG cells. Results: In vitro phenotyping showed that rtM204K conferred high-level resistance to adefovir and tenofovir but simultaneously impaired replication capacity. Its fitness could not be restored by rtL180M or rtL80I as described for rtM204I/V. rtM204L and rtM204Q conferred low-level reduced susceptibility to adefovir/tenofovir without loss of replication capacity. rtM204A/I/S/T reduced susceptibility to either drug substantially. Interestingly, the single mutation rtM204V showed significantly reduced susceptibility to both drugs but lost resistance in combination with the compensatory mutation rtL180M. By affecting the overlapping S-gene, rtM204 mutants except rtM204L showed reduced or diminished infectivity in HepaRG cells. Conclusions: We have established a time-and cost-effective phenotypic assay and identified novel rtM204 mutations conferring cross resistance to adefovir and tenofovir in vitro. Despite of their low frequency in the viral population, their clinical significance should not be underestimated due to the potential selection of compensatory mutations, which may restore viral fitness.