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Multi-drug resistant hepatitis B virus (HBV) has been reported in hepatitis B patients who received sequential antiviral therapy.In vitrostudies showed that HBV constructs with mutations resistant to lamivudine and adefovir have marked reduction in sensitivity to combination of lamivudine and adefovir, whereas constructs with mutations resistant to either drug remain sensitive to the other drug. We conducted this study to determine whether mutations conferring resistance to multiple antiviral agents co-locate on the same HBV genomein vivoand to describe the evolution of these mutations. Sera from six patients who had been found to have multi-drug resistant HBV mutations to lamivudine + adefovir, lamivudine + hepatitis B immunoglobulin (HBIG), or lamivudine + entecavir on direct sequencing were cloned after nested polymerase chain reaction (PCR). Analysis of 215 clones from 11 samples with multi-drug resistant mutations on direct sequencing showed that 183 (85%) clones had mutations to both therapies on the same genome; 31 clones had lamivudine-resistant mutants only. Clonal analysis of serial samples from three patients showed progressive evolution from all clones with lamivudine-resistant HBV mutations only to mixtures of clones that have multi-drug resistant mutations and clones that have lamivudine-resistant HBV mutations only, and ultimately all clones having multi-drug resistant HBV mutations.In conclusion,mutations conferring resistance to multiple antiviral agents co-locate on the same viral genome, suggesting that combination therapy directed against mutants resistant to each treatment may not be adequate in suppressing multi-drug resistant HBV.De novocombination therapy may prevent the emergence of multi-drug resistant mutants.