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The aim of this study was to examine the effects of ganciclovir and nalidixic acid either alone or in combination on duck hepatitis B virus DNA replication in vivo with particular reference to production of viral supercoiled DNA and RNA. The most effective antiviral response was observed in the livers of ducks treated by the combination therapy for 28 days, which resulted in a substantial decrease in the amounts of viral supercoiled DNA, relaxed circular and single-stranded DNA, and also viral RNA. This combination treatment was not hepatotoxic over the study period.