The in vitro antihepadnavirus activities of the purine nucleoside analogs ganciclovir {9-[2-hydroxy-1-(hydroxymethyl)ethoxymethyl]guanine} and penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine; BRL 39123] were compared in primary duck hepatocyte cultures congenitally infected with the duck hepatitis B virus (DHBV). Both compounds inhibited DHBV DNA replication to a comparable extent during continuous short-term treatment of the cultures. However penciclovir was more active both during longer-term continuous treatment (50% inhibitory concentrations: penciclovir, 0.7 ± 0.1 ,uM; ganciclovir, 4.0 ± 0.2 ,uM) and in washout experiments (50% inhibitory concentrations: penciclovir, 3.0 ± 0.4 ,uM; ganciclovir, 46 ± 1.5 ,uM) designed to compare the persistence of inhibitory activity after removal of the extracellular compound. The effects on viral protein synthesis were similar to the effects on viral DNA replication. These data suggest that penciclovir or its oral form, famciclovir, may have clinical utility in the treatment of chronic hepatitis B virus infection.Attempts to develop therapy against hepatitis B virus (HBV) have been hampered by its extremely narrow host range (only humans and chimpanzees are susceptible to HBV infection) and by the inability of cell lines to support complete autonomous HBV replication (22). These problems have been overcome to some extent by the recent development of assay systems for identifying potential antihepadnavirus agents. Animal models of HBV infection, particularly those of the woodchuck and duck, have been used for in vivo testing and evaluation, while continuous HBV-transfected cell lines and primary hepatocytes have been used for in vitro screening. The utilities and validities of these systems seem to have been vindicated by findings that agents previously found to be active in clinical studies (2,19,20) are generally active in the test systems (8,21,23,33,34) MATERLILS AND METHODSAnimals. One-day-old Pekin-Aylesbury cross ducks congenitally infected with an Australian strain of DHBV were obtained from a commercial supplier (33). Viremia was monitored by dot blot hybridization of serum, and 7-to 14-day-old ducklings with stable viral titers of 5 x 108 to 10 x 108 viral genome equivalents per ml were selected for hepatocyte isolation.Cell culture. Primary cultures of duckling hepatocytes were prepared as described previously (5, 30), except that feeder cell layers (a potential source of competing nucleosides) were not used. Six-well plastic culture plates (Greiner, Frickenhausen, Germany) were seeded with 2 x 106 to 2.5 x 106 hepatocytes per well, and cells were allowed to attach overnight before the first medium change (on day 1 postplating) and were maintained with medium changes every second day. Antiviral drug treatment. For each experiment, triplicate sets of PDH monolayers were exposed to drug concentrations in the range 0 to 500 ,uM beginning on day 1 postplating. One set was harvested after 5 days of drug treatment. Treatment of the second set was stopped aft...