Characterization of the Antiviral Effect of 2 9 ,3 9 -Dideoxy-2 9 , 3 9 -Didehydro- b - L -5-Fluorocytidine in the Duck Hepatitis B Virus Infection Model

A novel L -nucleoside analog of deoxycytidine, 2 * ,3 * -dideoxy-2 * ,3 * -didehydro- b - L -5-ﬂuorocytidine ( b - L -Fd4C), was recently shown to strongly inhibit hepatitis B virus (HBV) replication in the 2.2. 15 cell line. Therefore, its antiviral activity was evaluated in the duck HBV (DHBV) infection model. Using a cell-free system for the expression of the DHBV polymerase, b - L -Fd4C-TP exhibited a concentration-dependent inhibition of dCTP incorporation into viral minus-strand DNA with a 50% inhibitory concentration of 0.2 m M which was lower than that of other tested deoxycytidine analogs, i.e., lamivudine-TP, ddC-TP, and b - L -FddC-TP. Further analysis showed that b - L -Fd4C-TP is likely to be a competitive inhibitor of dCTP incorporation and to cause premature DNA chain termination. In primary duck hepatocyte cultures infected in vitro, b - L -Fd4C administration exhibited a long-lasting inhibitory effect on viral DNA synthesis but could not clear viral covalently closed circular DNA (CCC DNA). Results of short-term antiviral treatment in experimentally infected ducklings showed that b - L -Fd4C exhibited the most potent antiviral effect, followed by b - L -FddC, lamivudine, and ddC. Longer administration of b - L -Fd4C induced a sustained suppression of viremia (>95% of controls) and of viral DNA synthesis within the liver. However, the persistence of trace amounts of viral CCC DNA detected only by PCR was associated with a recurrence of viral replication after drug withdrawal. In parallel, b - L -Fd4C treatment suppressed viral antigen expression within the liver and decreased intrahepatic inﬂammation and was not associated with any sign of toxicity. Our data, therefore, demonstrate that in the duck model of HBV infection, b - L -Fd4C is a potent inhibitor of DHBV reverse transcriptase activity in vitro and suppresses viral replication in the liver in vivo. An in vitro assay for the expression of enzymatically active DHBV reverse transcriptase and the study of the inhibitory effect of nucleoside analog triphosphates. The enzymatically active DHBV polymerase polypeptide was synthesized from the plasmid pHP, which contains the viral polymerase gene under the control of the SP6 promoter, and the RNA template of reverse transcription, as previously described (39, 43). The DHBV polymerase gene was transcribed and translated in a coupled transcription-translation rabbit reticulocyte lysate system (TNT SP6 Coupled Reticulocyte Lysate System; Promega, Charbonnie`res, France), according to the manufacturer’s instructions. The reverse transcription assay was performed as previously described (42). After translation, the viral polymerase was incubated for 30 min at 30°C in a mixture containing 50 mM Tris-HCl (pH 7.5), 15 mM NaCl, 10 mM MgCl 2 , dATP, dGTP, dTTP (100 m M each), and 0.165 m M [ a - 32 P]dCTP (3,000 Ci/mmol). The inhibition of [ a - 32 P]d- CMP incorporation in DHBV minus-strand DNA was performed with the addition of various polymerase inhibitors ( b - L -Fd4C-TP, 3TC-TP, b - L -FddC-TP, and ddC-TP) at the concentrations indicated below. Radiolabelled viral DNA covalently attached to polymerase was subjected to electrophoresis through 0.1% sodium dodecyl sulfate (SDS)–10% polyacrylamide gels as already published (39, 43), and the dried gels were exposed to X-ray ﬁlm. A quantitative dot assay was performed after spotting 2 m l of the radiolabelled reverse Immunostaining of liver sections for DHBV pre-S antigen. Five-micrometer deparafﬁnized liver tissue sections were incubated overnight with an anti-DHBV pre-S murine monoclonal antibody (MAb), MAb 900, previously characterized by Chassot et al. (3). This step was followed by incubation with biotinylated goat anti-mouse immunoglobulin G (Dako, Trappes, France). The antigen-antibody complex was then revealed with streptavidin-horseradish peroxidase (Dako). The visualization was performed by using DAB chromogen substrate according to the manufacturer’s instructions (Dako) (35). All specimens were evaluated blind.