Inhibitory effect of 2'-fluoro-5-methyl-beta-L-arabinofuranosyl-uracil on duck hepatitis B virus replication

Abstract

The antiviral activity of 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU), a novel L-nucleoside analog of thymidine known to be an inhibitor of hepatitis B virus (HBV) replication in hepatoma cells (2.2.1.5 cell line), was evaluated in the duck HBV (DHBV) model. Short-term oral administration (5 days) of L-FMAU (40 mg/kg of body weight/day) to experimentally infected ducklings induced a significant decrease in the level of viremia. This antiviral effect was sustained in animals when therapy was prolonged for 8 days. The histological study showed no evidence of liver toxicity in the L-FMAU-treated group. By contrast, microvesicular steatosis was found in the livers of dideoxycytidine-treated animals. L-FMAU administration in primary duck hepatocyte cultures infected with DHBV induced a dose-dependent inhibition of both virion release in culture supernatants and intracellular viral DNA synthesis, without clearance of viral covalently closed circular DNA. By using a cell-free system for the expression of an enzymatically active DHBV reverse transcriptase, it was shown that L-FMAU triphosphate exhibits an inhibitory effect on the incorporation of dAMP in the viral DNA primer. Thus, our data demonstrate that L-FMAU inhibits DHBV replication in vitro and in vivo. Long-term administration of L-FMAU for the eradication of viral infection in animal models of HBV infection should be evaluated.

FIG. 1

Oral administration of l-FMAU decreases viral DNA synthesis in the livers of experimentally infected ducklings but does not clear viral CCC DNA. Protein-bound (A) and protein-free (B) viral DNAs were extracted from the livers of experimentally infected birds and were subjected to Southern blot analysis at the cessation of therapy. Liver samples from four control animals, four animals that received l-FMAU at 20 mg/kg twice a day, per os, for 4 days, and four animals that were treated with ddC at 50 mg/kg twice a day, per os, for 4 days were available for analysis. The positions of relaxed circular (RC), linear (L), CCC, and single-stranded (SS) DNAs are indicated, as are the therapeutic protocols.

FIG. 2

Oral administration of l-FMAU inhibits DHBV viremia in experimentally infected ducklings. Ducklings at 5 days of age were inoculated with a DHBV-positive serum specimen. l-FMAU was given orally at day 3 postinoculation by two different protocols. A group of four ducklings received l-FMAU at a dosage of 40 mg/kg once a day from day 3 to day 7 postinoculation (5 days of treatment). A second group of four ducklings was given l-FMAU at a dosage of 40 mg/kg once a day from day 3 postinoculation for 8 days. A third group of four ducklings served as controls. (A) Analysis of viremia levels. Viral DNA in serum was analyzed by a dot blot assay. The level of mean viral DNA in serum in each group of animals was plotted. (B) Analysis of lactic acid levels. The lactic acid levels in experimentally infected birds were determined before inoculation and at days 7, 10, 12, and 14 postinoculation. The mean serum lactic acid concentration in each group of animals was plotted.

FIG. 3

DHBV is not cleared from the livers of experimentally infected ducklings after l-FMAU withdrawal. (A) Analysis of intrahepatic viral DNA. A preparation of enriched protein-bound viral DNAs, extracted from the livers of experimentally infected birds, was subjected to Southern blot analysis 16 days after the cessation of therapy. Liver samples from three control animals, four animals that received l-FMAU for 8 days, and four animals that were treated for 5 days were available for analysis. The positions of relaxed circular (RC), linear (L), CCC, and single-stranded (SS) DNAs are indicated, as are the therapeutic protocols (controls, oral administration of l-FMAU for 5 or 8 days). (B) Analysis of intrahepatic viral proteins. Viral core proteins were analyzed by Western blotting of the same liver samples, as described in Material and Methods. MW, molecular mass.

FIG. 4

Analysis of liver histology revealing microvesicular steatosis in ddC-treated animals and no histological sign of toxicity during short-term administration of l-FMAU. (A) Liver histology for one control animal. Signs of viral hepatitis are observed: ballooning hepatocytes, portal tract infiltration, and lobular inflammation (obj., 25). (B) Liver histology for one l-FMAU (40 mg/kg every day)-treated animal. A pattern similar to that in the control animals is observed. (C) Liver histology for one ddC (50 mg/kg twice a day)-treated duckling. Typical signs of microvesicular steatosis (accumulation of lipid droplets in the cytoplasms of hepatocytes) and acidophilic necrosis of hepatocytes are shown. (D) Liver histology for the same ddC (50 mg/kg twice a day)-treated duckling but at a higher magnification (obj., 40), which shows intracytoplasmic lipid droplets.

FIG. 5

l-FMAU inhibits DHBV DNA synthesis in DHBV DNA-infected primary duck hepatocytes. l-FMAU or β-l-F-ddC was added at the indicated concentrations 3 days after plating for 6 days. Cells were harvested at the end of therapy (day 10 [D10]) and 3 days after drug release (day 13 [D13]). The viral DNA released in the supernatant of primary hepatocyte culture was analyzed by a dot blot assay (A). Intracellular protein-bound (B) and protein-free (C) viral DNAs were extracted and subjected to Southern blot analysis. The positions of relaxed circular (RC), linear (L), CCC, and single-stranded (SS) DNAs are indicated. The results of a typical experiment are shown.

FIG. 6

Inhibitory effect of l-FMAU-TP on the synthesis of the viral DNA primer for DHBV reverse transcription. The sequence of the short DNA primer covalently linked to the DHBV polymerase is 5′-GTAA-3′. (A) The DHBV polymerase was incubated with different concentrations of thymidine-TP analogs (TTP or ddTTP or l-FMAU-TP) together with dGTP and [α-³²P]dATP (3,000 Ci/mmol; final concentration, 0.3 μM) for 30 min. The incorporation of [α-³²P]dAMP was also analyzed in control reactions without TTP, without dGTP and TTP, or without DHBV polymerase. Reactions were analyzed with a 0.1% SDS–10% polyacrylamide gel and autoradiographed. The figure shows the results obtained with the TTP analog at a concentration of 50 μM. (B) Kinetics of [α-³²P]dAMP incorporation into primer DNA by the DHBV polymerase. The DHBV polymerase was incubated with thymidine-TP analogs at a concentration of 50 μM (⧫, TTP; ▪, ddTTP; ▴, l-FMAU-TP) together with dGTP (100 μM) and [α-³²P]dATP (3,000 Ci/mmol; final concentration, 0.3 μM). The incorporation of [α-³²P]dAMP in the viral primer was measured at different time points, as indicated. The activity at 30 min in the presence of TTP was arbitrarily defined as 100%.