Antiviral activity of beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine in woodchucks chronically infected with woodchuck hepatitis virus.

The L-nucleoside analog beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine (beta-L-Fd4C) was first shown to exhibit potent activity against hepatitis B virus (HBV) in tissue culture and then to significantly inhibit viral spread during acute infection in the duck HBV model (F. Le Guerhier et al., Antimicrob. Agents Chemother. 44:111-122, 2000). We have therefore examined its antiviral activity in a mammalian model of chronic HBV infection, the woodchuck chronically infected with woodchuck hepatitis virus (WHV). Side-by-side comparison of beta-L-Fd4C and lamivudine administered intraperitoneally during short-term and long-term protocols demonstrated a more profound inhibition of viremia in beta-L-Fd4C-treated groups. Moreover, beta-L-Fd4C induced a marked inhibition of intrahepatic viral DNA synthesis compared with that induced by lamivudine. Nevertheless, covalently closed circular (CCC) DNA persistence explained the lack of clearance of infected hepatocytes expressing viral antigens and the relapse of WHV replication after drug withdrawal. Liver histology showed a decrease in the inflammatory activity of chronic hepatitis in woodchucks receiving beta-L-Fd4C. An electron microscopy study showed the absence of ultrastructural changes of hepatic mitochondria, biliary canaliculi, and bile ducts. However, a loss of weight was observed in all animals, whatever the treatment, as was a transient skin pigmentation in all woodchucks during beta-L-Fd4C treatment. There was no evidence that lamivudine or beta-L-Fd4C could prevent the development of hepatocellular carcinoma with the protocols used. These results indicate that beta-L-Fd4C exhibits a more potent antiviral effect than lamivudine in the WHV model but was not able to eradicate CCC DNA and infected cells from the liver at the dosage and with the protocol used.

Characterization of the antiviral effect of 2',3'-dideoxy-2', 3'-didehydro-beta-L-5-fluorocytidine in the duck hepatitis B virus infection model.

A novel L-nucleoside analog of deoxycytidine, 2',3'-dideoxy-2', 3'-didehydro-beta-L-5-fluorocytidine (beta-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, beta-L-Fd4C-TP exhibited a concentration-dependent inhibition of dCTP incorporation into viral minus-strand DNA with a 50% inhibitory concentration of 0.2 microM which was lower than that of other tested deoxycytidine analogs, i.e. , lamivudine-TP, ddC-TP, and beta-L-FddC-TP. Further analysis showed that beta-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, beta-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 beta-L-Fd4C exhibited the most potent antiviral effect, followed by beta-L-FddC, lamivudine, and ddC. Longer administration of beta-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, beta-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, beta-L-Fd4C is a potent inhibitor of DHBV reverse transcriptase activity in vitro and suppresses viral replication in the liver in vivo.

Cloning and analysis of a Trichinella britovi gene encoding a cytoplasmic heat shock protein of 72 kDa.

A gene encoding a protein of 646 amino acid residues with a molecular mass of 71.3 kDa showing homology to the cytoplasmic form of the 70 kDa heat shock protein was cloned and sequenced from the nematode parasite Trichinella britovi (Tb). The gene was expressed in vitro as a protein of 71 kDa that was immunoprecipitated by a Trichinella-infected rabbit serum. Monospecific polyclonal antibodies raised against the recombinant Tb Hsp70 expressed in Escherichia coli, recognized a protein of 70 kDa by Western blot analysis of Tb soluble antigen (muscular stage). Tb Hsp70 was located in the nuclei of the muscle larvae as determined by the indirect immunofluorescent pattern on cross-sections of the worm. The expression of this protein was not detected in adult worm nuclei suggesting a differential expression of Hsp70 between the 2 stages of Trichinella.