Abstract
BackgroundWhile ribavirin mono-therapy regimens have minimal effect on patients with chronic hepatitis C virus (HCV) infections, they can be efficacious when combined with interferon. Clinical studies show that interferon-free combination therapies containing ribavirin are also efficacious, suggesting that an interferon-free therapy could be adopted in the near future. However, generation of drug resistant mutants and cross resistance to other drugs could impair the efficacy of the treatment. Therefore, understanding the mechanism of HCV resistance to ribavirin and cross resistance to other antiviral drugs could be of major importance.MethodsWe tested the ability of a J6/JFH1 derived HCV ribavirin resistant mutant to grow in tissue cultured Huh7D cells in the presence of the mutagen 5-Fluorouracil and the nucleoside analog 2′-C-Methylcytidine. Virus replication was assessed by detecting HCV antigens by immunofluorescence and by titrating virus present in the supernatants. Recovered viruses were amplified by RT-PCR and sequenced.ResultsThe sensitivity of HCV-RR relative to parental J6/JFH1 to the tested drugs varied. HCV-RR was more resistant than J6/JFH1 to 5-Fluorouracil but was not more resistant than J6/JFH1 to 2′-C-Methylcytidine. Growth of HCV-RR in 5-Fluorouracil allowed the selection of an HCV-RR derived mutant resistant to 5-Fluorouracil (HCV-5FU). HCV-5FU grows to moderate levels in the presence of high concentrations of 5-Fluorouracil and to parental levels in the absence of the drug. Sequence of its genome shows that HCV-5FU accumulated multiple synonymous and non-synonymous mutations.ConclusionsThese results indicate that determinants of resistance to ribavirin could also confer resistance to other anti-HCV drugs, shedding light toward understanding the mechanism of action of ribavirin and highlighting the importance of combination drug selection for HCV treatment. The results also show that it is possible to select a 5-Fluorouracil HCV resistant mutant that replicates to levels similar to parental virus when grown in the absence of 5-Fluorouracil.
Highlights
Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus member of the genus Hepacivirus of the Flaviviridae family
Several mechanisms of action of ribavirin against hepatitis C virus (HCV) were proposed including a direct effect against the HCV RNA dependent RNA polymerase (NS5b); induction of misincorporation of nucleotides leading to lethal mutagenesis; depletion of intracellular guanosine triphosphate pools; alteration in the cytokine balance from a Th2 profile to a Th1 profile; and up-regulation of genes involved in interferon signaling [9,10]
In this study we show that an HCV mutant resistant to ribavirin is more resistant than parental J6/JFH1 to 5-Fluorouracil, but is not more resistant than parental J6/JFH1 to 29-C-Methylcytidine
Summary
Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus member of the genus Hepacivirus of the Flaviviridae family. The standard therapy for the treatment of chronically HCV infected patients consists of a combination of pegylated interferon alpha and ribavirin [4]. Given the side effects associated with injections of interferon, an interferon-free regimen for the treatment of HCV infections is highly desirable. Recent studies have shown that ribavirin in combination with other antiviral drugs, without interferon, can be efficacious [7,8], suggesting that an interferon-free therapy containing ribavirin could be adopted in the near future. While ribavirin mono-therapy regimens have minimal effect on patients with chronic hepatitis C virus (HCV) infections, they can be efficacious when combined with interferon. Clinical studies show that interferon-free combination therapies containing ribavirin are efficacious, suggesting that an interferon-free therapy could be adopted in the near future. Understanding the mechanism of HCV resistance to ribavirin and cross resistance to other antiviral drugs could be of major importance
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