Nucleoside analogue 2'-C-methylcytidine inhibits hepatitis E virus replication but antagonizes ribavirin.

Hepatitis E virus (HEV) infection has emerged as a global health issue, but no approved medication is available. The nucleoside analogue 2'-C-methylcytidine (2CMC), a viral polymerase inhibitor, has been shown to inhibit infection with a variety of viruses, including hepatitis C virus (HCV). Here, we report that 2CMC significantly inhibits the replication of HEV in a subgenomic replication model and in a system using a full-length infectious virus. Importantly, long-term treatment with 2CMC did not result in a loss of antiviral potency, indicating a high barrier to drug resistance development. However, the combination of 2CMC with ribavirin, an off-label treatment for HEV, exerts antagonistic effects. Our results indicate that 2CMC serves as a potential antiviral drug against HEV infection.

Evaluation of the role of the antioxidant silymarin in modulating the in vivo genotoxicity of the antiviral drug ribavirin in mice.

Ribavirin (1-ß-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a widely used broad-spectrum antiviral drug. Recently, several reports revealed genotoxic effects of ribavirin in vivo and in vitro, which were correlated with the production of reactive oxygen species (ROS). This study aimed to evaluate the genotoxicity of ribavirin and to investigate the role of the natural antioxidant silymarin to modulate this genotoxicity. Male albino mice (age, 8-10 weeks) were injected intraperitoneally (i.p.) with ribavirin at three dose levels (20, 75 and 130 mg/kg bw) either in a single injection (acute treatment) or in multiple injections on five consecutive days (sub-acute treatment). Other comparable groups were treated with silymarin (70 mg/kg bw) 1h before the injection with ribavirin. Mice were sacrificed at different sampling times (24, 48 and 72 h) after the last ribavirin treatment. Micronucleus (MN) and single-strand conformation polymorphism (SSCP) assays were used to assess genotoxic and cytotoxic effects of ribavirin and to evaluate the protective effect of the pre-treatment with silymarin. Our results reveal genotoxic and cytotoxic effects of ribavirin in the MN assay. Pre-treatment with silymarin reduced the toxicity of ribavirin. In the SSCP assay, ribavirin treatment did not induce any mutations in the two selected sites in the D-loop of mitochondrial DNA (mtDNA).

Drug screening identified gemcitabine inhibiting hepatitis E virus by inducing interferon-like response via activation of STAT1 phosphorylation.

Exposure to hepatitis E virus (HEV) bears a high risk of developing chronic infection in immunocompromised patients, including organ transplant recipients and cancer patients. We aim to identify effective anti-HEV therapies through screening and repurposing safe-in-human broad-spectrum antiviral agents. In this study, a safe-in-human broad-spectrum antiviral drug library comprising of 94 agents was used. Upon screening, we identified gemcitabine, a widely used anti-cancer drug, as a potent inhibitor of HEV replication. The antiviral effect was confirmed in a range of cell culture models with genotype 1 and 3 HEV strains. As a cytidine analog, exogenous supplementation of pyrimidine nucleosides effectively reversed the antiviral activity of gemcitabine, but the level of pyrimidine nucleosides per se does not affect HEV replication. Surprisingly, similar to interferon-alpha (IFNα) treatment, gemcitabine activates STAT1 phosphorylation. This subsequently triggers activation of interferon-sensitive response element (ISRE) and transcription of interferon-stimulated genes (ISGs). Cytidine or uridine effectively inhibits gemcitabine-induced activation of ISRE and ISGs. As expected, JAK inhibitor 1 blocked IFNα, but not gemcitabine-induced STAT1 phosphorylation, ISRE/ISG activation, and anti-HEV activity. These effects of gemcitabine were completely lost in STAT1 knockout cells. In summary, gemcitabine potently inhibits HEV replication by triggering interferon-like response through STAT1 phosphorylation but independent of Janus kinases. This represents a non-canonical antiviral mechanism, which utilizes the innate defense machinery that is distinct from the classical interferon response. These results support repurposing gemcitabine for treating hepatitis E, especially for HEV-infected cancer patients, leading to dual anti-cancer and antiviral effects.

Reversal of the antiviral activity of ribavirin against Sindbis virus in Ae. albopictus mosquito cells.

Earlier work in our laboratory has shown that the replication of Sindbis virus in Aedes albopictus mosquito cells is inhibited by ribavirin (Rbv) and mycophenolic acid (MPA) (Sarver and Stollar (1978) Virology 91, 267-282; Malinoski and Stollar (1980) Virology 102, 473-476). We report here that the antiviral effect of Rbv and MPA can be reversed by depriving infected cells of methionine or isoleucine, or by treating them with fluorodeoxyuridine (FUdR) or cycloleucine. We suggest that, as was the case when the antiviral activity of Rbv was reversed by actinomycin D (Malinoski and Stollar (1981a) Virology 110, 281-291), these effects may be mediated by changes in the GTP pools of treated cells.

Synergistic action of tiazofurin and retinoic acid on differentiation and colony formation of HL-60 leukemia cells.

Tiazofurin and retinoic acid synergistically induced differentiation and inhibited colony formation in HL-60 human promyelocytic leukemia cells in cell culture. The synergism was the result of different mechanisms of action, since the effect of tiazofurin, unlike that of retinoic acid, was prevented by addition of guanosine. Since it has been shown that tiazofurin down-regulated the expression of c-Ki-ras oncogene, and retinoic acid that of the myc oncogene, the joint impact of these drugs is of clinical interest particularly in end-stage leukemia where the therapeutic usefulness of tiazofurin has recently been demonstrated.

Apoptosis induced by inhibitors of nucleotide synthesis in deoxyadenosine-resistant leukemia L1210 cells that lack p53 expression.

An L1210 cell line (Y8) selected for resistance to deoxyadenosine contains ribonucleotide reductase that is not subject to inhibition by dATP. In addition, the Y8 cells have other phenotypic expressions that include increased sensitivity to apoptosis induced by various agents such as radiation, doxorubicin, anisomycin and roscovitine. The Y8 cells were found to be more sensitive to apoptosis induced by methotrexate (MTX), tiazofurin (TZ), deoxyguanosine (dGuo) and N-(phosphonoacetyl)-L-aspartate (PALA). Deoxyguanosine, at concentrations that did not cause apoptosis in the Y8 cells, prevented the apoptotic response of the Y8 cells to MTX and TZ. Deoxycytidine had no effect. Since caspase-3 activation is involved in apoptotic pathways, the effects of the caspase-3 inhibitor, Ac-DEVD-CHO, were studied on the dGuo-, MTX- or TZ-induced apoptosis in the Y8 cells. Ac-DEVD-CHO caused a marked decrease in the fraction of cells in the early phase of apoptosis. However, there was a corresponding increase in the fraction of cells in the late apoptotic/necrotic stages of cell death. This is in marked contrast to the dGuo-induced decrease in apoptosis seen in the MTX- and TZ-treated Y8 cells in which there were no increases in the late apoptotic/necrotic fraction of cells. These data show that alterations of nucleotide pools in the Y8 cells cause marked increases in the apoptotic response which may indicate that the Y8 cells are much more susceptible to the effects of misincorporation of nucleotides into DNA than are the parental WT L1210 cells.

Bile acids promote the expression of hepatitis C virus in replicon-harboring cells.

Hepatitis C virus (HCV) is a cause of chronic liver disease, with more than 170 million persistently infected individuals worldwide. Although the combination therapy of alpha interferon (IFN-alpha) and ribavirin is effective for chronic HCV infection, around half of all patients infected with HCV genotype 1 fail to show sustained virologic responses and remain chronically infected. Previously, we demonstrated that bile acids were essential for growth of porcine enteric calicivirus in cell culture in association with down-regulation of IFN responses. Because hepatocytes are exposed to high concentrations of bile acids in the liver, we hypothesized that bile acids have similar effects on HCV replication. We incubated HCV replicon-harboring cells (genotype 1b, Con1) in the presence of various bile acids and monitored the expression of HCV RNA and protein (NS5B). The addition of an individual bile acid (deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, or glycochenodeoxycholic acid) in the medium increased the levels of HCV RNA and proteins up to fivefold at 48 h of incubation. An antagonist of bile acid receptor farnesoid X receptor (FXR), Z-guggulsterone, reduced the bile acid-mediated increase of HCV RNA. When IFN (alpha or gamma) and each bile acid were incubated together, we observed that bile acid significantly reduced the anti-HCV effect of IFN. These results indicated that bile acids are factors in the failure of IFN treatment for certain patients infected with HCV genotype 1. Our finding may also contribute to the establishment of better regimens for treatment of chronic HCV infections by including agents altering the bile acid-mediated FXR pathway.

Mechanism and specificity of action of ribavirin.

Ribavirin at a concentration of 30 mug/ml added immediately after infection completely inhibited influenza A/Port Chalmers/1/73 (H(3)N(2)) virus hemagglutinin production in infected MDCK cells. Under these conditions, host cell protein synthesis was inhibited by only 10 to 20%. Polyacrylamide gel electrophoresis of [(35)S]methionine-labeled material from virus-infected cultures confirmed that ribavirin inhibited viral but not host cell protein synthesis. In parallel experiments, actinomycin D also preferentially inhibited viral protein synthesis. The possibility that ribavirin inhibited viral protein synthesis as a result of general inhibition of ribonucleic acid synthesis was therefore examined. In uninfected cells, ribavirin at 30 mug/ml inhibited the incorporation of [(14)C]inosine or [(3)H]uridine into ribonucleic acid but stimulated the incorporation of [(3)H]guanosine. The effects noted are consistent with an inhibition of the host cell enzyme inosine 5'-monophosphate dehydrogenase. This suggestion is supported by the finding that addition of guanosine, but not inosine, to the culture medium substantially reversed the antiviral effect of ribavirin. There was no separation between the concentration of ribavirin causing inhibition of influenza A viral protein synthesis or inhibition of MDCK cell ribonucleic acid synthesis, suggesting that ribavirin is not specifically antiviral in this system but inhibits viral protein synthesis as a result of the general inhibition of ribonucleic acid synthesis.

Selective inhibition of functional lymphocyte subpopulations by ribavirin.

The present studies were designed to examine the effects of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), a broad-spectrum antiviral agent, on the generation of murine antibody responses in vitro. Whereas primary and secondary sheep erythrocyte-specific, plaque-forming cell responses by normal murine spleen cells were enhanced by low concentrations of ribavirin (1 microgram per culture), they were strongly inhibited by higher concentrations of ribavirin (5 to 10 micrograms per culture). Both phenomena occurred with the greatest magnitude when spleen cells were exposed to ribavirin 48 to 72 h after culture initiation. Enhancement appeared to result from selective interference with suppressor T cells, since ribavirin failed to augment lipopolysaccharide-specific plaque-forming cell responses in T cell-depleted spleen cell cultures but inhibited concanavalin A-induced lymphocyte proliferation and suppressor T cell generation in cultures of normal spleen cells. The immunosuppressive properties of ribavirin were mediated by a direct antiproliferative effect and, at higher concentrations, a cytotoxic effect for B lymphocytes, since the drug inhibited plaque-forming cell responses in T cell-depleted spleen cell cultures, suppressed lipopolysaccharide-induced lymphocyte proliferation and reduced viable spleen cell recoveries.

Ribavirin-induced behavioral, body temperature and electrocortical spectra effects in the rat.

Behavioral, electrocortical spectra and body temperature effects, following a single intracerebroventricular (i.c.v.) or intraperitoneal (i.p.) injection of ribavirin, were studied in male adult rats. Ribavirin, when administered i.p., induced squatting posture, sedation, electrocortical synchronization, piloerection and a slight decrease of tactile and/or auditory stimuli/responses lasting from 45 min to 16 hr, as well as a longer lasting hypothermic effect. Following i.c.v. administration, ribavirin (50, 100 and 200 micrograms) elicited a soporific effect and produced changes in the electrocortical spectra. Pretreatment, 15 min before, with either prazosin (10 mg/kg, i.p.), an alpha 1-adrenoceptor blocker, or piperoxan (20 mg/kg, i.p.) and yohimbine (1 mg/kg, i.p.), two alpha 2-adrenoceptor blockers, or naloxone (1 mg/kg, i.p.), an opioid antagonist, did not prevent the hypothermic and behavioral effects induced by i.p. or i.c.v. administration of ribavirin. The present findings exclude an involvement of alpha-adrenergic and opioid neurotransmission in mediating the hypothermic, electrocortical and behavioral effects induced by ribavirin.