Ribavirin: pharmacology, multiple modes of action and possible future perspectives

Abstract

Ribavirin is a unique guanosine analog with broad-spectrum activity against many RNA and DNA viruses. In addition to its mutational properties, ribavirin exerts extensive perturbation of cellular and viral gene expression. Furthermore, recent advances indicate that the impact of ribavirin on divergent cellular and viral pathways may be concentration dependent. This review aims at providing an overview of the pharmacology and multiple modes of action of ribavirin as well as pointing to possible novel future uses.

Ribavirin originally was synthesized as a guanosine analog (1) and was noted to exert unique broad-spectrum in vitro activity against both RNA and DNA viruses (2). However, unlike many other antiviral nucleoside/nucleotide analogs, incorporation of ribavirin triphosphate does not appear to lead to chain termination (3). Until recently, ribavirin was, together with IFN-α, a major therapeutic agent in HCV treatment (4-6), but ribavirin has also been indicated for respiratory syncytial virus infections (7), viral hemorrhagic fevers such as Lassa and Crimean-Congo (8,9), chronic hepatitis E virus infections (10), and many other severe, life-threatening viral infections (11). Additionally, ribavirin has been tested against a wide range of infectious agents, including poliovirus (12) and influenza virus (11,13). Despite ribavirin monotherapy only having modest effect on HCV RNA concentrations (14,15), the addition of ribavirin to IFN-α-based therapy markedly improved the likelihood of achieving a cure (4,5), also referred to as a sustained virologic response. Presently, the use of ribavirin in HCV therapy has diminished, but it continues to be recommended concomitantly with selected interferon-free, direct-acting antiviral treatments (16), especially for HCV genotype 3 infection. For example, among decompensated cirrhotic HCV patients enrolled in the ASTRAL-4 trial treated with sofosbuvir and velpatasvir, the highest sustained virologic response rate was observed among those also receiving ribavirin (17). Thus, ribavirin likely will remain a part of the HCV therapeutic arsenal, especially in the setting of decompensated cirrhosis (Child-Pugh B or C), as protease inhibitors are contraindicated in this setting (16,18).

Pharmacology

Pharmacokinetic studies in healthy volunteers who received 150mg of intravenous 13 C3 -ribavirin followed after 1 h by a 400mg oral dose showed that ribavirin had a mean bioavailability of 52%±22%, and a mean half-life of 37±14 h (19). Ribavirin has a large distribution volume, an elimination that is dependent on renal function, and thus requires more than 4 weeks to reach steady-state concentrations (20-22). Upon cellular entry, ribavirin is activated by intracellular phosphorylation into mono-, di- and triphosphates. In vitro studies have shown that polyphosphorylation irreversibly entraps the ribavirin in the erythrocytes, resulting in intracellular levels in excess of 550μM, as compared with plasma concentrations of 15μM (23).

At steady state, which often occurs after more than 4weeks of treatment in patients with normal renal function receiving weight-based ribavirin dosing for HCV infection, that is, 1g (<75kg bodyweight) or 1.2g daily (≥75kg bodyweight) divided into two oral doses, plasma concentrations of ribavirin of approximately 8-12μmol/l (2-3μg/ml) are achieved (15). However, it should be noted that although the optimal target trough concentration of ribavirin remains unclear, toxicity increases dramatically at plasma concentrations exceeding 15μmol/l (24,25). Despite this, considerably higher ribavirin dosing is recommended for many life-threatening viral infections, for example, for the treatment of Lassa hemorrhagic fever (26).

Shortening time to steady state

In a pilot study (RibaC), 2 weeks of ribavirin double dosing (i.e.,26mg/kg/day orally instead of standard 13mg/kg/day for HCV genotype 1 infection), resulted in high ribavirin plasma concentrations, but regrettably also in greater hemoglobin decline (15). The results of this study suggest that 1 week of double dosing (followed by standard dosing) may suffice to achieve steady state as well as decrease the risk of anemia.

Ribavirin in renal impairment

Ribavirin is mainly eliminated by the kidneys, and the pharmacokinetics of ribavirin are substantially altered in patients with renal impairment (27). Despite this, many patients with renal failure and/or ongoing hemodialysis have been successfully treated with ribavirin (21). A prerequisite, however, for the use of ribavirin in this setting is the initiation of therapy with a reduced dose, dependent upon the degree of kidney impairment (24,28), in addition to continuous monitoring of plasma ribavirin concentrations and hemoglobin levels.

Adverse effects

The major side effect of ribavirin is hemolytic anemia, with a mean decrease in hemoglobin of approximately 20g/l during treatment (24). This hemolysis is hypothesized to be secondary to oxidative stress induced by depletion of adenosine triphosphate (ATP) in erythrocytes (29,30). Because of the risks associated with anemia in patients with a history of or ongoing heart disease, ribavirin is contraindicated in these patients (24). Aside from anemia, ribavirin is also associated with increased risk of itching, rash, cough as well as neuropsychiatric side effects, for example, insomnia (28).

Teratogenicity

In light of the potential teratogenicity demonstrated in all animal models studied, ribavirin is contraindicated during pregnancy and breastfeeding. Avoidance of pregnancy for 4 months after ribavirin exposure for treated women and for 7 months for female partners of treated men is therefore recommended, although preliminary analysis of the‘Ribavirin Pregnancy Registry’ established in 2003 has not been able to demonstrate any clear evidence for human teratogenicity for ribavirin (31).

Impact of inosine triphosphate pyrophosphatase on ribavirin

Naturally occurring variants of the ITPase gene (ITPA) associated with reduced enzymatic activity, found in approximately a third of humans, have been demonstrated to protect against ribavirin-induced hemolytic anemia during ribavirin therapy in combination with peg-IFN-α (32,33) as well as with direct-acting antiviral therapy (34) for chronic HCV infection. Additionally, reduced ITPase activity has also been associated with improved efficacy of interferon-α and ribavirin therapy for HCV genotype 2 or 3 infection, mediated by a reduced relapse risk, in spite of lower ribavirin plasma concentrations (32). Recently ribavirin triphosphate (RTP) has been reported to be dephosphorylated intracellularly to ribavirin monophosphate (RMP) by ITPase (35). As ribavirin and RMP, but not RTP, can be transported across the plasma membrane through specific transporter proteins (ENT1, ENT2, CNT2, MRP4 and MRP5) (23,36-39), it is not surprising that reduced ITPase activity is associated both with higher intracellular RTP levels (35) and lower plasma ribavirin concentrations (Figure 1) (32).

Figure 1. Modes of action of ribavirin, ribavirin monophosphate and ribavirin triphosphate on cellular functions (red) and viral functions (blue). At lower concentrations (10μM), RMP inhibits inosine monophosphate dehydrogenase causing GTP depletion with secondary effects on mRNA-capping, G-protein signaling, ribosomal functioning, immune modulation (e.g.,favoring Th1 over Th2 responses), etc. RTP may directly inhibit viral RdRp. At higher concentrations (≥100μM), RTP causes mutations in the viral genome. RTP can be dephosphorylated to RMP by ITPase. Ribavirin can enter erythrocytes, which may lead to hemolytic anemia. RMP: Ribavirin monophosphate; RTP: Ribavirin triphosphate.

Modes of action

The precise molecular mechanism underlying the action of ribavirin on HCV infection remains unclear, but several mechanisms of action have been proposed and their impact may be ribavirin concentration dependent:

Inhibition of inosine monophosphate dehydrogenaseand effects on intracellular nucleotide concentrations

One of the first suggested mechanisms of action of ribavirin was the potent competitive inhibition of inosine monophosphate dehydrogenase (IMPDH) by RMP leading to reduced guanosine monophosphate biosynthesis by the diminution of the conversion of inosine monophosphate to xanthosine monophosphate, with subsequent intracellular depletion of GTP (40). This inhibition of IMPDH has been observed to occur even at relatively low ribavirin concentrations (10μM ribavirin), and leads to marked changes in the balance of intracellular nucleotide concentrations (35). These alterations in the nucleotide pool in cells may secondarily have major impact on host cell and viral gene expression as well as on viral replication (41-45).

Inhibition of mRNA capping

Eukaryotic mRNAs and many viral mRNAs contain extensive modification on the 5′ end (known as the ‘five-prime cap’), often utilizing guanine which is methylated in the 7-position, as this is essential for the stability and efficient translation of mRNA. Thus, RNA capping has major secondary impact on the translation of both viral and host cell mRNAs. Interestingly, RTP reportedly acts as a competitive inhibitor for the capping of mRNAs subsequently leading to impaired translation (46) by forming a covalent RMP-capping enzyme intermediate in place of the normally observed guanosine monophosphate-enzyme intermediate (47). This observation, however, has been disputed (48).

Impact on host cell gene expression, inflammation and immunomodulation

Aside from being an essential purine needed for RNA synthesis during transcription, GTP also acts as a source of energy used in protein synthesis as well as gluconeogenesis (49). For example, during the elongation stage of translation, GTP is essential for the binding of a new amino-bound tRNA to the A-site of the ribosome as well as for the translocation of the ribosome toward the 3′ end of the mRNA (49). Additionally, GTP is necessary for signal transduction in particular with G-proteins, in second-messenger mechanisms (49).

Thus, in light of the above-mentioned importance of maintaining adequate intracellular levels of GTP, it is understandable that ribavirin impacts on host cell and viral gene expression both in vivo (42,43) and in vitro (44,45), as well as on HCV-specific immune responses by modulating the TH1/2 subset balance (50), and enhancing TH1 immune response and IL-12 levels (41). Additionally, clinical HCV studies demonstrate that ribavirin monotherapy downregulates the expression of interferon-stimulated genes in addition to reducing systemic concentrations of liver enzymes (14) and IP-10 (also known as CXCL10) (15) in spite of only modest impact on viral levels. Similarly, ribavirin administration in mice infected with influenza virus significantly attenuated respiratory immune responses, as well as secretory and total IgA mucosal responses (51).

Inhibition of viralRdRPs

RTP has been reported to competitively inhibit the influenza virus RNA polymerase, with respect to ATP and GTP, whereas RMP has no such observable effect (52). However, for HCV there is conflicting data regarding the impact of ribavirin on theRdRP with reports of both no directinhibition (53), as well as observations that ribavirin containing RNA templates can cause a significant blockage of RNA elongation (54).

Enhancement of viral mutagenesis

It has been suggested that ribavirin enhances viral mutagenesis leading to error catastrophe by incorrect substitution of RTP for GTP (12,54,55) into viral RNA as most viral RdRPs lack proofreading, although this mechanism has been disputed for some viruses (56). For example, for poliovirus, a 9.7-fold increase in mutagenesis following ribavirin treatment resulted in 99.3% loss in infectivity (12). Unlike GTP, RTP has ambiguous base-pairing capacity and can form two hydrogen bonds with uridine triphosphate (UTP) or cytidine triphosphate (CTP) with equal efficiency (57), leading to a subsequent increase in G-to-A and C-to-U single nucleotide variations throughout the entire HCV open reading frame (58). Recent in vivo studies indicate that similar ribavirin-induced mutagenesis occurs in hepatitis E virus (59) and the tamarin GB virus B (60). Interestingly, the effects of ribavirin on in vitro HCV mutagenesis are observed at relatively high concentrations of ribavirin (≥100μM) (35).

Conclusion

Ribavirin is a unique guanosine analog with effect against a broad range of viruses, and there is now increasing support for the notion of multiple mechanisms of action for its antiviral effect (61). These differing modes, however, may vary across the ribavirin concentrations achieved as well as the virus studied. In the future ribavirin may have novel indications, especially when targeting both viral and host cell factors is desired. In light of the relatively long half-life of ribavirin, resulting in a considerable time to steady state when administered orally, this may be most appropriate in the setting of chronic rather than acute viral infections.

Future perspective

As ribavirin is unique in its broad antiviral effect, it likely will remain a potential drug for emerging viral diseases. It is also very possible that novel indications will materialize in the coming 5-10 years, especially for viral infections where targeting both viral and host responses is appropriate. In light of the extended time until achievement of steady state ribavirin concentrations when administered orally, this may be most appropriate in the setting of chronic viral infections where other therapeutic options are limited. Moreover, ribavirin is increasingly being studied as a therapeutic option in cancer. Currently, there are ongoing studies registered in ClinicalTrials.gov evaluating ribavirin in conjunction with follicular and mantle cell lymphoma, tonsil and/or tongue squamous cell carcinoma, and human papillomavirus-related malignancies.

Executive summary

Ribavirin is a unique guanosine analog with broad-spectrum activity against many viruses including:

• Hepatitis C virus.
• Hepatitis E virus.
• Respiratory syncytial virus.
• Lassa virus.
• Crimean-Congo hemorrhagic fever virus.
• Poliovirus.
• Influenza virus.

Pharmacology

Ribavirin:

• Has a long half-life resulting in more than 4 weeks to reach steady state ribavirin concentrations when administered orally in healthy individuals. Double dosing for 1 initial week may result in faster achievement of steady state.
• Has a large distribution volume.
• Has an elimination that is dependent on renal function requiring dose reduction in patients with renal impairment.
• Has adverse effects including hemolytic anemia, itching, rash, cough and insomnia.
• Is teratogenic.
• Is contraindicated in heart disease, pregnancy and breastfeeding.
• Ribavirin triphosphate is dephosphorylated to ribavirin monophosphate by inosine triphosphate pyrophosphatase. Reduced inosine triphosphate pyrophosphatase activity results in decreased anemia, lower plasma but higher intracellular ribavirin concentrations, and improved HCV therapeutic outcome.

Modes of action

• Inhibition of inosine monophosphate dehydrogenase (IMPDH) resulting in intracellular GTP depletion occurs at lower ribavirin concentrations (10μM ribavirin). Reduced intracellular GTP secondarily may result in:
  • Inhibition of mRNA capping.
  • Impact on host cell gene expression, inflammation and immunomodulation.
• Inhibition of viral RdRPs.
• Enhancement of viral mutagenesis, which occurs at higher ribavirin concentrations in vitro (≥100μM), by means of the incorrect substitution of ribavirin triphosphate for GTP. Ribavirin triphosphate can form two hydrogen bonds with UTP or CTP with equal efficiency, resulting in an increase in G-to-A and C-to-U single nucleotide variations.

Financial andcompeting interests’ disclosure

The Swedish Medical Research Council (Vetenskapsrådet; grant number: 2017-00855) and ALF Foundation (grant number: ALFGBG-717711) at Sahlgrenska University Hospital supported this study. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

References

1. Revankar GR, Robins RK. Synthesis and biological activity of some nucleosides resembling guanosine: imidazo(1,2-alpha)pyrimidine nucleosides. Ann. NY Acad. Sci. 255, 166–176 (1975). Reports the synthesis of ribavirin.

2. Sidwell RW, Huffman JH, Khare GP, Allen LB, Witkowski JT, Robins RK. Broad-spectrum antiviral activity of virazole: 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide. Science 177(4050), 705–706 (1972).

3. Toltzis P, Huang AS. Effect of ribavirin on macromolecular synthesis in vesicular stomatitis virus-infected cells. Antimicrob. Agents Chemother. 29(6), 1010–1016 (1986).

4. Mchutchison JG, Gordon SC, Schiff ER et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. N. Engl. J. Med. 339(21), 1485–1492 (1998).

5. Reichard O, Norkrans G, Fryden A, Braconier JH, Sonnerborg A, Weiland O. Randomised, double-blind, placebo-controlled trial of interferon alpha-2b with and without ribavirin for chronic hepatitis C. The Swedish Study Group. Lancet 351(9096), 83–87 (1998). First report of the therapeutic use of ribavirin in combination with interferon for hepatitis C virus infection.

6. Reichard O, Andersson J, Schvarcz R, Weiland O. Ribavirin treatment for chronic hepatitis C. Lancet 337(8749), 1058–1061 (1991).First report of the use of ribavirin mono-therapy for hepatitis C virus infection.

7. Randolph AG, Wang EE. Ribavirin for respiratory syncytial virus infection of the lower respiratory tract. Cochrane Database Syst. Rev. CD000181 (2000).

8. Ascioglu S, Leblebicioglu H, Vahaboglu H, Chan KA. Ribavirin for patients with Crimean-Congo haemorrhagic fever: a systematic review and meta-analysis. J. Antimicrob. Chemother. 66(6), 1215–1222 (2011).

9. Gowen BB, Bray M. Progress in the experimental therapy of severe arenaviral infections. Future Microbiol. 6(12), 1429–1441 (2011).

10. Lee KY, Hung CC. Ribavirin for chronic hepatitis E virus infection. N. Engl. J. Med. 370(25), 2447 (2014).Medline,

11. Riner A, Chan-Tack KM, Murray JS. Original research: intravenous ribavirin–review of the FDA's Emergency Investigational New Drug Database (1997–2008) and literature review. Postgrad. Med. 121(3), 139–146 (2009).

12. Crotty S, Cameron CE, Andino R. RNA virus error catastrophe: direct molecular test by using ribavirin. Proc. Natl Acad. Sci. USA 98(12), 6895–6900 (2001).

13. Chan-Tack KM, Murray JS, Birnkrant DB. Use of ribavirin to treat influenza. N. Engl. J. Med. 361(17), 1713–1714 (2009).

14. Pawlotsky JM, Dahari H, Neumann AU et al. Antiviral action of ribavirin in chronic hepatitis C. Gastroenterology 126(3), 703–714 (2004).

15. Waldenstrom J, Westin J, Nystrom K et al. Randomized trial evaluating the impact of ribavirin mono-therapy and double dosing on viral kinetics, ribavirin pharmacokinetics and anemia in hepatitis C virus genotype 1 infection. PLoS ONE 11(5), e0155142 (2016).Highlights the effects of 1 month of ribavirin mono-therapy as well as suggests that 1 week of double dosing (followed by standard dosing) may be appropriate if rapid achievement of steady state is desired.

16. AASLD/IDSA HCV Guidance Panel. Hepatitis C guidance: AASLD-IDSA recommendations for testing, managing, and treating adults infected with hepatitis C virus. Hepatology 62(3), 932–954 (2015).

17. Curry MP, O'Leary JG, Bzowej N et al. Sofosbuvir and velpatasvir for HCV in patients with decompensated cirrhosis. N. Engl. J. Med. 373(27), 2618–2628 (2015).

18. Waldenstrom J, Lagging M. Editorial: ribavirin continues to play a role in treatment with direct-acting antivirals for hepatitis C virus-infected patients with decompensated cirrhosis. Aliment. Pharmacol. Ther. 46(11–12), 1115–1116 (2017).

19. Preston SL, Drusano GL, Glue P, Nash J, Gupta SK, McNamara P. Pharmacokinetics and absolute bioavailability of ribavirin in healthy volunteers as determined by stable-isotope methodology. Antimicrob. Agents Chemother. 43(10), 2451–2456 (1999).

20. Morello J, Rodriguez-Novoa S, Jimenez-Nacher I, Soriano V. Usefulness of monitoring ribavirin plasma concentrations to improve treatment response in patients with chronic hepatitis C. J. Antimicrob. Chemother. 62(6), 1174–1180 (2008).

21. Bruchfeld A, Lindahl K, Schvarcz R, Stahle L. Dosage of ribavirin in patients with hepatitis C should be based on renal function: a population pharmacokinetic analysis. Therapeutic Drug Monit. 24(6), 701–708 (2002).Suggests appropriate dosing of ribavirin in patients with renal insufficiency.

22. Paroni R, Del Puppo M, Borghi C, Sirtori CR, Galli Kienle M. Pharmacokinetics of ribavirin and urinary excretion of the major metabolite 1,2,4-triazole-3-carboxamide in normal volunteers. Int. J. Clin. Pharmacol. Therapy Toxicol. 27(6), 302–307 (1989).Medline,

23. Endres CJ, Moss AM, Ke B et al. The role of the equilibrative nucleoside transporter 1 (ENT1) in transport and metabolism of ribavirin by human and wild-type or Ent1-/- mouse erythrocytes. J. Pharmacol. Exp. Ther. 329(1), 387–398 (2009).

24. Lagging M, Wejstal R, Norkrans G et al. Treatment of hepatitis C virus infection: updated Swedish Guidelines 2016. Infect. Dis. (Lond.) 49(8), 561–575 (2017).

25. Lindahl K, Hornfeld E, Stahle L et al. High-dose ribavirin enhances early virological response in hepatitis C genotype 1-infected patients. Ther. Drug Monit. 37(6), 745–750 (2015).Reports the virological response as well as adverse events associated with high-dosing of ribavirin.

26. Bausch DG, Hadi CM, Khan SH, Lertora JJ. Review of the literature and proposed guidelines for the use of oral ribavirin as postexposure prophylaxis for Lassa fever. Clin. Infect. Dis. 51(12), 1435–1441 (2010).

27. Gupta SK, Kantesaria B, Glue P. Exploring the influence of renal dysfunction on the pharmacokinetics of ribavirin after oral and intravenous dosing. Drug Discov. Ther. 8(2), 89–95 (2014).

28. Lagging M, Wejstal R, Duberg AS et al. Treatment of hepatitis C virus infection for adults and children: updated Swedish consensus guidelines 2017. Infect. Dis. (Lond.) 50(8), 569–583 (2018).

29. Brochot E, Castelain S, Duverlie G, Capron D, Nguyen-Khac E, Francois C. Ribavirin monitoring in chronic hepatitis C therapy: anaemia versus efficacy. Antiviral Therapy 15(5), 687–695 (2010).

30. De Franceschi L, Fattovich G, Turrini F et al. Hemolytic anemia induced by ribavirin therapy in patients with chronic hepatitis C virus infection: role of membrane oxidative damage. Hepatology 31(4), 997–1004 (2000).

31. Sinclair SM, Jones JK, Miller RK, Greene MF, Kwo PY, Maddrey WC. The ribavirin pregnancy registry: an interim analysis of potential teratogenicity at the mid-point of enrollment. Drug Saf. 40(12), 1205–1218 (2017).

32. Rembeck K, Waldenstrom J, Hellstrand K et al. Variants of the inosine triphosphate pyrophosphatase gene are associated with reduced relapse risk following treatment for HCV genotype 2/3. Hepatology 59(6), 2131–2139 (2014).

33. Fellay J, Thompson AJ, Ge D et al. ITPA gene variants protect against anaemia in patients treated for chronic hepatitis C. Nature 464(7287), 405–408 (2010).

34. Vasanthakumar A, Davis JW, Abunimeh M et al. Reduced ITPase activity and favorable IL28B genetic variant protect against ribavirin-induced anemia in interferon-free regimens. PLoS ONE 13(5), e0198296 (2018).

35. Nystrom K, Wanrooij PH, Waldenstrom J et al. Inosine triphosphate pyrophosphatase dephosphorylates ribavirin triphosphate and reduced enzymatic activity potentiates mutagenesis in hepatitis C virus. J. Virol. 92(19), pii:e01087-18 (2018).Reports that ribavirin triphosphate is dephosphorylated to ribavirin monophosphate by the enzyme ITPase, which explains the reduced relapse risk, in spite of lower ribavirin plasma concentrations, reported among hepatitis C virus genotype 2 or 3 infected patients with reduced ITPase activity following interferon and ribavirin therapy.

36. El Desoky ES, Abdelhafez AT, Cusato J et al. The role of ITPA and ribavirin transporter genes polymorphisms in prediction of ribavirin-induced anaemia in chronic hepatitis C Egyptian patients. Clin. Exp. Pharmacol. Physiol. 44(9), 965–968 (2017).

37. Koczor CA, Torres RA, Lewis W. The role of transporters in the toxicity of nucleoside and nucleotide analogs. Expert Opin. Drug Metab. Toxicol. 8(6), 665–676 (2012).

38. Pinilla-Macua I, Fernandez-Calotti P, Perez-Del-Pulgar S, Pastor-Anglada M. Ribavirin uptake into human hepatocyte HHL5 cells is enhanced by interferon-alpha via up-regulation of the human concentrative nucleoside transporter (hCNT2). Mol. Pharm. 11(9), 3223–3230 (2014).

39. Ibarra KD, Pfeiffer JK. Reduced ribavirin antiviral efficacy via nucleoside transporter-mediated drug resistance. J. Virol. 83(9), 4538–4547 (2009).

40. Streeter DG, Witkowski JT, Khare GP et al. Mechanism of action of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (Virazole), a new broad-spectrum antiviral agent. Proc. Natl Acad. Sci. USA 70(4), 1174–1178 (1973). Reports the first observed mechanism of action of ribavirin, in other words, the inhibition of inosine monophosphate dehydrogenase (IMPDH).

41. Fang SH, Hwang LH, Chen DS, Chiang BL. Ribavirin enhancement of hepatitis C virus core antigen-specific type 1 T helper cell response correlates with the increased IL-12 level. J. Hepatol. 33(5), 791–798 (2000).

42. Waldenstrom J, Farkkila M, Rembeck K et al. Short interferon and ribavirin treatment for HCV genotype 2 or 3 infection: NORDynamIC trial and real-life experience. Scand. J. Gastroenterol. 51(3), 337–343 (2016).

43. Feld JJ, Nanda S, Huang Y et al. Hepatic gene expression during treatment with peginterferon and ribavirin: identifying molecular pathways for treatment response. Hepatology 46(5), 1548–1563 (2007).

44. Thomas E, Feld JJ, Li Q, Hu Z, Fried MW, Liang TJ. Ribavirin potentiates interferon action by augmenting interferon-stimulated gene induction in hepatitis C virus cell culture models. Hepatology 53(1), 32–41 (2011).

45. Rotman Y, Noureddin M, Feld JJ et al. Effect of ribavirin on viral kinetics and liver gene expression in chronic hepatitis C. Gut 63(1), 161–169 (2014).

46. Sharma OK, Goswami BB, Borek E. Inhibition of mRNA methylation: an approach to specific inhibition of viral replication. Princess Takamatsu Symp. 12, 109–116 (1982).Medline,

47. Bougie I, Bisaillon M. The broad spectrum antiviral nucleoside ribavirin as a substrate for a viral RNA capping enzyme. J. Biol. Chem. 279(21), 22124–22130 (2004).

48. Yan Y, Svitkin Y, Lee JM, Bisaillon M, Pelletier J. Ribavirin is not a functional mimic of the 7-methyl guanosine mRNA cap. RNA 11(8), 1238–1244 (2005).

49. Solomon EP, Berg LR, Martin DW. Biology (7th Edition). Brooks/Cole Thomson Learning, Belmont, CA, USA (2005).

50. Hultgren C, Milich DR, Weiland O, Sallberg M. The antiviral compound ribavirin modulates the T helper (Th) 1/Th2 subset balance in hepatitis B and C virus-specific immune responses. J. Gen. Virol. 79(Pt 10), 2381–2391 (1998).

51. Liao SH, Li Y, Lai YN, Liu N, Zhang FX, Xu PP. Ribavirin attenuates the respiratory immune responses to influenza viral infection in mice. Arch. Virol. 162(6), 1661–1669 (2017).

52. Eriksson B, Helgstrand E, Johansson NG et al. Inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate. Antimicrob. Agents Chemother. 11(6), 946–951 (1977).

53. Lohmann V, Roos A, Korner F, Koch JO, Bartenschlager R. Biochemical and structural analysis of the NS5B RNA-dependent RNA polymerase of the hepatitis C virus. J. Viral Hepat. 7(3), 167–174 (2000).

54. Maag D, Castro C, Hong Z, Cameron CE. Hepatitis C virus RNA-dependent RNA polymerase (NS5B) as a mediator of the antiviral activity of ribavirin. J. Biol. Chem. 276(49), 46094–46098 (2001).

55. Crotty S, Maag D, Arnold JJ et al. The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen. Nat. Med. 6(12), 1375–1379 (2000).

56. Chevaliez S, Brillet R, Lazaro E, Hezode C, Pawlotsky JM. Analysis of ribavirin mutagenicity in human hepatitis C virus infection. J. Virol. 81(14), 7732–7741 (2007).

57. Crotty S, Maag D, Arnold JJ et al. The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen. Nat. Med. 6(12), 1375–1379 (2000).

58. Mejer N, Fahnoe U, Galli A, Ramirez S, Benfield T, Bukh J. Ribavirin-induced mutagenesis across the complete open reading frame of hepatitis C virus genotypes 1a and 3a. J. Gen. Virol. 99(8), 1066–1077 (2018).

59. Todt D, Gisa A, Radonic A et al. In vivo evidence for ribavirin-induced mutagenesis of the hepatitis E virus genome. Gut 65(10), 1733–1743 (2016).

60. Lanford RE, Chavez D, Guerra B et al. Ribavirin induces error-prone replication of GB virus B in primary tamarin hepatocytes. J. Virol. 75(17), 8074–8081 (2001).

61. Feld JJ, Hoofnagle JH. Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature 436(7053), 967–972 (2005).