West Nile virus (WNV) is a medically important emerging arbovirus causing serious neuroinfections in humans and against which no approved antiviral therapy is currently available. In this study, we demonstrate that 2'-C-methyl- or 4'-azido-modified nucleosides are highly effective inhibitors of WNV replication, showing nanomolar or low micromolar anti-WNV activity and negligible cytotoxicity in cell culture. One representative of C2'-methylated nucleosides, 7-deaza-2'-C-methyladenosine, significantly protected WNV-infected mice from disease progression and mortality. Twice daily treatment at 25 mg/kg starting at the time of infection resulted in 100% survival of the mice. This compound was highly effective, even if the treatment was initiated 3 days postinfection, at the time of a peak of viremia, which resulted in a 90% survival rate. However, the antiviral effect of 7-deaza-2'-C-methyladenosine was absent or negligible when the treatment was started 8 days postinfection (i.e., at the time of extensive brain infection). The 4'-azido moiety appears to be another important determinant for highly efficient inhibition of WNV replication in vitro However, the strong anti-WNV effect of 4'-azidocytidine and 4'-azido-aracytidine was cell type dependent and observed predominantly in porcine kidney stable (PS) cells. The effect was much less pronounced in Vero cells. Our results indicate that 2'-C-methylated or 4'-azidated nucleosides merit further investigation as potential therapeutic agents for treating WNV infections as well as infections caused by other medically important flaviviruses.

Department of Virology Veterinary Research Institute Brno Czech Republic

Institute of Organic Chemistry and Biochemistry The Czech Academy of Sciences Prague Czech Republic

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Ceske Budejovice Czech Republic

Masaryk University Department of Experimental Biology Brno Czech Republic

The Czech Academy of Sciences Institute of Vertebrate Biology Brno Czech Republic

Zobrazit více v PubMed

Baier A. 2011. Flaviviral infections and potential targets for antiviral therapy, p 89–104. In Ruzek D. (ed), Flavivirus encephalitis. InTech, Rijeka, Croatia.

Beasley DWC, Davis CT, Whiteman M, Granwehr B, Kinney RM, Barrett ADT. 2004. Molecular determinants of virulence of West Nile virus in North America. Arch Virol 18:35–41. PubMed

Chambers TJ, Hahn CS, Galler R, Rice CM. 1990. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44:649–688. doi:10.1146/annurev.mi.44.100190.003245. PubMed DOI

Khromykh AA, Sedlak PL, Guyatt KJ, Hall RA, Westaway EG. 1999. Efficient trans-complementation of the flavivirus Kunjin NS5 protein but not of the NS1 protein requires its coexpression with other components of the viral replicase. J Virol 73:10272–10280. PubMed PMC

Lin RJ, Chang BL, Yu HP, Liao CL, Lin YL. 2006. Blocking of interferon-induced Jak-Stat signaling by Japanese encephalitis virus NS5 through a protein tyrosine phosphatase-mediated mechanism. J Virol 80:5908–5918. doi:10.1128/JVI.02714-05. PubMed DOI PMC

Liu WJ, Wang XJ, Mokhonov VV, Shi PY, Randall R, Khromykh AA. 2005. Inhibition of interferon signaling by the New York 99 strain and Kunjin subtype of West Nile virus involves blockage of STAT1 and STAT2 activation by nonstructural proteins. J Virol 79:1934–1942. doi:10.1128/JVI.79.3.1934-1942.2005. PubMed DOI PMC

Hubalek Z. 2000. European experience with the West Nile virus ecology and epidemiology: could it be relevant for the new world? Viral Immunol 13:415–426. PubMed

Smithburn K, Hughes T, Burke A, Paul J. 1940. A neurotropic virus isolated from the blood of a native of Uganda. Am J Trop Med Hyg 20:471–492. doi:10.4269/ajtmh.1940.s1-20.471. DOI

Dauphin G, Zientara S, Zeller H, Murgue B. 2004. West Nile: worldwide current situation in animals and humans. Comp Immunol Microbiol Infect Dis 27:343–355. doi:10.1016/j.cimid.2004.03.009. PubMed DOI

Deardorff E, Estrada-Franco J, Brault AC, Navarro-Lopez R, Campomanes-Cortes A, Paz-Ramirez P, Solis-Hernandez M, Ramey WN, Davis CT, Beasley DW, Tesh RB, Barrett AD, Weaver SC. 2006. Introductions of West Nile virus strains to Mexico. Emerg Infect Dis 12:314–318. doi:10.3201/eid1202.050871. PubMed DOI PMC

Komar N, Clark GG. 2006. West Nile virus activity in Latin America and the Caribbean. Rev Panam Salud Publica 19:112–117. PubMed

Mostashari F, Bunning ML, Kitsutani PT, Singer DA, Nash D, Cooper MJ, Katz N, Liljebjelke KA, Biggerstaff BJ, Fine AD, Layton MC, Mullin SM, Johnson AJ, Martin DA, Hayes EB, Campbell GL. 2001. Epidemic West Nile encephalitis, New York, 1999: results of a household-based seroepidemiological survey. Lancet 358:261–264. doi:10.1016/S0140-6736(01)05480-0. PubMed DOI

Tsai TF, Popovici F, Cernescu C, Campbell GL, Nedelcu NI. 1998. West Nile encephalitis epidemic in southeastern Romania. Lancet 352:767–771. PubMed

Lim SM, Koraka P, Osterhaus ADME, Martina BEE. 2011. West Nile virus: immunity and pathogenesis. Viruses 3:811–828. doi:10.3390/v3060811. PubMed DOI PMC

Chowers MY, Lang R, Nassar F, Ben-David D, Giladi M, Rubinshtein E, Itzhaki A, Mishal J, Siegman-Igra Y, Kitzes R, Pick N, Landau Z, Wolf D, Bin H, Mendelson E, Pitlik SD, Weinberger M. 2001. Clinical characteristics of the West Nile fever outbreak, Israel, 2000. Emerg Infect Dis 7:675–678. doi:10.3201/eid0704.010414. PubMed DOI PMC

Nash D, Mostashari F, Fine A, Miller J, O'Leary D, Murray K, Huang A, Rosenberg A, Greenberg A, Sherman M, Wong S, Layton M, Campbell GL, Roehrig JT, Gubler DJ, Shieh WJ, Zaki S, Smith P. 2001. The outbreak of West Nile virus infection in the New York City area in 1999. N Engl J Med 344:1807–1814. doi:10.1056/NEJM200106143442401. PubMed DOI

Samuel MA, Diamond MS. 2006. Pathogenesis of West Nile virus infection: a balance between virulence, innate and adaptive immunity, and viral evasion. J Virol 80:9349–9360. doi:10.1128/JVI.01122-06. PubMed DOI PMC

De Clercq E. 2011. A 40-year journey in search of selective antiviral chemotherapy. Annu Rev Pharmacol Toxicol 51:1–24. doi:10.1146/annurev-pharmtox-010510-100228. PubMed DOI

De Clercq E. 2004. Antivirals and antiviral strategies. Nat Rev Microbiol 2:704–720. doi:10.1038/nrmicro975. PubMed DOI PMC

De Clercq E. 2008. Emerging antiviral drugs. Expert Opin Emerg Drugs 13:393–416. doi:10.1517/14728214.13.3.393. PubMed DOI

De Clercq E, Neyts J. 2009. Antiviral agents acting as DNA or RNA chain terminators. Handb Exp Pharmacol 189:53–84. doi:10.1007/978-3-540-79086-0_3. PubMed DOI

Benhamou Y, Tubiana R, Thibault V. 2003. Tenofovir disoproxil fumarate in patients with HIV and lamivudine-resistant hepatitis B virus. N Engl J Med 348:177–178. doi:10.1056/NEJM200301093480218. PubMed DOI

De Clercq E, Holý A. 2005. Acyclic nucleoside phosphonates: A key class of antiviral drugs. Nat Rev Drug Discov 4:928–940. doi:10.1038/nrd1877. PubMed DOI

Huang YS, Chang SY, Sheng WH, Sun HY, Lee KY, Chuang YC, Su YC, Liu WC, Hung CC, Chang SC. 2016. Virological response to tenofovir disoproxil fumarate in HIV-positive patients with lamivudine-resistant hepatitis B virus coinfection in an area hyperendemic for hepatitis B virus infection. PLoS One 11:e0169228. doi:10.1371/journal.pone.0169228. PubMed DOI PMC

Ray AS, Fordyce MW, Hitchcock MJM. 2016. Tenofovir alafenamide: a novel prodrug of tenofovir for the treatment of human immunodeficiency virus. Antiviral Res 125:63–70. doi:10.1016/j.antiviral.2015.11.009. PubMed DOI

Stedman C. 2014. Sofosbuvir, a NS5B polymerase inhibitor in the treatment of hepatitis C: a review of its clinical potential. Therap Adv Gastroenterol 7:131–140. doi:10.1177/1756283X13515825. PubMed DOI PMC

Eyer L, Šmídková M, Nencka R, Neča J, Kastl T, Palus M, De Clercq E, Růžek D. 2016. Structure-activity relationships of nucleoside analogues for inhibition of tick-borne encephalitis virus. Antiviral Res 133:119–129. doi:10.1016/j.antiviral.2016.07.018. PubMed DOI

Kumar M, O'Connell M, Namekar M, Nerurkar VR. 2014. Infection with non-lethal West Nile virus Eg101 strain induces immunity that protects mice against the lethal West Nile virus NY99 strain. Viruses 6:2328–2339. doi:10.3390/v6062328. PubMed DOI PMC

Eldrup AB, Allerson CR, Bennett CF, Bera S, Bhat B, Bhat N, Bosserman MR, Brooks J, Burlein C, Carroll SS, Cook PD, Getty KL, MacCoss M, McMasters DR, Olsen DB, Prakash TP, Prhavc M, Song QL, Tomassini JE, Xia J. 2004. Structure-activity relationship of purine ribonucleosides for inhibition of hepatitis C virus RNA-dependent RNA polymerase. J Med Chem 47:2283–2295. doi:10.1021/jm030424e. PubMed DOI

Klumpp K, Leveque V, Le PS, Ma H, Jiang WR, Kang HS, Granycome C, Singer M, Laxton C, Hang JQ, Sarma K, Smith DB, Heindl D, Hobbs CJ, Merrett JH, Symons J, Cammack N, Martin JA, Devos R, Najera I. 2006. The novel nucleoside analog R1479 (4'-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture. J Biol Chem 281:3793–3799. doi:10.1074/jbc.M510195200. PubMed DOI

Klumpp K, Kalayanov G, Ma H, Le Pogam S, Leveque V, Jiang WR, Inocencio N, De Witte A, Rajyaguru S, Tai E, Chanda S, Irwin MR, Sund C, Winqist A, Maltseva T, Eriksson S, Usova E, Smith M, Alker A, Najera I, Cammack N, Martin JA, Johansson NG, Smith DB. 2008. 2'-Deoxy-4'-azido nucleoside analogs are highly potent inhibitors of hepatitis C virus replication despite the lack of 2'-alpha-hydroxyl groups. J Biol Chem 283:2167–2175. doi:10.1074/jbc.M708929200. PubMed DOI

Migliaccio G, Tomassini JE, Carroll SS, Tomei L, Altamura S, Bhat B, Bartholomew L, Bosserman MR, Ceccacci A, Colwell LF, Cortese R, De Francesco R, Eldrup AB, Getty KL, Hou XS, LaFemina RL, Ludmerer SW, MacCoss M, McMasters DR, Stahlhut MW, Olsen DB, Hazuda DJ, Flores OA. 2003. Characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis C virus replication in vitro. J Biol Chem 278:49164–49170. doi:10.1074/jbc.M305041200. PubMed DOI

Olsen DB, Eldrup AB, Bartholomew L, Bhat B, Bosserman MR, Ceccacci A, Colwell LF, Fay JF, Flores OA, Getty KL, Grobler JA, LaFemina RL, Markel EJ, Migliaccio G, Prhavc M, Stahlhut MW, Tomassini JE, MacCoss M, Hazuda DJ, Carroll SS. 2004. A 7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis C virus replication with excellent pharmacokinetic properties. Antimicrob Agents Chemother 48:3944–3953. doi:10.1128/AAC.48.10.3944-3953.2004. PubMed DOI PMC

Smith DB, Kalayanov G, Sund C, Winqvist A, Pinho P, Maltseva T, Morisson V, Leveque V, Rajyaguru S, Le Pogam S, Najera I, Benkestock K, Zhou XX, Maag H, Cammack N, Martin JA, Swallow S, Johansson NG, Klumpp K, Smith M. 2009. The design, synthesis, and antiviral activity of 4'-azidocytidine analogues against hepatitis C virus replication: the discovery of 4'-azidoarabinocytidine. J Med Chem 52:219–223. doi:10.1021/jm800981y. PubMed DOI

Sofia MJ, Chang W, Furman PA, Mosley RT, Ross BS. 2012. Nucleoside, nucleotide, and non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA-polymerase. J Med Chem 55:2481–2531. doi:10.1021/jm201384j. PubMed DOI

Eyer L, Kondo H, Zouharova D, Hirano M, Valdés JJ, Muto M, Kastl T, Kobayashi S, Haviernik J, Igarashi M, Kariwa H, Vaculovicova M, Cerny J, Kizek R, Kröger A, Lienenklaus S, Dejmek M, Nencka R, Palus M, Salat J, De Clercq E, Yoshii K, Ruzek D. 2017. Escape of tick-borne flavivirus from 2'-C-methylated nucleoside antivirals is mediated by a single conservative mutation in NS5 that has a dramatic effect on viral fitness. J Virol 91:e01028-17. doi:10.1128/JVI.01028-17. PubMed DOI PMC

Eyer L, Nencka R, Huvarová I, Palus M, Joao Alves M, Gould EA, De Clercq E, Růžek D. 2016. Nucleoside inhibitors of Zika virus. J Infect Dis 214:707–711. doi:10.1093/infdis/jiw226. PubMed DOI

Eyer L, Valdés JJ, Gil VA, Nencka R, Hřebabecký H, Šála M, Salát J, Černý J, Palus M, De Clercq E, Růžek D. 2015. Nucleoside inhibitors of tick-borne encephalitis virus. Antimicrob Agents Chemother 59:5483–5493. doi:10.1128/AAC.00807-15. PubMed DOI PMC

Flint M, McMullan LK, Dodd KA, Dodd KA, Bird BH, Khristova ML, Nichol ST, Spiropoulou CF. 2014. Inhibitors of the tick-borne, hemorrhagic fever-associated flaviviruses. Antimicrob Agents Chemother 58:3206–3216. doi:10.1128/AAC.02393-14. PubMed DOI PMC

Julander JG, Jha AK, Choi JA, Jung KH, Smee DF, Morrey JD, Chu CK. 2010. Efficacy of 2'-C-methylcytidine against yellow fever virus in cell culture and in a hamster model. Antiviral Res 86:261–267. doi:10.1016/j.antiviral.2010.03.004. PubMed DOI PMC

Lanko K, Eggermont K, Patel A, Kaptein S, Delang L, Verfaillie CM, Neyts J. 2017. Replication of the Zika virus in different iPSC-derived neuronal cells and implications to assess efficacy of antivirals. Antiviral Res 145:82–86. doi:10.1016/j.antiviral.2017.07.010. PubMed DOI

Lee JC, Tseng CK, Wu YH, Kaushik-Basu N, Lin CK, Chen WC, Wu HN. 2015. Characterization of the activity of 2'-C-methylcytidine against dengue virus replication. Antiviral Res 116:1–9. doi:10.1016/j.antiviral.2015.01.002. PubMed DOI

Mateo R, Nagamine CM, Kirkegaard K. 2015. Suppression of drug resistance in dengue virus. mBio 6:e01960-15. doi:10.1128/mBio.01960-15. PubMed DOI PMC

Hercik K, Brynda J, Nencka R, Boura E. 2017. Structural basis of Zika virus methyltransferase inhibition by sinefungin. Arch Virol 162:2091–2096. doi:10.1007/s00705-017-3345-x. PubMed DOI

Zmurko J, Marques RE, Schols D, Verbeken E, Kaptein SJF, Neyts J. 2016. The viral polymerase inhibitor 7-deaza-2'-C-methyladenosine is a potent inhibitor of in vitro Zika virus replication and delays disease progression in a robust mouse infection model. PLoS Negl Trop Dis 10:e0004695. doi:10.1371/journal.pntd.0004695. PubMed DOI PMC

Lefebvre DJ, De Vleeschauwer AR, Goris N, Kollanur D, Billiet A, Murao L, Neyts J, De Clercq K. 2014. Proof of concept for the inhibition of foot-and-mouth disease virus replication by the anti-viral drug 2'-C-methylcytidine in severe combined immunodeficient mice. Transbound Emerg Dis 61:E89–E91. doi:10.1111/tbed.12069. PubMed DOI

Rocha-Pereira J, Jochmans D, Dallmeier K, Leyssen P, Cunha R, Costa I, Nascimento MS, Neyts J. 2012. Inhibition of norovirus replication by the nucleoside analogue 2'-C-methylcytidine. Biochem Bioph Res Commun 427:796–800. doi:10.1016/j.bbrc.2012.10.003. PubMed DOI

Rocha-Pereira J, Jochmans D, Debing Y, Verbeken E, Nascimento MSJ, Neyts J. 2013. The viral polymerase inhibitor 2'-C-methylcytidine inhibits Norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model. J Virol 87:11798–11805. doi:10.1128/JVI.02064-13. PubMed DOI PMC

Balestri F, Barsotti C, Lutzemberger L, Camici M, Ipata PL. 2007. Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain. Neurochem Int 51:517–523. doi:10.1016/j.neuint.2007.06.007. PubMed DOI

Lashkov AA, Shchekotikhin AA, Shtil AA, Sotnichenko SE, Mikhailov AM. 2016. Modified 5-fluorouracil: uridine phosphorylase inhibitor. Crystallogr Rep 61:826–829. doi:10.1134/S1063774516050138. DOI

Yin Z, Chen YL, Schul W, Wang QY, Gu F, Duraiswamy J, Kondreddi RR, Niyomrattanakit P, Lakshminarayana SB, Goh A, Xu HY, Liu W, Liu B, Lim JY, Ng CY, Qing M, Lim CC, Yip A, Wang G, Chan WL, Tan HP, Lin K, Zhang B, Zou G, Bernard KA, Garrett C, Beltz K, Dong M, Weaver M, He H, Pichota A, Dartois V, Keller TH, Shi PY. 2009. An adenosine nucleoside inhibitor of dengue virus. Proc Natl Acad Sci U S A 106:20435–20439. doi:10.1073/pnas.0907010106. PubMed DOI PMC

Eyer L, Nencka R, de Clercq E, Seley-Radtke K, Růžek D. 2018. Nucleoside analogs as a rich source of antiviral agents active against arthropod-borne flaviviruses. Antivir Chem Chemother 26:2040206618761299. doi:10.1177/2040206618761299. PubMed DOI PMC

Roe K, Kumar M, Lum S, Orillo B, Nerurkar VR, Verma S. 2012. West Nile virus-induced disruption of the blood-brain barrier in mice is characterized by the degradation of the junctional complex proteins and increase in multiple matrix metalloproteinases. J Gen Virol 93:1193–1203. doi:10.1099/vir.0.040899-0. PubMed DOI PMC

Styer LM, Lim PY, Louie KL, Albright RG, Kramer LD, Bernard KA. 2011. Mosquito saliva causes enhancement of West Nile virus infection in mice. J Virol 85:1517–1527. doi:10.1128/JVI.01112-10. PubMed DOI PMC

Schul W, Chen YL, Yin Z, Keller T, Shi PY. 2010. An adenosine nucleoside inhibitor of dengue virus. Antiviral Res 86:A24–A25. doi:10.1016/j.antiviral.2010.02.347. DOI

Arnold JJ, Sharma SD, Feng JY, Ray AS, Smidansky ED, Kireeva ML, Cho A, Perry J, Vela JE, Park Y, Xu Y, Tian Y, Babusis D, Barauskus O, Peterson BR, Gnatt A, Kashlev M, Zhong W, Cameron CE. 2012. Sensitivity of mitochondrial transcription and resistance of RNA polymerase II dependent nuclear transcription to antiviral ribonucleosides. PLoS Pathog 8:e1003030. doi:10.1371/journal.ppat.1003030. PubMed DOI PMC

Chen YL, Yin Z, Lakshminarayana SB, Qing M, Schul W, Duraiswamy J, Kondreddi RR, Goh A, Xu HY, Yip A, Liu B, Weaver M, Dartois V, Keller TH, Shi PY. 2010. Inhibition of dengue virus by an ester prodrug of an adenosine analog. Antimicrob Agents Chemother 54:3255–3261. doi:10.1128/AAC.00397-10. PubMed DOI PMC

Chen YL, Yin Z, Duraiswamy J, Schul W, Lim CC, Liu B, Xu HY, Qing M, Yip A, Wang G, Chan WL, Tan HP, Lo M, Liung S, Kondreddi RR, Rao R, Gu H, He H, Keller TH, Shi PY. 2010. Inhibition of dengue virus RNA synthesis by an adenosine nucleoside. Antimicrob Agents Chemother 54:2932–2939. doi:10.1128/AAC.00140-10. PubMed DOI PMC

Ferreira AC, Zaverucha-do-Valle C, Reis PA, Barbosa-Lima G, Vieira YR, Mattos M, Silva PP, Sacramento C, de Castro Faria Neto HC, Campanati L, Tanuri A, Brüning K, Bozza FA, Bozza PT, Souza TML. 2017. Sofosbuvir protects Zika vrius-infected mice from mortality, preventing short- and long-term sequelae. Sci Rep 7:9409. doi:10.1038/s41598-017-09797-8. PubMed DOI PMC

Kozuch O, Mayer V. 1975. Pig kidney epithelial (PS) cells: a perfect tool for study of flaviviruses and some other arboviruses. Acta Virol 19:498. PubMed

Melnick JL, Paul JR, Riordan JT, Barnett VH, Goldblum N, Zabin E. 1951. Isolation from human sera in Egypt of a virus apparently identical to West Nile virus. Proc Soc Exp Biol Med 77:661–665. PubMed

Rudolf I, Bakonyi T, Sebesta O, Mendel J, Peško J, Betášová L, Blažejová H, Venclíková K, Straková P, Nowotny N, Hubálek Z. 2014. West Nile virus lineage 2 isolated from Culex modestus mosquitoes in the Czech Republic, 2013: expansion of the European WNV endemic area to the North? Euro Surveill 19:2–5. https://www.eurosurveillance.org/content/10.2807/1560-7917.ES2014.19.31.20867. PubMed DOI

Eyer L, Zouharová D, Širmarová J, Fojtíková M, Štefánik M, Haviernik J, Nencka R, de Clercq E, Růžek D. 2017. Antiviral activity of the adenosine analogue BCX4430 against West Nile virus and tick-borne flaviviruses. Antiviral Res 142:63–67. doi:10.1016/j.antiviral.2017.03.012. PubMed DOI

De Madrid AT, Porterfield JS. 1969. A simple micro-culture method for study of group B arboviruses. Bull World Health Organ 40:113–121. PubMed PMC

History of Arbovirus Research in the Czech Republic

Broad-Spectrum Antiviral Activity of 3'-Deoxy-3'-Fluoroadenosine against Emerging Flaviviruses

FDA-Approved Drugs Efavirenz, Tipranavir, and Dasabuvir Inhibit Replication of Multiple Flaviviruses in Vero Cells