Tick-borne encephalitis virus (TBEV) is a leading cause of human neuroinfections in Europe and Northeast Asia. There are no antiviral therapies for treating TBEV infection. A series of nucleoside analogues was tested for the ability to inhibit the replication of TBEV in porcine kidney cells and human neuroblastoma cells. The interactions of three nucleoside analogues with viral polymerase were simulated using advanced computational methods. The nucleoside analogues 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), 2'-C-methyladenosine (2'-CMA), and 2'-C-methylcytidine (2'-CMC) inhibited TBEV replication. These compounds showed dose-dependent inhibition of TBEV-induced cytopathic effects, TBEV replication (50% effective concentrations [EC50]of 5.1 ± 0.4 μM for 7-deaza-2'-CMA, 7.1 ± 1.2 μM for 2'-CMA, and 14.2 ± 1.9 μM for 2'-CMC) and viral antigen production. Notably, 2'-CMC was relatively cytotoxic to porcine kidney cells (50% cytotoxic concentration [CC50] of ∼50 μM). The anti-TBEV effect of 2'-CMA in cell culture diminished gradually after day 3 posttreatment. 7-Deaza-2'-CMA showed no detectable cellular toxicity (CC50 > 50 μM), and the antiviral effect in culture was stable for >6 days posttreatment. Computational molecular analyses revealed that compared to the other two compounds, 7-deaza-2'-CMA formed a large cluster near the active site of the TBEV polymerase. High antiviral activity and low cytotoxicity suggest that 7-deaza-2'-CMA is a promising candidate for further investigation as a potential therapeutic agent in treating TBEV infection.

Department of Virology Veterinary Research Institute Brno Czech Republic

Department of Virology Veterinary Research Institute Brno Czech Republic Institute of Parasitology Biology Centre of the Czech Academy of Sciences České Budějovice Czech Republic Faculty of Science University of South Bohemia České Budějovice 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 České Budějovice Czech Republic

Joint BSC CRG IRB Research Program in Computational Biology Barcelona Supercomputing Center Barcelona Spain

Rega Institute for Medical Research Leuven Belgium

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.

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

Dumpis U, Crook D, Oksi J. 1999. Tick-borne encephalitis. Clin Infect Dis 28:882–890. doi:10.1086/515195. PubMed DOI

Heinz FX, Mandl CW. 1993. The molecular biology of tick-borne encephalitis virus. APMIS 101:735–745. doi:10.1111/j.1699-0463.1993.tb00174.x. PubMed DOI

Heinz FX, Stiasny K, Holzmann H, Grgic-Vitek M, Kriz B, Essl A, Kundi M. 2013. Vaccination and tick-borne encephalitis, Central Europe. Emerg Infect Dis 19:69–76. doi:10.3201/eid1901.120458. PubMed DOI PMC

Ruzek D, Dobler G, Mantke OD. 2010. Tick-borne encephalitis: pathogenesis and clinical implications. Travel Med Infect Dis 8:223–232. doi:10.1016/j.tmaid.2010.06.004. PubMed DOI

Zavadska D, Anca I, Andre F, Bakir M, Chlibek R, Cizman M, Ivaskeviciene I, Mangarov A, Meszner Z, Pokorn M, Prymula R, Richter D, Salman N, Simurka P, Tamm E, Tesovic G, Urbancikova I, Usonis V. 2013. Recommendations for tick-borne encephalitis vaccination from the Central European Vaccination Awareness Group (CEVAG). Hum Vaccin Immunother 9:362–374. doi:10.4161/hv.22766. PubMed DOI PMC

Puig-Basagoiti F, Tilgner M, Forshey BM, Philpott SM, Espina NG, Wentworth DE, Goebel SJ, Masters PS, Falgout B, Ren P, Ferguson DM, Shi PY. 2006. Triaryl pyrazoline compound inhibits flavivirus RNA replication. Antimicrob Agents Chemother 50:1320–1329. doi:10.1128/AAC.50.4.1320-1329.2006. PubMed DOI PMC

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

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

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

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

Klumpp K, Leveque V, Le Pogam S, 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

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 Thomas H, 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

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

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

Latour DR, Jekle A, Javanbakht H, Henningsen R, Gee P, Lee I, Tran P, Ren S, Kutach AK, Harris SF, Wang SM, Lok SJ, Shaw D, Li J, Heilek G, Klumpp K, Swinney DC, Deval J. 2010. Biochemical characterization of the inhibition of the dengue virus RNA polymerase by beta-D-2′-ethynyl-7-deaza-adenosine triphosphate. Antiviral Res 87:213–222. doi:10.1016/j.antiviral.2010.05.003. 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

Chen H, Liu L, Jones SA, Banavali N, Kass J, Li Z, Zhang J, Kramer LD, Ghosh AK, Li H. 2013. Selective inhibition of the West Nile virus methyltransferase by nucleoside analogs. Antiviral Res 97:232–239. doi:10.1016/j.antiviral.2012.12.012. 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

Smee DF, Morris JLB, Barnard DL, Vanaerschot A. 1992. Selective inhibition of arthropod-borne and arenaviruses in vitro by 3′-fluoro-3′-deoxyadenosine. Antiviral Res 18:151–162. doi:10.1016/0166-3542(92)90035-4. PubMed DOI

Smee DF, Alaghamandan HA, Ramasamy K, Revankar GR. 1995. Broad-spectrum activity of 8-chloro-7-deazaguanosine against RNA virus infections in mice and rats. Antiviral Res 26:203–209. doi:10.1016/0166-3542(94)00084-L. PubMed DOI PMC

Ojwang JO, Ali S, Smee DF, Morrey JD, Shimasaki CD, Sidwell RW. 2005. Broad-spectrum inhibitor of viruses in the Flaviviridae family. Antiviral Res 68:49–55. doi:10.1016/j.antiviral.2005.06.002. PubMed DOI

Chatelain G, Debing Y, De Burghgraeve T, Zmurko J, Saudi M, Rozenski J, Neyts J, Van Aerschot A. 2013. In search of flavivirus inhibitors: evaluation of different tritylated nucleoside analogues. Eur J Med Chem 65:249–255. doi:10.1016/j.ejmech.2013.04.034. PubMed DOI

Koonin EV, Dolja VV. 1993. Evolution and taxonomy of positive-strand RNA viruses—implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol 28:375–430. doi:10.3109/10409239309078440. PubMed DOI

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

Kozuch O, Mayer V. 1975. Pig kidney epithelial (ps) cells—perfect tool for study of flavi-viruses and some other arboviruses. Acta Virol 19:498. PubMed

Růzek D, Vancova M, Tesarova M, Ahantarig A, Kopecky J, Grubhoffer L. 2009. Morphological changes in human neural cells following tick-borne encephalitis virus infection. J Gen Virol 90:1649–1658. doi:10.1099/vir.0.010058-0. 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

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. doi:10.1038/nmeth.2019. PubMed DOI PMC

Sali A, Blundell TL. 1993. Comparative protein modeling by satisfaction of spatial restraints. J Mol Biol 234:779–815. doi:10.1006/jmbi.1993.1626. PubMed DOI

McGuffin LJ, Buenavista MT, Roche DB. 2013. The ModFOLD4 server for the quality assessment of 3D protein models. Nucleic Acids Res 41:W368–W372. doi:10.1093/nar/gkt294. PubMed DOI PMC

Benkert P, Kuenzli M, Schwede T. 2009. QMEAN server for protein model quality estimation. Nucleic Acids Res 37:W510–W514. doi:10.1093/nar/gkp322. PubMed DOI PMC

Berjanskii M, Zhou J, Liang Y, Lin G, Wishart DS. 2012. Resolution-by-proxy: a simple measure for assessing and comparing the overall quality of NMR protein structures. J Biomol NMR 53:167–180. doi:10.1007/s10858-012-9637-2. PubMed DOI

Li X, Jacobson MP, Zhu K, Zhao S, Friesner RA. 2007. Assignment of polar states for protein amino acid residues using an interaction cluster decomposition algorithm and its application to high resolution protein structure modeling. Proteins 66:824–837. PubMed

Schrödinger LLC. 2010. Maestro version 9.1. Schrödinger LLC, New York, NY.

Madadkar-Sobhani A, Guallar V. 2013. PELE web server: atomistic study of biomolecular systems at your fingertips. Nucleic Acids Res 41:W322–W328. doi:10.1093/nar/gkt454. PubMed DOI PMC

Takhampunya R, Ubol S, Houng HS, Cameron CE, Padmanabhan R. 2006. Inhibition of dengue virus replication by mycophenolic acid and ribavirin. J Gen Virol 87:1947–1952. doi:10.1099/vir.0.81655-0. PubMed DOI

Atilgan AR, Durell SR, Jernigan RL, Demirel MC, Keskin O, Bahar I. 2001. Anisotropy of fluctuation dynamics of proteins with an elastic network model. Biophys J 80:505–515. doi:10.1016/S0006-3495(01)76033-X. PubMed DOI PMC

Jacobson MP, Friesner RA, Xiang ZX, Honig B. 2002. On the role of the crystal environment in determining protein side-chain conformations. J Mol Biol 320:597–608. doi:10.1016/S0022-2836(02)00470-9. PubMed DOI

Still WC, Tempczyk A, Hawley RC, Hendrickson T. 1990. Semianalytical treatment of solvation for molecular mechanics and dynamics. J Am Chem Soc 112:6127–6129. doi:10.1021/ja00172a038. DOI

Borrelli KW, Vitalis A, Alcantara R, Guallar V. 2005. PELE: protein energy landscape exploration. A novel Monte Carlo based technique. J Chem Theory Comput 1:1304–1311. PubMed

Jorgensen WL, Tiradorives J. 1988. The Opls potential functions for proteins—energy minimizations for crystals of cyclic peptides and crambin. J Am Chem Soc 110:1657–1666. doi:10.1021/ja00214a001. PubMed DOI

Gil VA, Guallar V. 2014. pyProCT: automated cluster analysis for structural bioinformatics. J Chem Theory Comput 10:3236–3243. doi:10.1021/ct500306s. PubMed DOI

Osolodkin DI, Kozlovskaya LI, Dueva EV, Dotsenko VV, Rogova YV, Frolov KA, Krivokolysko SG, Romanova EG, Morozov AS, Karganova GG, Palyulin VA, Pentkovski VM, Zefirov NS. 2013. Inhibitors of tick-borne flavivirus reproduction from structure-based virtual screening. ACS Med Chem Lett 4:869–874. doi:10.1021/ml400226s. PubMed DOI PMC

Wallner G, Mandl CW, Ecker M, Holzmann H, Stiasny K, Kunz C, Heinz FX. 1996. Characterisation and complete genome sequences of high- and low-virulence variants of tick-borne encephalitis virus. J Gen Virol 77:1035–1042. doi:10.1099/0022-1317-77-5-1035. PubMed DOI

Crance JM, Scaramozzino N, Jouan A, Garin D. 2003. Interferon, ribavirin, 6-azauridine and glycyrrhizin: antiviral compounds active against pathogenic flaviviruses. Antiviral Res 58:73–79. doi:10.1016/S0166-3542(02)00185-7. PubMed DOI

Carroll SS, Tomassini JE, Bosserman M, Getty K, Stahlhut MW, Eldrup AB, Bhat B, Hall D, Simcoe AL, LaFemina R, Rutkowski CA, Wolanski B, Yang ZC, Migliaccio G, De Francesco R, Kuo LC, MacCoss M, Olsen DB. 2003. Inhibition of hepatitis C virus RNA replication by 2′-modified nucleoside analogs. J Biol Chem 278:11979–11984. doi:10.1074/jbc.M210914200. PubMed DOI

Cristalli G, Costanzi S, Lambertucci C, Lupidi G, Vittori S, Volpini R, Camaioni E. 2001. Adenosine deaminase: functional implications and different classes of inhibitors. Med Res Rev 21:105–128. doi:10.1002/1098-1128(200103)21:2<105::AID-MED1002>3.0.CO;2-U. PubMed DOI

Kinney RM, Huang CYH, Rose BC, Kroeker AD, Dreher TW, Iversen PL, Stein DA. 2005. Inhibition of dengue virus serotypes 1 to 4 in Vero cell cultures with morpholino oligomers. J Virol 79:5116–5128. doi:10.1128/JVI.79.8.5116-5128.2005. PubMed DOI PMC

Carroll SS, Ludmerer S, Handt L, Koeplinger K, Zhang NR, Graham D, Davies ME, MacCoss M, Hazuda D, Olsen DB. 2009. Robust antiviral efficacy upon administration of a nucleoside analog to hepatitis C virus-infected chimpanzees. Antimicrob Agents Chemother 53:926–934. doi:10.1128/AAC.01032-08. PubMed DOI PMC

Carroll SS, Koeplinger K, Vavrek M, Zhang NR, Handt L, MacCoss M, Olsen DB, Reddy KR, Sun ZL, van Poelje PD, Fujitaki JM, Boyer SH, Linemeyer DL, Hecker SJ, Erion MD. 2011. Antiviral efficacy upon administration of a HepDirect prodrug of 2′-c-methylcytidine to hepatitis C virus-infected chimpanzees. Antimicrob Agents Chemother 55:3854–3860. doi:10.1128/AAC.01152-10. PubMed DOI PMC

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

Methods for Evaluation of Antivirals and Neutralizing Antibodies Against Tick-Borne Encephalitis Virus

Mechanisms of Plasma Ozone and UV-C Sterilization of SARS-CoV-2 Explored through Atomic Force Microscopy

Membrane-Targeting Perylenylethynylphenols Inactivate Medically Important Coronaviruses via the Singlet Oxygen Photogeneration Mechanism

Guanine quadruplexes in the RNA genome of the tick-borne encephalitis virus: their role as a new antiviral target and in virus biology

A Helquat-like Compound as a Potent Inhibitor of Flaviviral and Coronaviral Polymerases

History of Arbovirus Research in the Czech Republic

Non-Nucleotide RNA-Dependent RNA Polymerase Inhibitor That Blocks SARS-CoV-2 Replication

Antiviral Activity of Vacuolar ATPase Blocker Diphyllin against SARS-CoV-2

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

Management Options for Ixodes ricinus-Associated Pathogens: A Review of Prevention Strategies

An E460D Substitution in the NS5 Protein of Tick-Borne Encephalitis Virus Confers Resistance to the Inhibitor Galidesivir (BCX4430) and Also Attenuates the Virus for Mice

Viral RNA-Dependent RNA Polymerase Inhibitor 7-Deaza-2'-C-Methyladenosine Prevents Death in a Mouse Model of West Nile Virus Infection

Arbidol (Umifenovir): A Broad-Spectrum Antiviral Drug That Inhibits Medically Important Arthropod-Borne Flaviviruses

Nucleoside analogs as a rich source of antiviral agents active against arthropod-borne flaviviruses

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

Pyrrolo[2,3-d]pyrimidine (7-deazapurine) as a privileged scaffold in design of antitumor and antiviral nucleosides