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Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2\u2032-\n            <jats:italic>C<\/jats:italic>\n            -methyladenosine (7-deaza-2\u2032-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2\u2032-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2\u2032-\n            <jats:italic>C<\/jats:italic>\n            -methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4\u2032-\n            <jats:italic>C<\/jats:italic>\n            -azidocytidine and 2\u2032-deoxy-2\u2032-beta-hydroxy-4\u2032-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2\u2032-\n            <jats:italic>C<\/jats:italic>\n            -methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2\u2032-\n            <jats:italic>C<\/jats:italic>\n            -methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus.\n          <\/jats:p>\n          <jats:p>\n            <jats:bold>IMPORTANCE<\/jats:bold>\n            This study found that the nucleoside analog 7-deaza-2\u2032-\n            <jats:italic>C<\/jats:italic>\n            -methyladenosine (7-deaza-2\u2032-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2\u2032-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2\u2032-CMA but also to a broad range of other 2\u2032-\n            <jats:italic>C<\/jats:italic>\n            -methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.\n          <\/jats:p>","DOI":"10.1128\/jvi.01028-17","type":"journal-article","created":{"date-parts":[[2017,8,17]],"date-time":"2017-08-17T00:30:18Z","timestamp":1502929818000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":46,"title":["Escape of Tick-Borne Flavivirus from 2\u2032-\n            <i>C<\/i>\n            -Methylated Nucleoside Antivirals Is Mediated by a Single Conservative Mutation in NS5 That Has a Dramatic Effect on Viral Fitness"],"prefix":"10.1128","volume":"91","author":[{"given":"Ludek","family":"Eyer","sequence":"first","affiliation":[{"name":"Department of Virology, Veterinary Research Institute, Brno, Czech Republic"},{"name":"Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Ceske 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