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J Virol. 2017 Aug 16. pii: JVI.01028-17. doi: 10.1128/JVI.01028-17. [Epub ahead of print]

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.

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Department of Virology, Veterinary Research Institute, Brno, Czech Republic.
Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic.
Laboratory of Public Health, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan.
Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic.
Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic.
Central Laboratories, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic.
Institute for Medical Microbiology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany.
Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany.
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic.
Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.
Department of Virology, Veterinary Research Institute, Brno, Czech Republic


Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2' -C-methyladenosine (7-deaza-2' -CMA), substantially improved disease outcome, 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' -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' -C-methylated nucleoside derivatives, but no cross-resistance was seen to other nucleoside analogs, such as 4' -C-azidocytidine and 2' -deoxy-2' -beta-hydroxy-4' -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' -C-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV (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 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' -C-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of viral NS5 RdRp and is associated with strong attenuation of the virus.Importance This study found that the nucleoside analog 7-deaza-2' -C-methyladenosine (7-deaza-2' -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' -CMA resulted in significantly higher survival rates, 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' -CMA but also to a broad range of other 2' -C-methylated antiviral medications. Our findings suggest that combination therapy could be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.

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