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 [EC ₅₀ ]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 [CC ₅₀ ] 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 (CC ₅₀ > 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.

Viral RNA dependent polymerases (vRdPs) are present in all RNA viruses; unfortunately, their sequence similarity is too low for phylogenetic studies. Nevertheless, vRdP protein structures are remarkably conserved. In this study, we used the structural similarity of vRdPs to reconstruct their evolutionary history. The major strength of this work is in unifying sequence and structural data into a single quantitative phylogenetic analysis, using powerful a Bayesian approach. The resulting phylogram of vRdPs demonstrates that RNA-dependent DNA polymerases (RdDPs) of viruses within Retroviridae family cluster in a clearly separated group of vRdPs, while RNA-dependent RNA polymerases (RdRPs) of dsRNA and +ssRNA viruses are mixed together. This evidence supports the hypothesis that RdRPs replicating +ssRNA viruses evolved multiple times from RdRPs replicating +dsRNA viruses, and vice versa. Moreover, our phylogram may be presented as a scheme for RNA virus evolution. The results are in concordance with the actual concept of RNA virus evolution. Finally, the methods used in our work provide a new direction for studying ancient virus evolution.

Ticks are important human and animal parasites and vectors of many infectious disease agents. Control of tick activity is an effective tool to reduce the risk of contracting tick-transmitted diseases. The castor bean tick (Ixodes ricinus) is the most common tick species in Europe. It is also a vector of the causative agents of Lyme borreliosis and tick-borne encephalitis, which are two of the most important arthropod-borne diseases in Europe. In recent years, increases in tick activity and incidence of tick-borne diseases have been observed in many European countries. These increases are linked to many ecological and anthropogenic factors such as landscape management, climate change, animal migration, and increased popularity of outdoor activities or changes in land usage. Tick activity is driven by many biotic and abiotic factors, some of which can be effectively managed to decrease risk of tick bites. In the USA, recommendations for landscape management, tick host control, and tick chemical control are well-defined for the applied purpose of reducing tick presence on private property. In Europe, where fewer studies have assessed tick management strategies, the similarity in ecological factors influencing vector presence suggests that approaches that work in USA may also be applicable. In this article we review key factors driving the tick exposure risk in Europe to select those most conducive to management for decreased tick-associated risk.

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 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′-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 with 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 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′- C -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. 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 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′-CMA but also to a broad range of other 2′- C -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.