Synthetic genomics-based generation of the tick-borne encephalitis virus Siberian subtype prototype strain and E51K-attenuated variant for vaccine development and antiviral screening.

Tick-borne encephalitis virus (TBEV) is a re-emerging pathogen in Kazakhstan, where the increasing risk of its spread underscores the need for improved healthcare preparedness, including the development of local vaccines. However, the absence of reference TBEV strains in the country presented a major challenge. To address this, we generated a prototype strain (Vasilchenko) of the Siberian TBEV genotype, predominant in Kazakhstan, using synthetic genome and molecular infectious clone technology. A DNA-launched TBEV molecular clone was assembled from DNA fragments, enabling virus rescue upon plasmid transfection. During the propagation of the post-transfection virus in cell culture, a single amino acid substitution (E51K) in the envelope protein emerged, resulting in a 100-fold increase in the titer of the mutant variant. In vivo, this mutation significantly attenuated virulence: while wild-type TBEV caused 100% mortality in BALB/c mice, the E51K variant was non-lethal and exhibited reduced viremia, suggesting impaired neuroinvasiveness. To further exploit this attenuated, high-titer virus, we developed a GFP-expressing reporter TBEV variant. Using this reporter system, we demonstrated that favipiravir possesses antiviral activity against TBEV, with inhibitory concentrations within a pharmacologically relevant range. In conclusion, synthetic genomics enabled the generation of a reference TBEV strain to replenish Kazakhstan's collections. The E51K mutation enhances viral replication in vitro while attenuating pathogenicity in vivo, and the derived reporter virus is suitable for antiviral compound screening.