Evolutionary dynamics and phylogeographic analysis of West Nile virus lineage 2 in the Russian Federation

West Nile virus is one of the most significant arboviruses circulating in the Russian Federation. Although WNV is classified into nine lineages, only lineage 2 has caused most outbreaks in Europe and Russia in recent years. Since 2018, molecular genetic surveillance has been conducted, but the evolutionary dynamics and routes of introduction and spread of WNV lineage 2 in Russia remain incompletely investigated. This study aimed to reconstruct the origin, migration routes, and evolutionary processes that drive the genetic diversity of WNV lineage 2 in Russia. The results showed that the WNV lineage 2 population circulating in Russia is genetically heterogeneous. It consists of four distinct genetic variants. Phylogeographic reconstruction allowed estimation of the migration routes and introduction dates for each variant. The viral diffusion rate was estimated at 92 km per year. Two major routes of introduction were identified: a southern route from Africa via the southern coast of the Black Sea, and a western route from Europe through Central and Eastern European countries, including Hungary and Romania. The observed dispersal patterns suggest that WNV lineage 2 circulation in Russia is maintained by repeated introductions along African and European transboundary routes. This contributes to the expansion of its geographic range. Selection pressure analysis showed that most codons were under strong purifying selection. Fourteen sites in structural and nonstructural proteins were under positive selection, including unique substitutions in NS4B, NS5, and E proteins. These may play a role in viral adaptation.