Dynamics of West Nile Virus Lineage 2 Spread in the Balkans in the Context of Global Spatio-Temporal Dispersal

West Nile Virus (WNV) is considered one of the most widely distributed arboviruses worldwide. In 2018, Serbia was among the European countries reporting the highest number of WNV cases. This study aimed to characterize WNV strains circulating in Serbia, and to estimate the pathways and dynamics of WNV-2 spread in the Balkans and globally through the phylogenetic approach. Fifty newly generated NS5 Serbian sequences were found to belong to WNV lineage 2. Phylodynamic analyses of the Balkan clade indicated the potential for an increase in genetic diversity and structure of virus populations. Presented phylogeographic analyses implied four instances of long-distance WNV-2 migration from Africa to Europe, during the 1940s to 1950s, while further dissemination of WNV-2 originated in Hungary in mid-1970s and subsequently spread to Austria and Serbia. Extensive dispersion took place during the 1990s–2000s, as the virus spread from Austria to central and western Europe, and from Hungary to the Balkans. Continuous phylogeography analysis of the Balkans WNV-2 clade implied the central role of Serbia for WNV dissemination across the Balkan peninsula. Furthermore, previously undocumented instance of intercontinental migration of WNV-2 from Europe to Asia was implied. In-depth phylogenetic investigation into the global distribution of WNV-2 may provide valuable insights into the prediction and prevention of potential epidemics.