First Detection of West Nile Virus (WNV) Lineage 2 in Mosquitoes in the Republic of Kosovo

West Nile virus (WNV, family <i>Flaviviridae</i>) is the most geographically widespread arbovirus affecting humans. It circulates between wild birds and mosquitoes, while humans and horses are dead-end hosts. In recent years, several outbreaks have been reported from European countries, including the Balkan Peninsula. In the Republic of Kosovo, a southern Balkan country, data on WNV are scarce, and neither mosquito monitoring nor WNV surveillance is established. To address this gap, we aimed to assess a first monitoring approach that should set the basis and support future large-scale activities in the country. Mosquito sampling was performed from May to September 2022 in a peri-urban area in the western part of the capital city Prishtina, Republic of Kosovo. Collected mosquitoes were pooled, homogenized, and total nucleic acid was extracted. A WNV-DENV-ZIKV-specific multiplex RT-qPCR was applied, and WNV-positive samples were confirmed by RT-PCR and whole-genome sequencing. Of 44 screened pools, one pool molecularly identified as <i>Culex pipiens</i> f. <i>pipiens</i> was positive for WNV RNA. Subsequent sequencing revealed WNV lineage 2, and phylogenetic analysis included our sample in a monophyletic clade consisting mostly of sequences from southeastern Europe. This finding represents the first detection of WNV in mosquitoes in Kosovo, and provides crucial baseline data for future vector-borne disease monitoring, and control efforts in Kosovo.