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

Abstract

West Nile virus (WNV, family Flaviviridae) 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 Culex pipiens f. pipiens 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.

Keywords: Culex; Kosovo; West Nile virus; monitoring; phylogeny.

Figure 1

Geographical position of the Republic of Kosovo (dark grey) in the Balkan Peninsula.

Figure 2

Weekly Cx. pipiens s.l. activity at the study site. WNV positive pool indicated by virus symbol. Temperature data were obtained from the Hydrometeorological Institute of Kosovo.

Figure 3

Maximum likelihood phylogeny of selected West Nile virus lineage 2 strains, including the sequence from a pool of Cx. pipiens f. pipiens captured in Prishtina, Kosovo, 2022. The consensus tree was inferred over 1000 ultrafast bootstrap replicates in IQTree2 using the TIM2+F+R2 substitution model on an alignment of the complete coding sequence. Bootstrap supports (>60%) are given above branches. Sequences originating from humans in Kosovo, 2018, are highlighted with black circles (previous studies) and from a pool of mosquitoes in Kosovo, 2022, with a black triangle (2022). Sequences indicated by grey circles were used as references to compare sequence similarity (Supporting Information 1: Appendix S1). The scale bar shows substitutions per site over the 10,302 bp open reading frame.