Detection and Characterization of West Nile Virus with evidence of Transovarial Transmission in the Coastal region, Kenya

Background West Nile virus (WNV) is a mosquito-borne flavivirus of global public health significance, maintained in an enzootic cycle between birds and mosquitoes, with humans and other mammals as incidental hosts. Understanding WNV circulation in diverse mosquito populations is critical for predicting and mitigating outbreaks. This study investigated mosquito populations from coastal Kenya, where WNV was detected, genetically characterized, and evidence of transovarial transmission in Aedes aegypti was observed. Methods Mosquitoes were collected from Kwale, Kilifi, Mombasa, and Isiolo counties (n=14,105) and pooled by species and location (1,596 pools). Pools were inoculated on Vero E6 cells, followed by RNA extraction, Illumina MiSeq sequencing, and preliminary analysis on CZ-ID. Reads were quality-controlled (PrinseqLite v0.20.4), assembled de novo (MEGAHIT v1.2.9), and analyzed via BLAST. Phylogenetic reconstruction used Maximum Likelihood, and codon-level selection pressure was evaluated using FEL, MEME, and FUBAR on Datamonkey. Results WNV was detected in ten pools: eight Lineage 1a and two Lineage 2. Virus isolates came from Culex pipiens , Culex univittatus , Anopheles funestus , Aedes aegypti , and Eretmapodites chrysogaster . Notably, one Lineage 1a isolate from a male Aedes aegypti confirmed transovarial transmission. Six codons were under diversifying selection, NS2B gene was found to carry the V103A mutation. Conclusion WNV in coastal Kenyan mosquitoes revealed transovarial transmission in Aedes aegypti . Lineage-specific evolution, codons under positive selection, and the NS2B:V103A mutation demonstrate ongoing viral adaptation. These findings highlight the need for continued genomic surveillance and targeted vector studies to guide WNV control strategies in Kenya and the wider region. Importance West Nile virus (WNV) remains a globally important arbovirus, yet genomic and experimental data from under-sampled regions such as coastal East Africa are limited. This study provides the first integrated molecular and genotypic characterization of WNV circulating along the Kenyan coast, revealing the co-detection of Lineage 1a and the first identification of Lineage 2 in this region. By combining field surveillance, whole-genome sequencing, evolutionary analyses, and lineage-specific replication assays across multiple vertebrate and mosquito cell lines, we demonstrate clear genetic and biological differences with implications for transmission and adaptation. Importantly, the detection of WNV in a male Aedes aegypti mosquito and recovery of full genomes offers compelling evidence of transovarial transmission, a mechanism that may support viral maintenance independent of vertebrate hosts. These findings expand current knowledge of WNV ecology in Africa and underscore the need for continued genomic surveillance to detect emerging variants and inform public health strategies.