Isolation and molecular characterization of West Nile Virus with evidence of vertical transmission in the Coastal region, Kenya

ABSTRACT West Nile virus (WNV) is a mosquito-borne flavivirus of global public health importance, maintained in an enzootic cycle between birds and mosquitoes, with humans and other mammals as incidental hosts. Although WNV has been documented in Kenya, surveillance remains inconsistent, geographically fragmented, and largely dependent on serological methods. This limits understanding of circulating lineages, vector diversity, and transmission dynamics, particularly in coastal regions. This study aimed to address these gaps through systematic arboviral surveillance of mosquito populations across four ecologically distinct Kenyan counties using an integrated genomic approach. Mosquitoes were collected using CDC miniature light traps 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. 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 vertical transmission. Six codons were under diversifying selection, and the NS2B gene was found to carry the V103A mutation. This study provides genomic evidence of active WNV circulation in the coastal region, revealing co-circulation of two lineages across ecologically diverse vector species. These findings underscore the need for sustained genomic surveillance, expanded vector competence studies, and integrated arbovirus monitoring to inform targeted prevention and control strategies in Kenya and the broader 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 vertical 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.