Characterization of West Nile virus Koutango lineage from Phlebotomine Sandflies in Kenya 2021

The West Nile virus (WNV), primarily transmitted by mosquitoes, is one of the most widespread flaviviruses globally, with past outbreaks occurring in the USA and Europe. Recent studies in parts of Africa, including Kenya, have identified the West Nile virus Koutango lineage (WN-KOUTV) among phlebotomine sandfly populations, however, our understanding of this virus remains limited. Hence, this study aimed to characterize WN-KOUTV from phlebotomine sandflies. Sandflies were sampled between 12-16 ^th March 2021 from six villages in Baringo South, Kenya, using CDC light traps. Female sandflies were taxonomically identified and pooled based on genus. Virus isolation was performed in Vero cells. Viral genome was determined using next-generation sequencing. Phylogenetic and molecular clock analyses were done to decipher the virus’s evolutionary relationships. Comparative analyses of amino acid sequences were performed to determine variations. Protein modeling in Pymol was conducted to elucidate variations in key protein regions. Evolutionary pressure analysis investigated the selection pressures on the virus. In vitro experiments were done to investigate the virus growth kinetics in mammalian (Vero-E6) and mosquito (C636) cells. We report the isolation of WN-KOUTV from Salabani Baringo South, Kenya. The isolated WN-KOUTV clustered with previously identified WN-KOUTV strains. Comparative analysis revealed unique amino acid at NS5 653. Diversifying pressure was acting NS3 267 of the WN-KOUTV lineage. WN-KOUTV replicates efficiently in Vero-E6 and C636 cells comparable to West Nile virus Lineage 1a, isolated from mosquitoes. The isolation of WN-KOUTV in sandflies points to them as potential vectors, however, vector competence studies would confirm this. The efficient replication in mammalian and mosquito cell lines elucidated its adaptability to host and vector. We speculate the close genetic relationship of WN-KOUTV strains is enabled by the bird migratory route between East and West Africa. If proven, this may point to a potential future pandemic pathway for this virus.