Molecular Characterization of RNA Viruses in Sandflies from Select Regions in Kenya

Abstract

Phlebotomine sandflies are vectors of arboviruses from genera Phlebovirus, Vesiculovirus, and Orbivirus that cause febrile and neurological illnesses in humans and animals. However, the diversity and public health significance of Phelebotomine sandfly-associated viruses in Sub-Saharan Africa remain poorly understood, representing a critical surveillance gap. This retrospective study used metagenomic, virus isolation, next-generation sequencing, and metabarcoding to address this gap by characterizing RNA viruses and their sandfly vectors in Kenya, where unexplained febrile illnesses persist. Female sandflies, previously collected between September 2020 and July 2022 from Baringo, West Pokot, Nakuru, Kisumu, Kilifi, and Kwale counties, had been stored at –80°C to preserve RNA integrity prior to pooling and homogenization. Virus isolation was performed in Vero cells, with cytopathic effect

(CPE)-positive samples subjected to next-generation sequencing. Supernatant super- pools were screened for RNA viruses using metagenomics, while genomic DNA

from homogenate pellets of arbovirus-positive pools enabled sandfly species identification via metabarcoding. Metagenomic analysis revealed a diverse virome, including, West Nile virus Koutango lineage (WN-KOUTV), Bogoria virus (BOGV), Phlebotomine rhabdovirus and several insect-specific viruses from five families: Dicistroviridae, Chuviridae, Iflaviridae, Nodaviridae, and Negevirus. Genomic characterization of isolated WN-KOUTV and BOGV provided new insights into their phylogeny and potential public health relevance. Additionally, metabarcoding identified Sergentomyia species, including Sergentomyia schwetzi, Sergentomyia inermis, Sergentomyia sp. SERG.UNK. F. 1- 8, Sergentomyia squamipleuris, and other Phlebotominae species within each pool. The results highlight the ongoing circulation of medically important and insect-specific viruses in Kenyan sandflies, emphasizing the need for continued surveillance and vector control strategies to address undiagnosed febrile illnesses. These findings also demonstrate the utility of combining metagenomics and metabarcoding with traditional virological methods for comprehensive arbovirus surveillance.