Isolation and characterisation of novel fruit bat alphaherpesvirus from Rousettus aegyptiacus bats in Coastal Kenya

Background: Herpesviruses are a group of double-stranded DNA viruses known to infect a wide range of vertebrates and establish life-long latent infections. While bats serve as natural reservoir hosts for numerous viral families, relatively few bat herpesviruses have been successfully isolated. In this study, we report the isolation and characterization of two novel alphaherpesvirus strains obtained from Rousettus aegyptiacus bats in Coastal Kenya. Methods: The samples of oral and rectal swabs were collected from three different species of bats from coastal Kenya between October 2024 and April 2025; the bat species collected include Hipposideros spp., Coleura afra, and Rousettus aegyptiacus. Virus isolation was performed by inoculation of samples in Vero E6 cells and subsequent monitoring for cytopathic effects (CPE). Total nucleic acids were extracted from CPE positive cultures and subjected to library preparation to enable unbiased detection of both RNA and DNA viruses. The libraries were sequenced using next-generation sequencing with Illumina MiSeq platform. Subsequently, bioinformatic analysis was carried out to identify the virus, generate consensus genomes as well as phylogenetic analysis to determine the placement of identified viruses. Results: Two samples from R. aegyptiacus (KIK_460_O and KIK_465_O) induced typical CPE within five days. Sequencing and assembly yielded partial consensus sequences of approximately 60 kb (KIK_460_O) and 70 kb (KIK_465_O), representing extended genomic data for a bat-associated alphaherpesvirus. This virus has a genome of about 140kb, indicating that our partial assemblies account for about 43-50% of the total genome. Both isolates were found to be closely related to Dzifa herpesvirus, an alphaherpesvirus previously identified in Kilifi, Kenya. Alphaherpesvirus was identified based on partial sequencing of UL19 (3,787 bp) and UL30 (2,846 bp) genes. The two isolates were found to be identical at the UL19 gene, showing that they belonged to the same virus strain. Phylogenetic analysis showed that the novel alphaherpesvirus belongs to primate alphaherpesviruses under the subfamily Alphaherpesvirinae. Conclusion: This study reports the isolation and genomic characterization of a novel fruit bat alphaherpesvirus from Kenyan Rousettus aegyptiacus bats. The partial genome assembly (60-70 kb) represent the first extended genomic data for this virus, covering approximately 43-50% of the estimated 140 kb complete genome. The phylogenetic placement of this alphaherpesvirus near primate viruses, especially Pteropodid alphaherpesvirus 1, suggests bat-association and needs further investigation into its zoonotic potential.