Whole-Genome Sequencing and Phylogeographic Analysis of Lassa Virus Strains Circulating in the South West and South East Zones of Nigeria

Lassa virus (LASV) is endemic across West Africa, but its phylogeographic structure and within-host evolutionary dynamics in Nigeria's non-traditionally endemic South West and South East zones remain poorly characterized, limiting understanding of LASV expansion and spillover risk. This study performed whole-genome sequencing and phylogeographic analysis on LASV isolates from confirmed Lassa fever cases in Edo, Ondo, and Ebonyi States, spanning the South West and South East geopolitical zones, over a 36-month surveillance period. Serum RNA from 40 PCR-confirmed LASV cases was subjected to amplicon-based whole-genome sequencing on Illumina and Oxford Nanopore platforms. Maximum likelihood and Bayesian phylogenetic analyses were conducted. Discrete trait ancestral state reconstruction was performed to map spatial diffusion of viral lineages. Temporal signal assessment and molecular clock analysis estimated evolutionary rates and time to most recent common ancestor. All sequenced strains clustered within LASV lineage II, the historically dominant Nigerian clade, with two novel sub-lineages unique to the South East. Bayesian skyline analysis revealed LASV effective population size expansions coinciding with periods of high rodent Mastomys natalensis density estimated from environmental data. Phylogeographic diffusion modeling identified Edo State as a major source for South West dissemination, with three independent introduction events into Ebonyi State. These findings redefine the phylogeographic landscape of Nigerian LASV and identify critical source zones for targeted rodent control and healthcare preparedness. Keywords: Lassa virus, phylogeography, whole-genome sequencing, Nigeria, Mastomys natalensis.