Background: Equine influenza virus (EIV) H3N8 is a highly contagious respiratory pathogen that poses significant health and economic risks to equids globally. In southeastern Nigeria where equids are sold and slaughtered, limited data exist on EIV epidemiology and circulating lineages.

Methods: To address this gap, an active surveillance was conducted between January 2022 and October 2023. A total of 400 nasal swabs were collected from horses and donkeys at slaughterhouses and animal markets. The swabs were screened for equine influenza virus (EIV) using quantitative Reverse-Transcription Polymerase Chain Reaction (RT-qPCR), and positive samples underwent whole-genome sequencing. A spatiotemporal Bayesian phylogeographic analysis was performed. Amino acid comparisons were carried out against the World Organization for Animal Health (WOAH) recommended Florida clade-1 (Fc-1) vaccine strains (accession numbers GU447312, DQ124192) and mutations were mapped onto 3D H3 hemagglutinin structure with protein data bank 4UO0 using PyMOL.

Results: Two samples (0.5%) from non clinical signs and deceased donkeys tested positive for the H3N8 virus. A spatiotemporal Bayesian phylogeographic analysis, which included sequences from outbreaks in Africa between 2018 and 2023, revealed multiple introductions of the virus into Africa. The introductions of Fc-1 lineage into Africa may have originated from Argentina (2018/2019) and the UK (2021), while Florida clade-2 seems to have originated from Ireland (2019). The 2022 H3N8 strains identified in this study may be a result of persistence from the 2018/2019 epizootic in northern Nigeria. Additionally, we discovered previously unreported hemagglutinin substitutions compared to the WOAH recommended Fc-1 vaccine strain, along with novel changes adjacent to antigenic sites and four distinct glycosylation profiles in the virus, which underscores their potential epidemiological significance.

Conclusions: Our findings revealed multiple introductions of EIV probably from South America and Western Europe, rapid virus evolution, and significant transboundary spread facilitated by livestock trade, particularly involving donkeys and subclinical infections in the transmission of the virus. These results underscore the persistence and evolution of EIV H3N8 (Fc-1) in Nigeria and emphasize the need for improved genomic surveillance, control measures, and vaccination strategies against EIV in Africa. Additionally, regulating transboundary livestock trade is essential to mitigate the risk of future outbreaks.