First report of West Nile virus infections in horses in Tunisia from 2018 to 2023.

Background: West Nile virus (WNV) is an arthropod borne virus, the most widely distributed of the encephalitic Orthoflaviviruses. It can cause severe neurological symptoms in both humans and horses. It poses an emerging threat to both public and animal health. In this study, we retrospectively screened 25 suspected WNV samples collected from 2018 to 2023.

Materials and methods: A total number of 25 samples (brain tissue and blood) were collected from clinically affected horses and those already deceased across several locations in Tunisia. All samples were tested for the presence of WNV NS2A gene using qRT-PCR. Eleven positive samples underwent virus isolation in Vero cells and partial sequencing of their envelope (E) glycoprotein gene. The resulting sequences were analyzed to gain molecular and phylogenetic insights.

Results: Eleven of the 25 (44%) samples were WNV positive and 11 partial E protein sequences were taken. The phylogenetic analysis revealed that all Tunisian isolates belonged to lineage 1a and were closely related to each other and formed a distinct group within the Mediterranean subtype of clade 1a. Tunisian WNV were characterized by the presence of a potential N-linked glycosylation site at residue 154-156 (Asn-Tyr-Ser) (NYS) and did not have the mutation E-I159V or E-I159A.

Conclusion: Genomic monitoring of horses has revealed the circulation of WNV in several regions of Tunisia. Our findings highlight the critical need for prompt investigation of WNV infections in horses, as these cases may precede outbreaks of human cases in the country.