Tracing the 2021 equine influenza Outbreak: First characterization and phylogeographic analysis of H3N8 Florida clade 1 virus in Tunisia.

Overview

• This study examined the 2021 outbreak of equine influenza (EI) in Tunisia by identifying the virus strain, analyzing its genetic makeup, and tracing its geographic origins to understand how it spread and to inform future surveillance efforts.

Background

• Equine influenza (EI) is a highly contagious respiratory disease in horses and related animals (equids), capable of causing major outbreaks worldwide.
• In spring 2021, an EI outbreak occurred in Tunisia in a farm with purebred Arabian horses, showing clinical signs such as respiratory distress.
• Due to this outbreak, national restrictions on horse movements and cancellation of equestrian events were implemented to control the spread.

Sample Collection and Testing

• A total of 432 serum samples collected from 2018 to 2021 were tested for antibodies against EI using an ELISA competition assay to assess prior exposure or infection.
• During the 2021 outbreak, 100 nasal swab samples were collected from symptomatic horses and tested for the presence of EI viral RNA using quantitative reverse transcription PCR (qRT-PCR), a method to detect viral genetic material.

Genetic Sequencing and Analysis

• Five positive samples from 2021 underwent full genetic sequencing, focusing on two key viral genes: hemagglutinin (HA) and neuraminidase (NA), which are important for virus infectivity and antigenic properties.
• Phylogenetic analysis was performed using Bayesian statistical methods. This allowed researchers to understand the evolutionary relationships of the Tunisian virus strains compared to others worldwide.
• Phylogeographic analysis integrated genetic data with geographical information, helping to trace the probable origin and spread of the virus into Tunisia.

Findings

• Out of 432 serum samples tested over four years, 80 (18.51%) were positive for EI antibodies, indicating exposure in the horse population, with peak seroprevalence in 2018 and 2021 correlating with outbreak occurrences.
• The virus strain responsible for the 2021 outbreak was identified as H3N8 subtype, belonging to the American lineage, specifically Florida clade 1.
• Genetic sequences of Tunisian strains showed close similarity to strains isolated in Algeria, the USA, France, Saudi Arabia, and the UK, indicating potential multiple sources or shared transmission routes.
• Phylogeographic analysis suggested that the virus likely entered Tunisia from the USA and France around 2017-2018, before causing the 2021 outbreak.

Implications

• The identification of the H3N8 Florida clade 1 strain highlights the importance of continuous monitoring of EI virus strains to detect and respond to emerging variants promptly.
• Understanding the geographic origin and genetic relationships of EI viruses aids in tracking virus evolution and spread, which is critical for controlling outbreaks and preventing international dissemination.
• Implementing molecular surveillance and rapid diagnostic testing supports timely interventions such as movement restrictions and vaccination strategies to curb virus transmission.

Conclusion

• This comprehensive study presents the first genetic characterization and phylogeographic tracing of the 2021 EI outbreak in Tunisia, providing insights into the virus’s origin and spread patterns.
• The findings underscore the necessity for ongoing surveillance and molecular characterization of EI viruses, especially considering their high contagion potential and the risk they pose to equine populations worldwide.