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Virology journal2025; 22(1); 318; doi: 10.1186/s12985-025-02918-0

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

Abstract: 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. 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. Conclusions: 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.
© 2025. The Author(s).
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Publication Date: 2025-10-01 PubMed ID: 41035042PubMed Central: PMC12486666DOI: 10.1186/s12985-025-02918-0Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study reports the first detection and genetic analysis of West Nile virus (WNV) infections in horses in Tunisia between 2018 and 2023.
  • It emphasizes the presence and genetic characteristics of WNV in Tunisian horses, demonstrating its potential as an early warning for human outbreaks.

Introduction to West Nile Virus and its Significance

  • West Nile virus (WNV) is an arthropod-borne virus, primarily transmitted by mosquitoes.
  • It is part of the encephalitic Orthoflaviviruses group and is widely distributed across different geographical regions.
  • WNV infection can lead to severe neurological disease in both humans and horses.
  • Due to its impact on both public health (humans) and veterinary health (animals such as horses), WNV is considered an emerging infectious disease threat.
  • Monitoring WNV in horses can act as an important sentinel system to predict and control potential human outbreaks.

Research Aim and Sample Collection

  • The study aimed to investigate suspected cases of WNV infections in horses in Tunisia over five years, from 2018 to 2023.
  • A total of 25 samples were gathered from clinically affected horses or those that had died; the samples included blood and brain tissue.
  • These samples were collected from various locations across Tunisia to provide a broad representation of the virus’s local circulation.

Methodology

  • Each of the 25 horse samples was screened for WNV genetic material by targeting the NS2A gene using quantitative reverse transcription polymerase chain reaction (qRT-PCR).
  • Samples that tested positive (11 out of 25) underwent further analysis: virus isolation using Vero cells, a standard cell line to grow viruses.
  • Partial sequencing was performed on the envelope (E) glycoprotein gene of the virus from these positive isolates.
  • The E protein gene sequences were then used for molecular and phylogenetic analyses to determine the genetic relationships among the isolates and compare them to known WNV strains globally.

Results

  • Out of the 25 samples tested, 11 (44%) were positive for WNV infection.
  • Sequencing of the envelope protein gene revealed all Tunisian isolates belonged to lineage 1a of WNV.
  • Phylogenetic analyses showed that these isolates formed a distinct cluster within the Mediterranean subtype of clade 1a, suggesting a locally circulating virus strain.
  • The Tunisian isolates had a potential N-linked glycosylation site at amino acid residues 154-156 (Asn-Tyr-Ser, referred to as NYS), which is a biochemical feature possibly influencing viral infectivity or immune evasion.
  • Importantly, none of the isolates carried mutations E-I159V or E-I159A, which are mutations observed in some other WNV strains and may affect viral properties.

Conclusions and Implications

  • This is the first reported surveillance confirming the circulation of WNV in horses throughout several Tunisian regions.
  • The study highlights the significance of genomic monitoring in horses as an early indicator of potential WNV activity in the environment.
  • Because WNV infection in horses can precede human cases, prompt investigation and continued surveillance are critical for public health preparedness and response.
  • The genetic characterization of the virus provides valuable data to understand viral evolution and aids in designing diagnostic tools, vaccines, and control measures specific to the Tunisian context.

Cite This Article

APA
Badr C, Handous M, Nsiri J, ElBehi I, Arbi M, Maaroufi A, Bennour MA, Ben Osman R, Dachraoui K, Abbes M, Mahmoudi A, Khosrof I, Abrougui S, Lachheb J, Zhioua E, Larbi I. (2025). First report of West Nile virus infections in horses in Tunisia from 2018 to 2023. Virol J, 22(1), 318. https://doi.org/10.1186/s12985-025-02918-0

Publication

Virology journal
ISSN: 1743-422X
NlmUniqueID: 101231645
Country: England
Language: English
Volume: 22
Issue: 1
Pages: 318
PII: 318

Researcher Affiliations

Badr, Chaima
  • Institut Pasteur de Tunis, Tunis, Tunisia.
  • Tunis El Manar University, Tunis, Tunisia.
Handous, Mariem
  • Institut Pasteur de Tunis, Tunis, Tunisia.
  • Tunis El Manar University, Tunis, Tunisia.
Nsiri, Jihene
  • Institut Pasteur de Tunis, Tunis, Tunisia.
  • Tunis El Manar University, Tunis, Tunisia.
ElBehi, Imen
  • Institut Pasteur de Tunis, Tunis, Tunisia.
  • Tunis El Manar University, Tunis, Tunisia.
Arbi, Marwa
  • Institut Pasteur de Tunis, Tunis, Tunisia.
  • Tunis El Manar University, Tunis, Tunisia.
Maaroufi, Abderrazak
  • Institut Pasteur de Tunis, Tunis, Tunisia.
  • Tunis El Manar University, Tunis, Tunisia.
Bennour, Mohamed Ali
  • Ministry of Agriculture, Tunis, Tunisia.
Ben Osman, Raja
  • National Drug Control Laboratory, Vaccine Control Unit, Tunis, National Drug Control Laboratory, Vaccine Control Unit, Tunis, Tunisia.
Dachraoui, Khalil
  • Institut Pasteur de Tunis, Tunis, Tunisia.
  • Tunis El Manar University, Tunis, Tunisia.
Abbes, Mondher
  • Minsitry of the Interior, Tunis, Directorate of Specialized Brigades, Ministry of the Interior, Tunis, Tunisia.
Mahmoudi, Anis
  • Minsitry of the Interior, Tunis, Directorate of Specialized Brigades, Ministry of the Interior, Tunis, Tunisia.
Khosrof, Ines
  • Horse Clinic, El Mbarka, Sidi Thabet, Horse Clinic, El Mbarka, Sidi Thabet, Tunisia.
Abrougui, Soufiene
  • Institut Pasteur de Tunis, Tunis, Tunisia.
  • Tunis El Manar University, Tunis, Tunisia.
Lachheb, Jihene
  • Institut Pasteur de Tunis, Tunis, Tunisia.
  • Tunis El Manar University, Tunis, Tunisia.
Zhioua, Elyes
  • Institut Pasteur de Tunis, Tunis, Tunisia.
  • Tunis El Manar University, Tunis, Tunisia.
Larbi, Imen
  • Institut Pasteur de Tunis, Tunis, Tunisia. imen.larbi@pasteur.tn.
  • Tunis El Manar University, Tunis, Tunisia. imen.larbi@pasteur.tn.

MeSH Terms

  • Horses
  • Animals
  • Tunisia / epidemiology
  • West Nile Fever / veterinary
  • West Nile Fever / epidemiology
  • West Nile Fever / virology
  • Horse Diseases / virology
  • Horse Diseases / epidemiology
  • West Nile virus / genetics
  • West Nile virus / isolation & purification
  • West Nile virus / classification
  • Phylogeny
  • Retrospective Studies
  • Viral Envelope Proteins / genetics
  • Chlorocebus aethiops
  • Vero Cells
  • Viral Nonstructural Proteins / genetics

Grant Funding

  • LR 16IPT03 / Tunisian Ministry of Higher Education and Scientific Research and was a part of the research program of the Laboratory of Epidemiology and Veterinary Microbiology. Institut Pasteur de Tunis.
  • LR 16IPT03 / Tunisian Ministry of Higher Education and Scientific Research and was a part of the research program of the Laboratory of Epidemiology and Veterinary Microbiology. Institut Pasteur de Tunis.
  • LR 16IPT03 / Tunisian Ministry of Higher Education and Scientific Research and was a part of the research program of the Laboratory of Epidemiology and Veterinary Microbiology. Institut Pasteur de Tunis.
  • LR 16IPT03 / Tunisian Ministry of Higher Education and Scientific Research and was a part of the research program of the Laboratory of Epidemiology and Veterinary Microbiology. Institut Pasteur de Tunis.
  • LR 16IPT03 / Tunisian Ministry of Higher Education and Scientific Research and was a part of the research program of the Laboratory of Epidemiology and Veterinary Microbiology. Institut Pasteur de Tunis.
  • LR 16IPT03 / Tunisian Ministry of Higher Education and Scientific Research and was a part of the research program of the Laboratory of Epidemiology and Veterinary Microbiology. Institut Pasteur de Tunis.
  • LR 16IPT03 / Tunisian Ministry of Higher Education and Scientific Research and was a part of the research program of the Laboratory of Epidemiology and Veterinary Microbiology. Institut Pasteur de Tunis.
  • LR 16IPT03 / Tunisian Ministry of Higher Education and Scientific Research and was a part of the research program of the Laboratory of Epidemiology and Veterinary Microbiology. Institut Pasteur de Tunis.
  • LR 16IPT03 / Tunisian Ministry of Higher Education and Scientific Research and was a part of the research program of the Laboratory of Epidemiology and Veterinary Microbiology. Institut Pasteur de Tunis.
  • LR 16IPT03 / Tunisian Ministry of Higher Education and Scientific Research and was a part of the research program of the Laboratory of Epidemiology and Veterinary Microbiology. Institut Pasteur de Tunis.

Conflict of Interest Statement

Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests. Ethics declarations: Not applicable.

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