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Enhancing African horse sickness virus detection: comparing and adapting PCR assays.
Abstract: African horse sickness (AHS) is the only equine disease for which the World Organisation for Animal Health (WOAH) gives official disease-free status, given that it poses a major threat to the equine industry. The disease is caused by AHS virus (AHSV; family , taxon species ), which is endemic in sub-Saharan Africa. Reverse-transcription quantitative real-time PCR (RT-qPCR) is a rapid, sensitive detection method used in the diagnosis of AHS and the certification of animals as negative for AHSV for the purpose of movement. Genetic variability of AHSV may influence the accuracy of RT-qPCR detection methods because of possible mispriming and/or probe binding failures. We evaluated the diagnostic accuracy of the current WOAH-recommended RT-qPCR assays for the detection of AHSV, namely the Agüero et al. and Guthrie et al. methods. Utilizing 150 AHSV-positive diagnostic samples, we performed in vitro analysis using both assays. The Agüero assay failed to detect AHSV in 13 samples (8.7% false-negative rate). The AHSV VP7 genes of the 13 negative samples, and publicly archived sequences were used to perform in silico analysis, and we incorporated minor changes into the primers and probes of modified Guthrie and modified Agüero assays. A second in vitro analysis yielded 100% sensitivity for both assays. Differences in both the in silico and in vitro analyses highlight the need for continuous monitoring of the efficacy of molecular protocols used for the detection of AHSV.
Publication Date: 2026-02-07 PubMed ID: 41653013PubMed Central: PMC12882837DOI: 10.1177/10406387261417355Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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Overview
- This research compares and improves PCR tests for detecting African horse sickness virus (AHSV), a serious disease affecting horses, by addressing genetic variability that causes false negatives in current diagnostic assays.
Background
- African horse sickness (AHS): A severe viral disease affecting horses, with significant economic and animal health implications.
- World Organisation for Animal Health (WOAH): Recognizes AHS as the only equine disease with official disease-free status certification.
- Virus: African horse sickness virus (AHSV) belongs to a specific virus family and taxon species and is endemic primarily in sub-Saharan Africa.
- Importance of detection: Rapid and sensitive methods are critical to diagnose AHS and certify animals as free from infection, particularly for animal movement and trade.
Current Detection Methods and Limitations
- Reverse-transcription quantitative real-time PCR (RT-qPCR): The standard diagnostic tool for AHSV detection, recommended by WOAH.
- Assays tested: The study focused on two WOAH-recommended RT-qPCR assays designed by Agüero et al. and Guthrie et al.
- False negatives due to genetic variability: Mutations in the virus’s VP7 gene can affect primer and probe binding in RT-qPCR, leading to missed detections (false negatives).
Study Methods
- Sample collection: 150 AHSV-positive diagnostic samples were used to evaluate assay performance.
- Initial in vitro testing: Both Agüero and Guthrie assays were performed on the samples to assess detection sensitivity.
- Observation of failures: The Agüero assay failed to detect AHSV in 13 samples, revealing an 8.7% false-negative rate.
- Genetic analysis: VP7 gene sequences from the 13 false-negative samples and publicly available AHSV sequences were analyzed in silico (computer-based simulation) to identify sequence variations causing failure.
Assay Modifications and Reassessment
- Primer and probe redesign: Minor modifications were made to primers and probes in both the Agüero and Guthrie RT-qPCR assays to better match the viral genetic variations.
- Second in vitro testing: The modified assays were again evaluated on the sample set.
- Improved sensitivity: Both modified assays achieved 100% sensitivity, successfully detecting AHSV in all samples.
Conclusions and Implications
- Genetic variability impact: Viral mutations can compromise diagnostic PCR accuracy, leading to false negatives with existing protocols.
- Need for continual assay evaluation: The study highlights the importance of regularly monitoring and updating molecular diagnostic tools to maintain high detection sensitivity.
- Enhanced detection reliability: By adapting primers and probes, the accuracy of AHSV detection can be significantly improved, supporting effective disease surveillance and control.
- Broader impact: The approach may serve as a model for maintaining robust detection methods for other viruses with genetic variability.
Cite This Article
APA
Penzhorn L, Crafford JE, Guthrie AJ.
(2026).
Enhancing African horse sickness virus detection: comparing and adapting PCR assays.
J Vet Diagn Invest, 10406387261417355.
https://doi.org/10.1177/10406387261417355 Publication
Researcher Affiliations
- Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa.
- Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa.
- Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa.
Conflict of Interest Statement
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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