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Microorganisms2025; 13(12); 2684; doi: 10.3390/microorganisms13122684

Modification and Validation of a Reference Real-Time RT-PCR Method for the Detection of a New African Horse Sickness Virus Variant.

Abstract: African horse sickness (AHS) is a disease affecting equids caused by the AHS virus (AHSV). The World Organisation for Animal Health (WOAH) includes AHS as a notifiable disease and, upon detection within the European Union, immediate control and eradication measures are mandated. Thus, validated diagnostic methods for rapid AHSV detection are essential. The Agüero 2008 and Guthrie 2013 rRT-PCR methods have been widely validated for detection of any AHSV strain and are included as reference rRT-PCRs in the WOAH manual. However, the WOAH Reference Laboratory for AHS in the Republic of South Africa (RSA) reported an AHSV variant undetected by the Agüero 2008 rRT-PCR. Therefore, a set of modified primers and probe, containing degenerate positions to avoid mismatches with the sequence of the new RSA strain, was developed. The modified-Agüero method was validated by the WOAH Reference Laboratories in Spain and the UK, employing a broad collection of AHSV strains and clinical samples as well as a synthetic RNA mimicking the target sequence of the new RSA AHSV variant (AHSV-sRNA-RSA). Comparative assessment of the modified-Agüero versus the WOAH reference rRT-PCRs showed that the modified method exhibited good diagnostic performance and enabled detection of the new RSA AHSV variant nucleic acid.
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Publication Date: 2025-11-25 PubMed ID: 41471886PubMed Central: PMC12735099DOI: 10.3390/microorganisms13122684Google Scholar: Lookup
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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 research focused on improving and validating a real-time RT-PCR diagnostic method to detect a newly discovered variant of the African horse sickness virus (AHSV) that previous tests failed to identify.
  • The modified test was validated by international laboratories and demonstrated reliable detection of various AHSV strains including the new variant, supporting its use for rapid diagnosis essential for disease control.

Background on African Horse Sickness and Diagnostic Challenges

  • African Horse Sickness (AHS): A fatal viral disease affecting horses and other equids, caused by the African Horse Sickness Virus (AHSV).
  • Regulatory Importance: The World Organisation for Animal Health (WOAH) classifies AHS as a notifiable disease, requiring immediate measures to control and eradicate the virus if detected, especially within the European Union.
  • Need for Rapid Detection: Quick and accurate identification of AHSV is crucial for timely quarantine and outbreak management to prevent spread.

Existing Diagnostic Methods and their Limitations

  • Agüero 2008 and Guthrie 2013 rRT-PCR assays: These are established real-time reverse transcription PCR methods widely validated and recommended by WOAH for detecting AHSV strains.
  • Detection Issue: The WOAH Reference Laboratory in South Africa identified a novel AHSV variant that was not detected by the Agüero 2008 assay due to genetic mismatches in the viral sequence.
  • Impact: This raised concerns about the assay’s sensitivity and the risk of false negatives, which could compromise outbreak control efforts.

Modification of the Agüero 2008 rRT-PCR Method

  • Primer and Probe Redesign: The researchers introduced degenerate nucleotide positions into the primers and probe sequences to accommodate genetic variations found in the new RSA strain.
  • Purpose of Degenerate Positions: These allow the assay to hybridize effectively with multiple viral genetic sequences, including the new variant, enhancing detection capability.
  • Termed “Modified-Agüero Method”: This updated assay was tailored specifically to detect both existing and new strains of AHSV.

Validation Process

  • Collaborations with WOAH Reference Laboratories: Two international reference labs in Spain and the UK conducted thorough assessments of the modified assay.
  • Sample Diversity: Validation used a wide collection of AHSV strains, including diverse clinical samples representing naturally infected horses.
  • Synthetic RNA Control: A synthetic RNA (AHSV-sRNA-RSA) mimicking the new RSA variant’s target sequence was employed to test and confirm assay sensitivity and specificity.

Results and Implications

  • Diagnostic Performance: The modified-Agüero assay showed strong performance characteristics, including sensitivity and specificity, comparable to or exceeding existing reference tests.
  • Detection of New Variant: It successfully detected nucleic acids from the new RSA variant that the original Agüero 2008 assay missed.
  • Broader Application: The assay retained its ability to detect a broad range of AHSV strains, ensuring broad usability for surveillance and diagnosis worldwide.
  • Regulatory and Practical Impact: This validated update enhances the global toolkit for controlling African horse sickness by providing a reliable, rapid diagnostic test tuned to emerging viral variants.

Cite This Article

APA
Morales J, Ruano MJ, Tena-Tomás C, van Schalkwyk A, Loundras EA, Valero-Lorenzo M, López-Herranz A, Romito M, Batten C, Villalba R, Agüero M. (2025). Modification and Validation of a Reference Real-Time RT-PCR Method for the Detection of a New African Horse Sickness Virus Variant. Microorganisms, 13(12), 2684. https://doi.org/10.3390/microorganisms13122684

Publication

Microorganisms
ISSN: 2076-2607
NlmUniqueID: 101625893
Country: Switzerland
Language: English
Volume: 13
Issue: 12
PII: 2684

Researcher Affiliations

Morales, Jorge
  • Laboratorio Central de Veterinaria, Ministry of Agriculture, Fisheries and Food, 28110 Algete, Spain.
Ruano, María José
  • Laboratorio Central de Veterinaria, Ministry of Agriculture, Fisheries and Food, 28110 Algete, Spain.
Tena-Tomás, Cristina
  • Tecnologías y Servicios Agrarios, S.A. (TRAGSATEC), 28037 Madrid, Spain.
van Schalkwyk, Antoinette
  • Agricultural Research Council, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa.
Loundras, Eleni-Anna
  • The Pirbright Institute, Woking GU24 0NF, UK.
Valero-Lorenzo, Marta
  • Laboratorio Central de Veterinaria, Ministry of Agriculture, Fisheries and Food, 28110 Algete, Spain.
López-Herranz, Ana
  • Laboratorio Central de Veterinaria, Ministry of Agriculture, Fisheries and Food, 28110 Algete, Spain.
Romito, Marco
  • Agricultural Research Council, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa.
Batten, Carrie
  • The Pirbright Institute, Woking GU24 0NF, UK.
Villalba, Rubén
  • Laboratorio Central de Veterinaria, Ministry of Agriculture, Fisheries and Food, 28110 Algete, Spain.
Agüero, Montserrat
  • Laboratorio Central de Veterinaria, Ministry of Agriculture, Fisheries and Food, 28110 Algete, Spain.

Grant Funding

  • Project: 101200696 - EURL-AHS-BT 2025-2027 - SMP-FOOD-2025-EURL-EURC-PJG-IBA. / This research was funded by the Spanish Ministry of Agriculture (MAPA) and the European Health and Digital Executive Agency (HADEA), EU executive agency under the powers delegated by the European Commission.

Conflict of Interest Statement

Author Cristina Tena-Tomás was employed by the Tecnologías y Servicios Agrarios, S.A. (TRAGSATEC). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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