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Home / Equine Research Bank / Viruses / Development and Validation of Three Triplex Real-Time RT-PCR...
Viruses2024; 16(3); 470; doi: 10.3390/v16030470

Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications.

Abstract: The African horse sickness virus (AHSV) belongs to the Genus Orbivirus, family Sedoreoviridae, and nine serotypes of the virus have been described to date. The AHSV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in decreasing size order (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable AHSV protein and the primary target for neutralizing antibodies. Consequently, Seg-2 determines the identity of the virus serotype. An African horse sickness (AHS) outbreak in an AHS-free status country requires identifying the serotype as soon as possible to implement a serotype-specific vaccination program. Considering that nowadays 'polyvalent live attenuated' is the only commercially available vaccination strategy to control the disease, field and vaccine strains of different serotypes could co-circulate. Additionally, in AHS-endemic countries, more than one serotype is often circulating at the same time. Therefore, a strategy to rapidly determine the virus serotype in an AHS-positive sample is strongly recommended in both epidemiological situations. The main objective of this study is to describe the development and validation of three triplex real-time RT-PCR (rRT-PCR) methods for rapid AHSV serotype detection. Samples from recent AHS outbreaks in Kenia (2015-2017), Thailand (2020), and Nigeria (2023), and from the AHS outbreak in Spain (1987-1990), were included in the study for the validation of these methods.
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Publication Date: 2024-03-20 PubMed ID: 38543834PubMed Central: PMC10974454DOI: 10.3390/v16030470Google 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.

African horse sickness virus (AHSV) serotyping is crucial for disease control and vaccination strategies, especially during outbreaks. This study develops and validates three triplex real-time RT-PCR assays to rapidly identify serotypes of AHSV from clinical samples, aiding in effective epidemiological surveillance and targeted vaccination.

Background and Importance

  • African horse sickness virus (AHSV) is a member of the genus Orbivirus within the Sedoreoviridae family and has nine known serotypes.
  • The genome consists of ten double-stranded RNA segments; segment 2 (Seg-2) encodes the outer capsid protein VP2, which is the most variable region and determines serotype.
  • VP2 is the primary target for neutralizing antibodies, making it critical for serotype identification and vaccine development.
  • Rapid serotyping of AHSV is essential during outbreaks to implement serotype-specific vaccine programs efficiently.
  • Currently, only polyvalent live attenuated vaccines are commercially available, which means multiple serotypes can co-circulate, complicating disease control.
  • In endemic countries, simultaneous circulation of several serotypes increases the need for rapid and accurate serotyping tools.

Objectives of the Study

  • Develop three triplex real-time reverse transcription PCR (rRT-PCR) assays that can detect and differentiate AHSV serotypes rapidly.
  • Validate these assays with samples collected from various outbreaks including Kenya (2015-2017), Thailand (2020), Nigeria (2023), and Spain (1987-1990).
  • Provide a tool to facilitate quick epidemiological diagnosis to improve outbreak response and vaccination strategies.

Methodological Approach

  • The assays target segment 2 (Seg-2) of the viral genome responsible for VP2, to exploit its serotype-specific variability.
  • Three triplex assays were designed, likely each capable of identifying three AHSV serotypes in a single reaction, enhancing testing throughput.
  • Validation involved using both recent outbreak samples and archived samples to test the reliability, specificity, and sensitivity of the assays.
  • Real-time RT-PCR was chosen due to its high sensitivity, specificity, and quantitative capabilities for viral RNA detection.

Key Findings and Validation

  • The triplex rRT-PCR assays successfully distinguished between various AHSV serotypes from outbreak samples in different geographic regions and time periods.
  • The assays showed potential for rapid and accurate typing in field and laboratory conditions, essential for controlling outbreak spread.
  • Application to diverse outbreak samples demonstrated robustness against different viral strains, indicating wide utility.
  • Assays can handle co-circulating serotypes’ detection, an important feature for endemic regions and vaccination monitoring.

Implications for Disease Control and Laboratory Applications

  • Rapid serotyping allows for timely deployment of serotype-specific vaccines, critical to reduce morbidity and mortality in horses.
  • Improves outbreak management by enabling epidemiologists and veterinarians to understand serotype distribution promptly.
  • Useful for quality control and differentiation between vaccine strains and field strains during surveillance.
  • Provides a practical molecular diagnostic tool adaptable to various laboratory settings worldwide.
  • Supports global efforts to monitor AHSV spread and emergence of new serotypes or reassortants by facilitating routine screening.

Conclusion

  • This study presents validated triplex rRT-PCR assays that streamline AHSV serotyping from clinical samples.
  • These molecular tools enhance the ability to respond quickly to African horse sickness outbreaks through targeted vaccination and epidemiological tracking.
  • The assays’ broad validation with diverse outbreak samples supports their integration into both endemic and AHS-free country diagnostic workflows.

Cite This Article

APA
Villalba R, Tena-Tomás C, Ruano MJ, Valero-Lorenzo M, López-Herranz A, Cano-Gómez C, Agüero M. (2024). Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications. Viruses, 16(3), 470. https://doi.org/10.3390/v16030470

Publication

Viruses
ISSN: 1999-4915
NlmUniqueID: 101509722
Country: Switzerland
Language: English
Volume: 16
Issue: 3
PII: 470

Researcher Affiliations

Villalba, Rubén
  • 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.
Ruano, María José
  • Laboratorio Central de Veterinaria, Ministry of Agriculture, Fisheries and Food, 28110 Algete, Spain.
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.
Cano-Gómez, Cristina
  • 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.

MeSH Terms

  • Animals
  • Horses
  • African Horse Sickness Virus
  • Reverse Transcriptase Polymerase Chain Reaction
  • African Horse Sickness / diagnosis
  • African Horse Sickness / epidemiology
  • African Horse Sickness / prevention & control
  • Orbivirus / genetics
  • Antibodies, Neutralizing
  • Viral Vaccines

Conflict of Interest Statement

Author Cristina Tena-Tomás was employed by the company Tecnologías y Servicios Agrarios. 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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Citations

This article has been cited 2 times.
  1. 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. Modification and Validation of a Reference Real-Time RT-PCR Method for the Detection of a New African Horse Sickness Virus Variant.. Microorganisms 2025 Nov 25;13(12).
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  2. Ashby M, Moore R, King S, Newbrook K, Flannery J, Batten C. Designing a Multiplex PCR-xMAP Assay for the Detection and Differentiation of African Horse Sickness Virus, Serotypes 1-9.. Microorganisms 2024 May 3;12(5).
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