Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine serotypes of African horse sickness virus.
Abstract: Blood samples collected as part of routine diagnostic investigations from South African horses with clinical signs suggestive of African horse sickness (AHS) were subjected to analysis with an AHS virus (AHSV) group specific reverse transcription quantitative polymerase chain reaction (AHSV RT-qPCR) assay and virus isolation (VI) with subsequent serotyping by plaque inhibition (PI) assays using AHSV serotype-specific antisera. Blood samples that tested positive by AHSV RT-qPCR were then selected for analysis using AHSV type specific RT-qPCR (AHSV TS RT-qPCR) assays. The TS RT-qPCR assays were evaluated using both historic stocks of the South African reference strains of each of the 9 AHSV serotypes, as well as recently derived stocks of these same viruses. Of the 503 horse blood samples tested, 156 were positive by both AHSV RT-qPCR and VI assays, whereas 135 samples that were VI negative were positive by AHSV RT-qPCR assay. The virus isolates made from the various blood samples included all 9 AHSV serotypes, and there was 100% agreement between the results of conventional serotyping of individual virus isolates by PI assay and AHSV TS RT-qPCR typing results. Results of the current study confirm that the AHSV TS RT-qPCR assays for the identification of individual AHSV serotypes are applicable and practicable and therefore are potentially highly useful and appropriate for virus typing in AHS outbreak situations in endemic or sporadic incursion areas, which can be crucial in determining appropriate and timely vaccination and control strategies.
Copyright © 2015 Elsevier B.V. All rights reserved.
Publication Date: 2015-07-29 PubMed ID: 26232526DOI: 10.1016/j.jviromet.2015.07.015Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research focused on the development of three triplex real-time reverse transcription PCR assays, designed to detect and distinguish between the nine serotypes of African horse sickness virus in blood samples of South African horses. The developed assays were found to have high accuracy and are potentially highly useful for managing outbreak situations of the disease.
Research Method
- The research team conducted tests on blood samples drawn from South African horses who showed symptoms of African Horse Sickness (AHS).
- The samples were initially tested using a virus group-specific reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay and virus isolation (VI) methods.
- Those samples that were found positive by the AHSV RT-qPCR were selected for further testing using type-specific RT-qPCR (AHSV TS RT-qPCR) assays.
- The effectiveness of these TS RT-qPCR assays was then evaluated using reference strains of all nine AHSV serotypes.
Results
- Out of 503 horse blood samples tested, 156 were identified as positive for AHSV using both RT-qPCR and VI assays.
- However, there were 135 samples that tested negative through VI but were detected as positive by the AHSZ RT-qPCR method.
- The virus isolates drawn from the tested blood samples included all the 9 AHSV serotypes.
- A comparison between traditional serotyping methods and the developed RT-qPCR assays showed a 100% match in the results, confirming the high accuracy of these newly developed methods.
Conclusion
- The research concluded that the developed AHSV TS RT-qPCR assays can efficiently identify and distinguish the various AHSV serotypes in horse blood samples.
- Their heightened accuracy and the ability to identify specific serotypes make these assays a potentially useful tool for managing AHS outbreaks, playing a critical role in effective disease control and timely vaccinations.
Cite This Article
APA
Weyer CT, Joone C, Lourens CW, Monyai MS, Koekemoer O, Grewar JD, van Schalkwyk A, Majiwa PO, MacLachlan NJ, Guthrie AJ.
(2015).
Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine serotypes of African horse sickness virus.
J Virol Methods, 223, 69-74.
https://doi.org/10.1016/j.jviromet.2015.07.015 Publication
Researcher Affiliations
- Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa. Electronic address: equineresearchcentre@gmail.com.
- Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
- Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
- Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
- Agricultural Research Council, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa; Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
- Epidemiology, Veterinary Services (Animal Health) Western Cape Department of Agriculture, Private Bag X1, Elsenburg 7607, South Africa.
- Agricultural Research Council, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa.
- Agricultural Research Council, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa; Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
- Equine Viral Disease Laboratory, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA; Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
- Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
MeSH Terms
- African Horse Sickness Virus / classification
- African Horse Sickness Virus / genetics
- Animals
- Genotyping Techniques / methods
- Horses
- Molecular Typing / methods
- Multiplex Polymerase Chain Reaction / methods
- RNA, Viral / genetics
- Real-Time Polymerase Chain Reaction / methods
- Reverse Transcriptase Polymerase Chain Reaction / methods
- Sensitivity and Specificity
- Serogroup
Citations
This article has been cited 9 times.- 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).
- 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).
- Villalba R, Tena-Tomás C, Ruano MJ, Valero-Lorenzo M, López-Herranz A, Cano-Gómez C, Agüero M. 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 2024 Mar 20;16(3).
- Wang Y, Ong J, Ng OW, Songkasupa T, Koh EY, Wong JPS, Puangjinda K, Fernandez CJ, Huangfu T, Ng LC, Chang SF, Yap HH. Development of Differentiating Infected from Vaccinated Animals (DIVA) Real-Time PCR for African Horse Sickness Virus Serotype 1. Emerg Infect Dis 2022 Dec;28(12):2446-2454.
- Dennis SJ, Meyers AE, Hitzeroth II, Rybicki EP. African Horse Sickness: A Review of Current Understanding and Vaccine Development. Viruses 2019 Sep 11;11(9).
- Ngoveni HG, van Schalkwyk A, Koekemoer JJO. Evidence of Intragenic Recombination in African Horse Sickness Virus. Viruses 2019 Jul 18;11(7).
- Brister H, Barnum SM, Reedy S, Chambers TM, Pusterla N. Validation of two multiplex real-time PCR assays based on single nucleotide polymorphisms of the HA1 gene of equine influenza A virus in order to differentiate between clade 1 and clade 2 Florida sublineage isolates. J Vet Diagn Invest 2019 Jan;31(1):137-141.
- Durán-Ferrer M, Agüero M, Zientara S, Beck C, Lecollinet S, Sailleau C, Smith S, Potgieter C, Rueda P, Sastre P, Monaco F, Villalba R, Tena-Tomás C, Batten C, Frost L, Flannery J, Gubbins S, Lubisi BA, Sánchez-Vizcaíno JM, Emery M, Sturgill T, Ostlund E, Castillo-Olivares J. Assessment of reproducibility of a VP7 Blocking ELISA diagnostic test for African horse sickness. Transbound Emerg Dis 2019 Jan;66(1):83-90.
- Weyer CT, Grewar JD, Burger P, Rossouw E, Lourens C, Joone C, le Grange M, Coetzee P, Venter E, Martin DP, MacLachlan NJ, Guthrie AJ. African Horse Sickness Caused by Genome Reassortment and Reversion to Virulence of Live, Attenuated Vaccine Viruses, South Africa, 2004-2014. Emerg Infect Dis 2016 Dec;22(12):2087-2096.
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