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Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus.
Abstract: Nucleotide sequences of 52 South African isolates of African horse sickness virus (AHSV) collected during 2004-2005 and including viruses of all nine AHSV serotypes, were used to design and develop a duplex real-time reverse transcription quantitative PCR (RT-PCR) assay targeting the VP7 (S8) and NS2 (S9) genes of AHSV. The assay was optimized for detection of AHSV in fresh and frozen blood of naturally infected horses. Assay performance was enhanced using random hexamers rather than gene-specific primers for RT, and with denaturation of double-stranded RNA in the presence of random hexamers. The assay was efficient with a linear range of at least five orders of magnitude. The analytical sensitivity of the assay was 132 copies of the target genes (4125 copies per ml of blood), and the assay was at least 10-fold more sensitive than virus isolation on BHK-21 cells. The assay was also highly specific because it did not detect related orbiviruses, such as bluetongue and equine encephalosis viruses.
Copyright 2010 Elsevier B.V. All rights reserved.
Publication Date: 2010-03-19 PubMed ID: 20304015DOI: 10.1016/j.jviromet.2010.03.009Google 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.
- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
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.
The research developed and improved a diagnostic test that accurately identifies the African horse sickness virus (AHSV) in a horse’s blood samples using real-time reverse transcription quantitative PCR (RT-PCR) technology, targeting two distinctive genes of the AHSV.
Research Context
- African horse sickness (AHS) is a lethal viral disease affecting horses, caused by the African horse sickness virus (AHSV).
- For this research, the researchers used nucleotide sequences of 52 South African strains of AHSV collected during 2004-2005, representing all nine known AHSV serotypes.
- The aim of the research was to develop and optimize a diagnostic assay, more specifically, a duplex real-time reverse transcription quantitative PCR (RT-PCR) assay that targets two specific genes of the AHSV: the VP7 (S8) and NS2 (S9) genes.
Methodology
- The assay was developed for the detection of AHSV in fresh and frozen blood samples collected from naturally infected horses.
- The researchers found that using random hexamers as opposed to gene-specific primers for reverse transcription, and denaturing double-stranded RNA in the presence of random hexamers enhanced the performance of the assay.
Results
- The optimized assay proved to be highly efficient, with a linear range of at least five orders of magnitude.
- The assay’s analytical sensitivity was measured at 132 copies of the targeted virus genes, which is equivalent to 4125 copies of the virus per ml of blood.
- Importantly, the sensitivity of the assay was at least 10 times more than the previous virus isolation system using BHK-21 cells, a traditional cell line used for virus detection.
- The specificity of the test was also validated, as it did not detect related orbiviruses such as bluetongue and equine encephalosis viruses, confirming its selectiveness for detecting AHSV only.
Conclusion
- In summary, the research successfully developed and optimized an advanced RT-PCR assay that accurately identifies the presence of AHSV in horse’s blood, displaying significant improvements in terms of sensitivity and specificity compared to the previous diagnostic methods.
Cite This Article
APA
Quan M, Lourens CW, MacLachlan NJ, Gardner IA, Guthrie AJ.
(2010).
Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus.
J Virol Methods, 167(1), 45-52.
https://doi.org/10.1016/j.jviromet.2010.03.009 Publication
Researcher Affiliations
- Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa. melvyn.quan@up.ac.za
MeSH Terms
- African Horse Sickness / diagnosis
- African Horse Sickness / virology
- African Horse Sickness Virus / genetics
- African Horse Sickness Virus / isolation & purification
- Animals
- Antigens, Viral / genetics
- Blood / virology
- DNA Primers / genetics
- Horses
- Molecular Sequence Data
- RNA, Viral / genetics
- Reverse Transcriptase Polymerase Chain Reaction / methods
- Sensitivity and Specificity
- Sequence Analysis, DNA
- Viral Core Proteins / genetics
- Viral Nonstructural Proteins / genetics
Citations
This article has been cited 13 times.- Penzhorn L, Crafford JE, Guthrie AJ. Enhancing African horse sickness virus detection: comparing and adapting PCR assays. J Vet Diagn Invest 2026 Feb 7;:10406387261417355.
- 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).
- Huang C, Wang J, Ruan Z, Wu J, Lin Y, Cao C, Yang J, Weng Q, Jin Y, Chen P, Hua Q. Real-Time Reverse Transcription Multienzyme Isothermal Rapid Amplification for Rapid Detection of African Horse Sickness Virus. Transbound Emerg Dis 2025;2025:1852368.
- 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.
- Bekker S, Huismans H, van Staden V. Generation of a Soluble African Horse Sickness Virus VP7 Protein Capable of Forming Core-like Particles. Viruses 2022 Jul 26;14(8).
- Fairbanks EL, Brennan ML, Mertens PPC, Tildesley MJ, Daly JM. Re-parameterization of a mathematical model of African horse sickness virus using data from a systematic literature search. Transbound Emerg Dis 2022 Jul;69(4):e671-e681.
- 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).
- Fowler VL, Howson ELA, Flannery J, Romito M, Lubisi A, Agüero M, Mertens P, Batten CA, Warren HR, Castillo-Olivares J. Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of African Horse Sickness Virus. Transbound Emerg Dis 2017 Oct;64(5):1579-1588.
- Lulla V, Lulla A, Wernike K, Aebischer A, Beer M, Roy P. Assembly of Replication-Incompetent African Horse Sickness Virus Particles: Rational Design of Vaccines for All Serotypes. J Virol 2016 Aug 15;90(16):7405-7414.
- Alberca B, Bachanek-Bankowska K, Cabana M, Calvo-Pinilla E, Viaplana E, Frost L, Gubbins S, Urniza A, Mertens P, Castillo-Olivares J. Vaccination of horses with a recombinant modified vaccinia Ankara virus (MVA) expressing African horse sickness (AHS) virus major capsid protein VP2 provides complete clinical protection against challenge. Vaccine 2014 Jun 17;32(29):3670-4.
- Bachanek-Bankowska K, Maan S, Castillo-Olivares J, Manning NM, Maan NS, Potgieter AC, Di Nardo A, Sutton G, Batten C, Mertens PP. Real time RT-PCR assays for detection and typing of African horse sickness virus. PLoS One 2014;9(4):e93758.
- Johnson N, Voller K, Phipps LP, Mansfield K, Fooks AR. Rapid molecular detection methods for arboviruses of livestock of importance to northern Europe. J Biomed Biotechnol 2012;2012:719402.
- Belaganahalli MN, Maan S, Maan NS, Tesh R, Attoui H, Mertens PP. Umatilla virus genome sequencing and phylogenetic analysis: identification of stretch lagoon orbivirus as a new member of the Umatilla virus species. PLoS One 2011;6(8):e23605.
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