Use of reverse transcriptase-polymerase chain reaction (RT-PCR) and dot-blot hybridisation for the detection and identification of African horse sickness virus nucleic acids.
Abstract: A coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) for the detection of African horse sickness virus (AHSV) dsRNA, has been developed using genome segment 7 as the target template for primers. RNA from isolates of all nine AHSV serotypes were readily detected. The potential inhibitory effects of either ethylene diamine tetra acetic acid (EDTA) or heparin on the RT-PCR were eliminated by washing blood samples before lysis of the red blood cells and storage. There was a close agreement in the sensitivity and the specificity of the RT-PCR and an indirect sandwich ELISA. Confirmation of the presence of AHSV using RT-PCR and dot-blot hybridization on blood samples collected from horses experimentally infected with AHSV serotype 4 (AHSV 4) and AHSV serotype 9 (AHSV 9), was achieved within 24 hours, compared to the period of several days required for virus isolation. The RT-PCR and virus isolation methods showed similar levels of sensitivity when used for the detection of AHSV in 3 horses infected with AHSV 4, and in 2 out of 3 horses infected with a less virulent isolate of AHSV 9. Although viraemia was detected in the third horse by virus isolation, from 6 to 14 days after infection, this animal remained consistently negative by RT-PCR. Conversely, AHSV viral RNA was detected by RT-PCR in the blood of 4 donkeys and 4 mules up to 55 days after their experimental infection despite the absence of any detectable infectious virus. RT-PCR is a sensitive and rapid method for detecting AHSV nucleic acids during either the incubation period at the start of an African horse sickness (AHS) epizootic, or for epidemiological investigations in species where clinical signs may be inapparent.
Publication Date: 1998-10-24 PubMed ID: 9785517DOI: 10.1007/978-3-7091-6823-3_28Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study shares the development of a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect and confirm the presence of African horse sickness virus (AHSV) in horses, donkeys, and mules in a quick and sensitive manner.
Research Purpose
- The main objective of this research is to develop a reliable and efficient testing method for detecting the AHSV. This would be used to eliminate the viral disease or control its spread, especially at the start of an African Horse Sickness (AHS) epizootic (an outbreak of the disease in animal populations).
Methodology
- The researchers created an RT-PCR test using genome segment 7 as the target template for primers. This test allows for the detection of AHSV double-stranded RNA from isolates of all nine known AHSV serotypes.
- Two potential inhibitors, EDTA and heparin, were eliminated from the samples by washing blood samples before lysis (breaking down) of the red blood cells.
- The sensitivity and specificity of the new test were closely aligned with an indirect sandwich ELISA, another testing method.
- The team confirmed the presence of AHSV by using both RT-PCR and dot-blot hybridization from blood samples taken from horses infected with AHSV serotype 4 (AHSV 4) and AHSV serotype 9 (AHSV 9). This process was completed within 24 hours compared to the several days required for virus isolation.
Findings
- RT-PCR performed comparably to virus isolation methods when used on horses infected with different serotypes (AHSV 4 and AHSV 9). One outlier was a horse infected with AHSV 9 which showed a viraemia (presence of virus in the blood) detectable by virus isolation but not by RT-PCR.
- In contrast, RT-PCR effectively detected AHSV viral RNA in blood of donkeys and mules up to 55 days post-infection, despite those animals showing no signs of active infectious virus.
Implications
- The findings from this study suggest that RT-PCR is an effective tool for detecting AHSV, providing quick and accurate identification of the virus. This is critical for controlling and preventing the spread of African horse sickness, especially during an outbreak. This research may also be valuable in studying viral diseases in other species where symptoms may not be evident.
Cite This Article
APA
Zientara S, Sailleau C, Moulay S, Crucière C, el-Harrak M, Laegreid WW, Hamblin C.
(1998).
Use of reverse transcriptase-polymerase chain reaction (RT-PCR) and dot-blot hybridisation for the detection and identification of African horse sickness virus nucleic acids.
Arch Virol Suppl, 14, 317-327.
https://doi.org/10.1007/978-3-7091-6823-3_28 Publication
Researcher Affiliations
- Centre National d'Etudes Veterinaires et Alimentaires, Laboratoire Central de Recherches Veterinaires, Maisons-Alfort, France.
MeSH Terms
- African Horse Sickness / diagnosis
- African Horse Sickness Virus / genetics
- African Horse Sickness Virus / immunology
- African Horse Sickness Virus / isolation & purification
- Animals
- Antibodies, Viral / blood
- Bluetongue virus / genetics
- Bluetongue virus / immunology
- Enzyme-Linked Immunosorbent Assay / veterinary
- Equidae
- Polymerase Chain Reaction / veterinary
- RNA, Double-Stranded / blood
- RNA, Viral / blood
- Sensitivity and Specificity
- Viremia / diagnosis
- Viremia / veterinary
Citations
This article has been cited 4 times.- Ndebé MMF, Mouiche MMM, Moffo F, Poueme RNS, Awah-Ndukum J. Seroprevalence and Risk Factors of African Horse Sickness in Three Agroecological Zones of Cameroon. Vet Med Int 2022;2022:2457772.
- Karamalla ST, Gubran AI, Adam IA, Abdalla TM, Sinada RO, Haroun EM, Aradaib IE. Sero-epidemioloical survey on African horse sickness virus among horses in Khartoum State, Central Sudan. BMC Vet Res 2018 Aug 1;14(1):230.
- Maan NS, Maan S, Nomikou K, Belaganahalli MN, Bachanek-Bankowska K, Mertens PP. Serotype specific primers and gel-based RT-PCR assays for 'typing' African horse sickness virus: identification of strains from Africa. PLoS One 2011;6(10):e25686.
- Aradaib IE, Mohemmed ME, Sarr JA, Idris SH, Ali NO, Majid AA, Karrar AE. A simple and rapid method for detection of African horse sickness virus serogroup in cell cultures using RT-PCR. Vet Res Commun 2006 Apr;30(3):319-24.
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