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
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.
KeywordsEthylene Diamine Tetra Acetic Acid Virus Isolation Genome Segment Ethylene Diamine Tetra Acetic Acid Ethylene Diamine Tetra Acetic Acid
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- 1.Barker RH, Trairat B, Courval JM, Suwonkerd W, Rimwungtragoon K, Wirth DF (1992) A simple method to detect Plasmodium falciparum from blood samples using the polymerase chain reaction. Am J Med Hyg 46: 416–426Google Scholar
- 2.Beutler E, Gelbart T, Kuhl W (1990) Interference of heparin with the polymerase chain reaction. Bio Techniques 9: 166Google Scholar
- 3.Borden EC, Shope RE, Murphy FA (1971) Physiochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus — a new taxonomic group. Physiochemical and serological studies. J Gen Virol 13: 261–271Google Scholar
- 7.Coetzar JAW, Erasmus BJ (1994) African horse sickness. In: Coetzar JAW, Thomson GR,Tustin RC (eds) Infectious diseases of livestock. Oxford University Press, Oxford, pp 460–475Google Scholar
- 9.Du Toit RM (1944) The transmission of bluetongue and horse sickness by Culicoides. Onderstepoort J Vet Res 50: 536–541Google Scholar
- 13.Herniman KAJ, Gumm ID, Owen L, Taylor WP, Sellers RF (1980) Distribution of bluetongue virus and antibodies in some countries of the eastern hemisphere. Bull Off Int Epiz 92: 581–586Google Scholar
- 23.Mizukoshi N, Sakamoto K, Iwata A, Tsuchiya T, Ueda S, Watanabe T, Kamada M, Fukusho A (1992) The complete sequence of African horse sickness virus serotype 4 (vaccine strain) RNA segment 5 and its predicted polypeptide compared with NS 1 of bluetongue virus. J Gen Virol 73: 2425–2428PubMedCrossRefGoogle Scholar