Molecular applications of the African horsesickness virus genome segment 2 in diagnostics and epidemiology
Koekemoer, Jacobus Johannes Otto
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The mortality rate of AHS is more than 90 % in fully susceptible horses. Outbreaks of AHS can spread rapidly and have severe economic consequences. Therefore, the disease is classified in "list A" by the OIE and the international movement of horses is subject to stringent zoosanitary regulations. Apart from prevention by large scale vaccinations, a good knowledge of the epidemiology of the disease together with accurate and rapid diagnostic procedures are the most important means of disease control. The use of genome segment 2 of the virus, in both diagnostic and epidemiological applications, was investigated. During this study it was shown that cDNA copies of genome segment 2 of the nine reference strain viruses of AHSV hybridize exclusively to viral RNA from their corresponding serotypes. This result provided proof of concept for the feasibility of developing nucleic acid based serotyping procedures. It was followed up by the development of the first two genome segment 2 based serotyping methods. The first is a method that uses a set of nine full-length AHSV genome segment 2 clones as probes to hybridize serotype-specifically to denatured dsRNA. The second method makes use of a universal RT-PCR primer set that was developed for the partial amplification of genome segment 2 of any AHSV serotype. The RT-PCR amplification product is labelled and used as a probe to hybridize with immobilized segment 2 cDNA of the nine serotypes of AHSV in a reverse line blot format. The reverse line blot hybridization method proved to be highly sensitive and accurate and can be used to serotype any AHSV with a single RTPCR and hybridization within one day. It was further shown that the method is sufficiently sensitive to be performed directly on clinical organ samples. A set of genome segment 2 clones was also used for the first ever segment 2-based AHSV phylogenetic analysis and molecular epidemiological investigation. Genome segment 2 sequence data was generated from AHSV 7 field cases that occurred in South Africa over a two year period which included an important outbreak in the African horsesickness free area in the Western Cape. The phylogenetic, classification was compared with historical and geographical data of the different disease cases. Results indicated that segment 2 nucleotide sequence data can be used to distinguish between different isolates/strains of one serotype and that the phylogenetic grouping of the isolates reflected their historical and geographical groupings. From this it can be concluded that genome segment 2 sequence data can be used in molecular epidemiological investigations to classify AHSVs according to geographical origin, or topotype. This information can be used to track the movement of a particular virus strain to its origin and implicate possible ways by which the disease is spread from endemic to non-endemic areas. With the new experimental diagnostic methods that have been developed, the virus can be serotyped directly from clinical organ samples within one day, compared to serological methods which take 2-3 weeks. The phylogenetic analysis demonstrated the advantage of a molecular epidemiological approach to provide definitive data for determining the origin of an AHS outbreak. In future, the use of these new serotyping procedures in conjunction with comprehensive molecular epidemiological data should significantly speed up AHSV diagnostics and contribute to more effective prevention, control and surveillance systems for AHS.