Abstract
African horse sickness is a devastating, transboundary animal disease, that is ‘listed’ by the Office International des Epizooties (OIE). Although attenuated, inactivated and subunit vaccines have been developed for African horse sickness virus (AHSV), these are serotype-specific and their effective deployment therefore relies on rapid and reliable identification of virus type. AHSV serotype is controlled by the specificity of interactions between neutralising antibodies, and components of the outer-capsid, particularly protein VP2 (encoded by AHSV genome segment 2 (Seg-2)). We report the development and evaluation of novel gel based reverse transcription-PCR (RT–PCR) assays targeting AHSV Seg-2, which can be used to very significantly increase the speed and reliability of detection and identification (compared to virus neutralisation tests) of the nine serotypes of AHSV. Primer sets were designed targeting regions of Seg-2 that are conserved between strains within each of the AHSV serotype (types 1 to 9). These assays were evaluated using multiple AHSV strains from the orbivirus reference collection at IAH (www.reoviridae.org/dsRNA_virus_proteins/ReoID/AHSV-isolates.htm). In each case the Seg-2 primers showed a high level of specificity and failed to cross-amplify the most closely related heterologous AHSV types, or other related orbiviruses (such as bluetongue virus (BTV), or equine encephalosis virus (EEV)). The assays are rapid and sensitive, and can be used to detect and type viral RNA in blood, tissue samples, or cultivated viral suspensions within 24 h. They were used to identify AHSV strains from recent outbreaks in sub-Saharan African countries. These methods also generate cDNAs suitable for sequencing and phylogenetic analyses of Seg-2, identifying distinct virus lineages within each virus-type and helping to identify strain movements/origins. The RT-PCR methods described here provide a robust and versatile tool for rapid and specific detection and identification of AHSV serotypes 1 to 9.
Highlights
African horse sickness virus is a distinct virus species within the genus Orbivirus, within the family Reoviridae
Comparisons of full-length Seg-2 sequences for reference strains of the nine African horse sickness virus (AHSV) serotypes and other field isolates [31,43,52,53, Bankowska et al – in preparation], were used to identify regions of conservation within individual AHSV serotypes and variability between different types. These regions were used to design a minimum of two serotype-specific primer-pairs for use in RT-PCR assays to detect each AHSV serotype (Table S1)
The RT-PCR primers were tested in reactions containing RNA extracted from BHK-21 cells infected with the different AHSV serotypes (Table 1)
Summary
African horse sickness virus is a distinct virus species within the genus Orbivirus (of which Bluetongue virus is the ‘type species’), within the family Reoviridae. African horsesickness (AHS) is an important transboundary disease that is ‘listed’ by the Office International des Epizooties (OIE). It is an acute or sub-acute, non-contagious, arthropod-borne viral disease, characterized by severe pyrexia, widespread haemorrhages and oedematous exudations [1]. The African horse sickness virus (AHSV) can infect large carnivores via an oral route and may be a factor in the viability of endangered wildlife populations [4,5]. AHS is endemic in sub-Saharan Africa [6] but single AHSV serotypes have occasionally expanded outside these boundaries, persisting in newly affected areas for a number of years [7].
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