Serotype Specific Primers and Gel-Based RT-PCR Assays for ‘Typing’ African Horse Sickness Virus: Identification of Strains from Africa

et al. (2011) Serotype Specific Primers and Gel-Based RT-PCR Assays for 'Typing' African Horse Sickness Virus: Identification of Strains from Africa. PLoS ONE 6(10): e25686. doi:10.1371/journal.pone.0025686 Serotype Specific Primers and Gel-Based RT-PCR Assays for 'Typing' African Horse Sickness Virus: Identification of Strains from Africa Narender S. Maan 0 1 Sushila Maan 0 1 Kyriaki Nomikou 0 1 Manjunatha N. Belaganahalli 0 1 Katarzyna Bachanek- 0 1 Bankowska 0 1 Peter P. C. Mertens 0 1 Rudy A. Hartskeerl, Royal Tropical Institute, Netherlands 0 Current address: Department of Animal Biotechnology, LLR University of Veterinary and Animal Sciences , Hisar, Haryana , India 1 Vector-borne Disease Programme, Institute for Animal Health, Pirbright Laboratory , Pirbright, Woking, Surrey , United Kingdom 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. - Funding: This work was partly funded by Biotechnology and Biological Sciences Research Council (BBSRC) and the European Union (EU) (SANCO/940/2002) awarded to NSM and PM. SM and KN were part of this work funded by the Department for Environment, Food and Rural Affairs (DEFRA)(SE2617). For part of this work, MB was funded by Commonwealth Scholarship Commission (http://cscuk.dfid.gov.uk/), and KB was funded by an Institute for Animal Health (IAH) studentship (http://www.bbsrc.ac.uk/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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]. AHS has case fatality levels as high as 95% in horses and 80% in mules, while donkeys and zebras show few if any clinical signs and are thought to act as reservoir hosts [2,3]. The African horse sickness virus (AHSV) can also 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]. In 195961 an outbreak caused by AHSV serotype 9 (AHSV-9) occurred in the Middle East, spreading as far as Pakistan and India, causing heavy mortality in equids [8]. In 1965, AHSV-9 caused an outbreak that started in North Africa but spread as far as southern Spain [7,9]. In the late 1980s importation of a zebra infected with AHSV-4 from Namibia, caused an outbreak in Spain and Portugal [7,10,11,12]. These incursions demonstrate that the geographic area suitabl (...truncated)