title: Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine serotypes of African horse sickness virus.
abstract: Blood samples collected as part of routine diagnostic investigations from South African horses with clinical signs suggestive of African horse sickness (AHS) were subjected to analysis with an AHS virus (AHSV) group specific reverse transcription quantitative polymerase chain reaction (AHSV RT-qPCR) assay and virus isolation (VI) with subsequent serotyping by plaque inhibition (PI) assays using AHSV serotype-specific antisera. Blood samples that tested positive by AHSV RT-qPCR were then selected for analysis using AHSV type specific RT-qPCR (AHSV TS RT-qPCR) assays. The TS RT-qPCR assays were evaluated using both historic stocks of the South African reference strains of each of the 9 AHSV serotypes, as well as recently derived stocks of these same viruses. Of the 503 horse blood samples tested, 156 were positive by both AHSV RT-qPCR and VI assays, whereas 135 samples that were VI negative were positive by AHSV RT-qPCR assay. The virus isolates made from the various blood samples included all 9 AHSV serotypes, and there was 100% agreement between the results of conventional serotyping of individual virus isolates by PI assay and AHSV TS RT-qPCR typing results. Results of the current study confirm that the AHSV TS RT-qPCR assays for the identification of individual AHSV serotypes are applicable and practicable and therefore are potentially highly useful and appropriate for virus typing in AHS outbreak situations in endemic or sporadic incursion areas, which can be crucial in determining appropriate and timely vaccination and control strategies.
authors.affiliation: Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa. Electronic address: equineresearchcentre@gmail.com.
authors.forename: Camilla T
authors.initials: CT
authors.lastname: Weyer
authors.affiliation: Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
authors.forename: Christopher
authors.initials: C
authors.lastname: Joone
authors.affiliation: Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
authors.forename: Carina W
authors.initials: CW
authors.lastname: Lourens
authors.affiliation: Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
authors.forename: Mpho S
authors.initials: MS
authors.lastname: Monyai
authors.affiliation: Agricultural Research Council, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa; Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
authors.forename: Otto
authors.initials: O
authors.lastname: Koekemoer
authors.affiliation: Epidemiology, Veterinary Services (Animal Health) Western Cape Department of Agriculture, Private Bag X1, Elsenburg 7607, South Africa.
authors.forename: John D
authors.initials: JD
authors.lastname: Grewar
authors.affiliation: Agricultural Research Council, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa.
authors.forename: Antoinette
authors.initials: A
authors.lastname: van Schalkwyk
authors.affiliation: Agricultural Research Council, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa; Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
authors.forename: Phelix O A
authors.initials: PO
authors.lastname: Majiwa
authors.affiliation: Equine Viral Disease Laboratory, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA; Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
authors.forename: N James
authors.initials: NJ
authors.lastname: MacLachlan
authors.affiliation: Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Guthrie
mesh_terms: D000356:African Horse Sickness Virus / Q000145:classification* / Q000235:genetics*; D000818:Animals; D060005:Genotyping Techniques / Q000379:methods*; D006736:Horses; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D065288:Serogroup
chemical_list: D012367:RNA, Viral
keywords: AHS; Equine; Field diagnosis; RT-qPCR; Serotype