title: A quantitative real-time reverse transcription PCR (qRT-PCR) assay to detect genome segment 9 of all 26 bluetongue virus serotypes.
abstract: Bluetongue (BT) is an arboviral disease, which can often be fatal in naïve sheep and white tailed deer, but is usually less severe, or unapparent in other ruminants. Twenty-six bluetongue virus (BTV) serotypes have been recognised so far, two of which (BTV-25 and BTV-26) were recently identified by phylogenetic comparisons of genome-segment/outer-capsid protein VP2 (subsequently confirmed by serological 'virus-neutralisation' assays). Rapid, sensitive, reliable and quantitative diagnostic-assays for detection and identification of BTV represent important components of effective surveillance and control strategies. The BTV genome comprises 10 linear segments of dsRNA. We describe a 'TaqMan' fluorescence-probe based quantitative real-time RT-PCR assay, targeting the highly conserved genome-segment-9 (encoding the viral-helicase 'VP6' and NS4). The assay detected Seg-9 from isolates of all 26 BTV types, as well as from clinical samples derived from BTV-6w and BTV-8w outbreaks (in Europe), BTV-25 from Switzerland, BTV-26 from Kuwait, BTV-1w, BTV-4w and BTV-8w from Spain, BTV-4w, BTV-8, BTV-10 and BTV-16 from Brazil. Assay efficiency was evaluated with RNA derived from the reference strain of BTV-1w [RSArrrr/01] and was 99.6%, detecting down to 4 copies per reaction. Samples from uninfected insect or mammalian cell-cultures, hosts-species (uninfected sheep blood) or vector-insects, all gave negative results. The assay failed to detect RNA from heterologous but related Orbivirus species (including the nine African horse sickness virus [AHSV] and seven epizootic haemorrhagic disease virus [EHDV] serotypes).
authors.affiliation: The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom.
authors.forename: Narender S
authors.initials: NS
authors.lastname: Maan
authors.affiliation: The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom.
authors.forename: Sushila
authors.initials: S
authors.lastname: Maan
authors.affiliation: The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom.
authors.forename: Manjunatha
authors.initials: M
authors.lastname: Belaganahalli
authors.affiliation: The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom.
authors.forename: Gillian
authors.initials: G
authors.lastname: Pullinger
authors.affiliation: The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom; Animal Health Department, University of Cordoba, Cordoba, Spain.
authors.forename: Antonio J Arenas
authors.initials: AJ
authors.lastname: Montes
authors.affiliation: The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom; Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
authors.forename: Marcela R
authors.initials: MR
authors.lastname: Gasparini
authors.affiliation: The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom.
authors.forename: Marc
authors.initials: M
authors.lastname: Guimera
authors.affiliation: The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom.
authors.forename: Kyriaki
authors.initials: K
authors.lastname: Nomikou
authors.affiliation: The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom. Electronic address: peter.mertens@pirbright.ac.uk.
authors.forename: Peter P C
authors.initials: PP
authors.lastname: Mertens
mesh_terms: D000818:Animals; D001819:Bluetongue / Q000175:diagnosis* / Q000821:virology; D001820:Bluetongue virus / Q000235:genetics / Q000302:isolation & purification*; D016679:Genome, Viral; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D065288:Serogroup; D012756:Sheep; D014730:Veterinary Medicine / Q000379:methods
chemical_list: D012367:RNA, Viral
keywords: Bluetongue virus; Diagnosis; Quantitative PCR; Real-time RT-PCR; Seg-9; VP6 and NS4 protein