Real time RT-PCR assays for detection and typing of African horse sickness virus.
Although African horse sickness (AHS) can cause up to 95% mortality in horses, naïve animals can be protected by vaccination against the homologous AHSV serotype. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the most variable of the AHSV proteins. VP2 is also a primary target for AHSV specific neutralising antibodies, and consequently determines the identity of the nine AHSV serotypes. In contrast VP1 (the viral polymerase) and VP3 (the sub-core shell protein), encoded by Seg-1 and Seg-3 respectively, are highly conserved, representing virus species/orbivirus-serogroup-specific antigens. We report development and evaluation of real-time RT-PCR assays targeting AHSV Seg-1 or Seg-3, that can detect any AHSV type (virus species/serogroup-specific assays), as well as type-specific assays targeting Seg-2 of the nine AHSV serotypes. These assays were evaluated using isolates of different AHSV serotypes and other closely related orbiviruses, from the 'Orbivirus Reference Collection' (ORC) at The Pirbright Institute. The assays were shown to be AHSV virus-species-specific, or type-specific (as designed) and can be used for rapid, sensitive and reliable detection and identification (typing) of AHSV RNA in infected blood, tissue samples, homogenised Culicoides, or tissue culture supernatant. None of the assays amplified cDNAs from closely related heterologous orbiviruses, or from uninfected host animals or cell cultures.
Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Surrey, United Kingdom.
Katarzyna
K
Bachanek-Bankowska
Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Surrey, United Kingdom.
Sushila
S
Maan
Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Surrey, United Kingdom.
Javier
J
Castillo-Olivares
Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Surrey, United Kingdom.
Nicola M
NM
Manning
Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Surrey, United Kingdom.
Narender Singh
NS
Maan
Deltamune (Pty) Ltd, Lyttelton, Centurion, South Africa; Department of Biochemistry, Centre for Human Metabonomics, North-West University, Private Bag X6001, Potchefstroom, South Africa.
Abraham C
AC
Potgieter
Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Surrey, United Kingdom.
Antonello
A
Di Nardo
Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
Geoff
G
Sutton
Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Surrey, United Kingdom.
Carrie
C
Batten
Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Surrey, United Kingdom.
Peter P C
PP
Mertens
D000355:African Horse Sickness / Q000175:diagnosis / Q000821:virology*; D000356:African Horse Sickness Virus / Q000145:classification* / Q000235:genetics; D000818:Animals; D057134:Antibodies, Neutralizing / Q000737:chemistry; D002460:Cell Line; D002519:Ceratopogonidae / Q000821:virology*; D002522:Chlorocebus aethiops; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D018163:Orbivirus / Q000235:genetics; D012367:RNA, Viral / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D014709:Vero Cells
D057134:Antibodies, Neutralizing; D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral