Detection of Toggenburg Orbivirus by a segment 2-specific quantitative RT-PCR.
Toggenburg Orbivirus (TOV) has been detected recently in healthy goats in Switzerland. The virus is related closely to bluetongue virus (BTV) and is considered tentatively as a 25th serotype of BTV. Upon detection of additional TOV-positive goats in Switzerland, Germany, and Italy, these TOV isolates were characterized genetically by partial sequencing of the viral genome segment 2 which encodes VP2, the major outer capsid protein of orbiviruses. A TOV-specific RT-qPCR was developed, targeting conserved areas within segment 2. Since TOV cannot be propagated up to now outside its natural host, a synthetic positive control for the RT-qPCR was constructed by cloning the entire coding region of segment 2 and subsequent in vitro transcription of RNA from both ends to obtain double-stranded RNA. The TOV-specific RT-qPCR was able to detect as few as 30 dsRNA copies and proved to be equally sensitive as a pan BTV assay that was shown previously to have a detection limit of 0.001 TCID(50).
Hofmann
Martin A
MA
Institute of Virology and Immunoprophylaxis, CH-3147 Mittelhaeusern, Switzerland. martin.hofmann@ivi.admin.ch
Renzullo
Sandra
S
Planzer
Jeannette
J
Mader
Markus
M
Chaignat
Valérie
V
Thuer
Barbara
B
D000818:Animals; D001483:Base Sequence; D016679:Genome, Viral / Q000235:genetics*; D015511:Goat Diseases / Q000175:diagnosis* / Q000821:virology; D006041:Goats; D008969:Molecular Sequence Data; D018163:Orbivirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D010802:Phylogeny; D012088:Reoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D015678:Viral Structural Proteins / Q000235:genetics
D015678:Viral Structural Proteins