Assessment of bluetongue viraemia in sheep by real-time PCR and correlation with viral infectivity.
Inoculation of embryonated chicken eggs is the standard method for the titration of infectious Bluetongue virus (BTV). Here, six RNA extraction methods coupled with optimised dsRNA denaturation and real-time RT-PCR were evaluated for the quantitation of BTV in blood samples from experimentally infected sheep and results were correlated to infectious virus titres. An exogenous dsRNA internal control (IC) from the closely related Epizootic hemorrhagic disease virus (EHDV) was used to assess the efficiency of BTV genome extraction, dsRNA denaturation, RT, and PCR amplification. Recovery rates of IC and BTV dsRNA copies from extracted blood samples were highly correlated. Adjustment of BTV concentrations according to the IC recovery reduced variation in sample analyses among the different extraction methods and improved the accuracy of BTV quantitation. The EID(50)/ml titre, determined in blood samples from sheep infected experimentally with BTV-1 or BTV-9, correlated highly with the assessed concentration of BTV dsRNA copies. However, this correlation was consistent only during the first 28 days post-infection. The optimised extraction methods and quantitative RT-PCR could be useful for experimental studies of BTV transmission, pathogenesis and vaccine efficacy, or adapted further for the detection and quantitation of EHDV, African horse sickness virus and other dsRNA viruses.
Chatzinasiou
E
E
Laboratory of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece.
Dovas
C I
CI
Papanastassopoulou
M
M
Georgiadis
M
M
Psychas
V
V
Bouzalas
I
I
Koumbati
M
M
Koptopoulos
G
G
Papadopoulos
O
O
D000356:African Horse Sickness Virus / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D001819:Bluetongue / Q000821:virology*; D001820:Bluetongue virus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity*; D002642:Chick Embryo; D005260:Female; D018164:Hemorrhagic Disease Virus, Epizootic / Q000235:genetics / Q000302:isolation & purification; D009691:Nucleic Acid Denaturation; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012756:Sheep; D019562:Viral Load / Q000379:methods* / Q000592:standards; D014766:Viremia / Q000821:virology*
D012367:RNA, Viral