Specific detection of Rinderpest virus by real-time reverse transcription-PCR in preclinical and clinical samples from experimentally infected cattle.
A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (rRT-PCR)system, was developed. Five different RPV genomic targets were examined, and one was selected and optimized to detect viral RNA in infected tissue culture fluid with a level of detection ranging from 0.59 to 87.5 50% tissue culture infectious doses (TCID(50)) per reaction depending on the viral isolate. The strain sensitivity of the test was validated on 16 RPV strains belonging to all three phylogenetic branches described for RPV. No cross-reactivity was detected with closely related peste des petit ruminants or with symptomatically similar viruses, including all seven serotypes of foot-and-mouth disease virus, two serotypes of vesicular stomatitis virus, bluetongue virus, and bovine herpes virus type 2. In samples from experimentally infected cattle, our real-time RT-PCR test was significantly more sensitive than the gold standard test of virus isolation, allowing the detection of the disease 2 to 4 days prior to the appearance of clinical signs. The comparison of clinical samples with putative diagnostic value from live animals showed that conjunctival swabs and blood buffy coat were the samples of choice for epidemiological surveillance, while lymph nodes performed the best as postmortem specimens. This portable and rapid real-time RT-PCR has the capability of the preclinical detection of RPV and provides differential diagnosis from look-alike diseases of cattle. As RPV is declared globally eradicated, this test provides an important rapid virus detection tool that does not require the use of infectious virus and allows the processing of a large number of samples.
Carrillo
C
C
Foreign Animal Disease Diagnostic Laboratory, Animal Plant Health Inspection Service, Foreign Animal Disease Research Unit, Agricultural Research Service, US Department of Agriculture, Plum Island Animal Disease Center, Orient Pt, New York 11957, USA. consuelo.carrillo@APHIS.USDA.GOV
Prarat
M
M
Vagnozzi
A
A
Calahan
J D
JD
Smoliga
G
G
Nelson
W M
WM
Rodriguez
L L
LL
D000818:Animals; D001820:Bluetongue virus; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology; D003429:Cross Reactions; D052687:Deltapapillomavirus; D004195:Disease Models, Animal; D029544:Foot-and-Mouth Disease Virus; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012301:Rinderpest / Q000175:diagnosis* / Q000821:virology; D012302:Rinderpest virus / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013997:Time Factors; D018116:Vesiculovirus; D014773:Virology / Q000379:methods*