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Title: Specific detection of rinderpest virus by real-time reverse transcription-PCR in preclincal and clinical samples of experimentally infected cattle

Author
item CARRILLO, CONSUELO - Animal And Plant Health Inspection Service (APHIS)
item PRARAT, MELANIE - Former ARS Employee
item VAGNOZZI, ARIEL - Oak Ridge Institute For Science And Education (ORISE)
item CALAHAN, JOHNNY - Tetracore, Inc
item Smoliga, George
item NELSON, WILLIAM - Tetracore, Inc
item Rodriguez, Luis

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/8/2010
Publication Date: 11/1/2010
Citation: Carrillo, C., Prarat, M.V., Vagnozzi, A., Calahan, J.D., Smoliga, G.R., Nelson, W.D., Rodriguez, L.L. 2010. Specific detection of rinderpest virus by real-time reverse transcription-PCR in preclincal and clinical samples of experimentally infected cattle. Journal of Clinical Microbiology. 11:4094-4101.

Interpretive Summary: Rinderpest is a devastating disease of cattle that, until recently caused serious economic hardship to various parts of the world, especially in Africa. Through a global vaccination effort we are at the brink of eradicating the disease from the world. It is very important to have rapid and sensitive diagnostic methods to detect any accidental or deliberate reintroduction of this disease. Here we report the development and validation in the laboratory of a highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (RT-PCR). The test was able to detect all 16 RPV strains representing the genetic and geographic diversity of this virus. No cross-reactivity was detected with closely related viruses or viruses that cause similar disease, 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 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. Comparison of different clinical samples from infected animals showed that conjunctiva swabs and blood 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 detecting RPV before clinical disease is evident 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.

Technical Abstract: A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (RT-PR) 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 (TCID50) 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 clincal signs. The comparison of clincal 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.