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Title: Rapid detection of classical swine fever virus by a portable real time reverse transcriptase-PCR asssay

Author
item Risatti, Guillermo
item CALLAHAN, JOHNNY - TETRACORE, INC.
item NELSON, WILLIAM - TETRACORE, INC.
item Borca, Manuel

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/22/2002
Publication Date: 1/10/2003
Citation: Risatti, G.R., Callahan, J.D., Nelson, W.M., Borca, M.V. 2003. Rapid detection of classical swine fever virus by a portable real time reverse transcriptase-PCR asssay. Journal of Clinical Microbiology. 41(1):500-505. doi: 10.1128/jcm.41.1.500-505.2003.

Interpretive Summary: Classical Swine Fever (CSF) is a highly infectious disease of swine. Morbidity and mortality rates in infected herds can be high. Widely distributed around the world, CSF is enzootic in Eastern Europe, South-East Asia, South and Central America, southern Mexico and the Caribbean. Recent CSF outbreaks in Europe and the Caribbean (1997-2001) have had devastating economic consequences for the countries involved, and the same is expected should the disease be introduced into the United States. Improvements in CSF diagnostics and vaccines are needed to reduce the risk of disease introduction and to better manage a disease outbreak should it occur. Routine diagnosis of CSF includes the combined use different techniques, which are time-consuming thereby delaying disease diagnosis. Here this issue was addressed: a real time reverse transcriptase PCR assay for CSF virus (CSFV) was developed and evaluated in experimentally infected swine. The assay was shown to specifically detected CSFV representing different phylogenetic groupings but did not amplify viral RNA from related Pestiviruses. The assay was shown to be at least as sensitive of virus isolation when tested on clinical samples. Significantly, the assay detected the presence of the virus in clinical samples 2 to 4 days prior to onset of clinical disease. Importantly, the assay can be performed in only two hours, providing a rapid method for the diagnosis of CSF. The availability of these type of assay will reduce the risk of disease introduction and provide new tools to more effectively manage a disease outbreak in the U.S. should it occur.

Technical Abstract: Improvements in Classical Swine Fever (CSF) diagnostics are needed to reduce the risk of disease introduction and to better manage a disease outbreak should it occur. Routine diagnosis of CSF includes the combined use of ELISA, direct immunofluorescence assay (IFA) on tonsil samples, reverse transcription polymerase chain reaction (RT-PCR), and virus isolation. Although these techniques are not complex, they are time-consuming thereby delaying disease diagnosis. To address this need, a fluorogenic probe hydrolysis (TaqMan®) reverse transcriptase PCR assay for CSF virus (CSFV) was developed and evaluated in experimentally infected swine. The assay detected CSFV representing different phylogenetic groupings (Brescia, Haiti96, CS) but did not amplify viral RNA from related Pestiviruses, Bovine Viral Diarrhea Virus and Border Disease Virus. The assay met or exceeded the sensitivity (1-100 TCID50/ml) of viral cultures on samples from experimentally infected animals. Significantly, the assay detected viral RNA in nasal and tonsil scraping samples 2 to 4 days prior to onset of clinical disease. The assay can be performed in two hours or less, thus providing a rapid method for the diagnosis of CSF. The assay will have application for CSF surveillance and management of emergency responses during a disease outbreak, allowing rapid identification of infected herds.