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United States Department of Agriculture

Agricultural Research Service

Title: Use of Polymerase Chain Reaction to Simultaneously Detect and Type Bovine Viral Diarrhoea Viruses Isolated from Clinical Specimens

Authors
item El-Kholy, A - VET SERA & VACCINES RES
item Bolin, Steven
item Ridpath, Julia
item Arab, R - CAIRO UNIVERSITY
item Abou-Zeid, A - CAIRO UNIVERSITY
item Hammam, H - VET SERA & VACCINES RES
item Platt, K - IOWA STATE UNIVERSITY

Submitted to: Office of International Epizootics Scientific and Technical Review
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 17, 1998
Publication Date: N/A

Interpretive Summary: Bovine viral diarrhea (BVD) virus is a common viral pathogen of cattle. The virus induces reproductive failure, respiratory disease, and enteric disease. Most BVD viruses are noncytopathic when grown in cell culture. Noncytopathic means that the virus does not visibly harm the cell it grows in; therefore, laboratory diagnosticians cannot see that they have isolated the virus unless they perform an indirect test. Most of the indirect tests used to detect BVD virus rely on antibody that reacts with viral antigen in the infected cell cultures. Antibody tests for detection of BVD virus work well, but are time consuming and expensive. A more rapid method for detection of BVD virus was developed that used polymerase chain reaction (PCR) amplification of the viral genome. This procedure was found reliable for detection of virus in cell cultures and for detection of virus in clinical specimens. The ability to detect virus directly in clinical specimens eliminates the need for, and expense of, cell cultures to isolate virus. Veterinary diagnostic laboratories and vaccine manufacturers can use this PCR test to rapidly detect BVD virus for diagnostic purposes or for quality control. Improved diagnostic tests for BVD virus benefit cattle producers and help to maintain a safe, wholesome food supply.

Technical Abstract: The techniques of indirect immunofluorescence (IF), immunoperoxidase (IP) staining, and the one-step reverse transcriptase polymerase chain reaction (RT-PCR) were compared for detection of 102 isolates of bovine viral diarrhoea virus (BVDV) in infected cell cultures. The BVDV was obtained from bovine clinical specimens, including sera, buffy coats and tissues, submitted from farms located in the states of Iowa and Wisconsin, United States of America. The IF technique detected 88/102 (86.3%) of the viral isolates, whereas IP staining detected an additional 4 isolates (92/102; 90%). The one-step RT-PCR using primers derived from the 5' untranslated region of the BVDV genome detected 102/102 (100%) of the BVDV isolates. A second-round PCR utilizing another pair of PCR primers from the 5' untranslated region, allowed rapid genotyping of BVDV. The procedure used showed that the PCR assay based on the 5' untranslated region of the virus genome is the most sensitive indicator for BVDV detection in cell culture, and is also of considerable epidemiological importance since it allowed rapid genotyping of BVDV isolated from clinical specimens. In addition to detection and genotyping of BVDV isolated form clinical specimens, the RT-PCR procedure can be used for routine screening of locally produced and imported biologicals for BVDV contamination. However, the procedure requires further refinement to enable direct application on the clinical specimen.

Last Modified: 8/27/2014
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