Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 4, 2003
Publication Date: February 1, 2004
Citation: Mecham, J.O., Wilson, W.C. 2004. Antigen capture competitive enzyme-linked immunosorbent assays using baculovirus-expressed antigens for diagnosis of bluetongue virus and epizootic hemorrhagic disease virus. Journal of Clinical Microbiology. 42(2):518-523. Interpretive Summary: Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are arthropod-borne viruses that infect both domestic and wild ruminants. Bluetongue is classified as a list A disease by the Office of International Epizootics (OIE). Infections caused by this virus cause considerable economic loss due to both disease and trade restrictions on the movement of livestock and livestock products. Regulatory restrictions often require that animals be certified as BTV and/or antibody free before allowing import or export of these animals or their products. Since livestock can be infected with both BTV and EHDV, especially in areas where livestock co-mingle with wildlife, it is important to distinguish between infections caused by these two viruses. However, missed diagnosis has been a problem because of antigenic similarity between these viruses. To overcome this problem, competitive enzyme-linked immunosorbent assay (c-ELISA) procedures have been developed for the serologic diagnosis of infections caused by these two viruses. These assays depend on the incorporation of a suitable antigen. Typically, antigens for BTV and EHDV ELISA tests are produced by infection of susceptible cultured cells. This process is time consuming, requires large volumes of cells and reagents, can vary in quality and quantity from preparation to preparation, and such antigen preparations may still be infectious. Baculoviruses have been widely used as vehicles to produce quantities of specific proteins of interest in insect cells. This paper reports the production of viral proteins using baculovirus that are used in novel antigen capture c-ELISA (Ag Cap c-ELISA) tests for the diagnosis of infections caused by BTV and EHDV. The Ag Cap c-ELISA tests offer the advantages of using easily produced, high tittered, non-infectious, viral antigen directly from baculovirus infected insect cell culture without further purification or concentration.
Technical Abstract: Bluetongue virus (BTV) and Epizootic hemorrhagic disease virus (EHDV) are orbiviruses that infect both livestock and wild ruminants. Antigenic cross-reactivity between BTV and EHDV often results in serologic misdiagnosis. Competitive enzyme-linked immunosorbent assays (c-ELISA) show increased sensitivity and specificity for the identification of these viral diseases; however, the preparation of cell culture derived viral antigen, for these tests, is laborious, variable from batch to batch and the resulting antigen may be infectious. To overcome these problems, the genes coding for a structural protein VP7, of BTV and EHDV, were cloned into baculovirus and the recombinant proteins expressed in Sf9 cultured insect cells. Recombinant viral proteins released into the baculovirus infected Sf9 cell culture supernatant were used in antigen capture c-ELISA (Ag Cap c-ELISA) tests that specifically detected antibody in the serum of cattle experimentally infected with BTV and EHDV. The Ag Cap c-ELISA offers the advantages of using an easily produced, easily standardized, non-infectious antigen that does not require further purification or concentration.