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Classical swine fever (CSF) is a highly contagious viral disease of pigs that has a tremendous socioeconomic impact. Vaccines are available for disease control. However, most industrialized countries are implementing stamping-out strategies to eliminate the disease and avoid trade restrictions. These restrictions can be avoided through the use of marker vaccines such as CP7_E2alf. Marker vaccines have to be accompanied by reliable and robust discriminatory assays. In this context, a multiplex microsphere immunoassay for serological differentiation of infected from vaccinated animals (DIVA) was developed to distinguish CSF virus (CSFV)-infected animals from CP7_E2alf-vaccinated animals. To this end, three viral proteins, namely, CSFV E2, CSFV E(rns), and bovine viral diarrhea virus (BVDV) E2, were produced in insect cells using a baculovirus expression system; they were used as antigens in a microsphere immunoassay, which was further evaluated by testing a large panel of pig sera and compared to a well-characterized commercial CSFV E2 antibody enzyme-linked immunosorbent assays (ELISAs) and a test version of an improved CSFV E(rns) antibody ELISA. Under a cutoff median fluorescence intensity value of 5,522, the multiplex microsphere immunoassay had a sensitivity of 98.5% and a specificity of 98.9% for the detection of antibodies against CSFV E2. The microsphere immunoassay and the CSFV E(rns) ELISA gave the same results for 155 out of 187 samples (82.8%) for the presence of CSFV E(rns) antibodies. This novel multiplex immunoassay is a valuable tool for measuring and differentiating immune responses to vaccination and/or infection in animals.

Differentiation of Classical Swine Fever Virus Infection from CP7_E2alf Marker Vaccination by a Multiplex Microsphere Immunoassay