The implementation of an effective control plan for PRRS will require safe and effective vaccines. Future modified live virus (MLV) vaccines will require a mechanism that can be used to 1) identify vaccinated pigs, 2) identify naturally infected pigs, and 3) detect vaccination failure (the presence of a wild-type infection in a vaccinated pig). One approach, used successfully for other vaccines, is to prepare an attenuated virus with a deletion in a non-essential protein, a so-called deletion marker vaccine. An immune response to the deleted region would indicate a natural infection. We modified an infectious PRRS virus that contains a 132 amino acid deletion between amino acids 628 and 759 in the nsp2 region of PRRSV. The deleted region was replaced with either a large EGFP tag or with a much smaller TRIP tag. The overall objectives of the project were to study the replication properties of nsp2 deleted viruses, 2)evaluate the antibody response to the nsp(628-759) peptide and the inserted tags, 3) determine if the modified virus can re-acquire the deleted peptide by recombination with VR-2332, and 4) develop an assay for measuring antibody against the nsp2(628-659). The results showed that deleted viruses replicate in pigs, but were attenuated relative to the parent wild-type virus. The response of wild-type virus infected pigs to nsp2(628-759) was demonstrated experimentally, but variable results were obtained when antibodies were from pigs infected with a variety of viruses or from sera from field cases. Several modifications of the assay to detect nsp2(628-759) were tried including the use of a novel microsphere immunoassay (MIA) approach. Interestingly, pigs infected with the PRRSV-EGFP isolate produced a robust response to the EGFP protein. The results show that viruses can support deletions in nsp2. However, the current peptide candidate nsp2(628-759) has limitations when used as a marker. The expression of foreign tags provides a marker than can be used for compliance. One important outcome is that deletions in nsp2 provide the means for the one-step attenuation of PRRSV for the preparation of strain-specific live vaccines. Raymond (Bob) R. R. Rowland, Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS 66506, 785-532-4631"", [email protected]