Currently, in the absence of effective vaccines and therapeutic drugs, one of the key approaches to achieve the “National PRRS Elimination” is to identify PRRSV infected pigs, so such pigs can be quarantined, isolated or removed from herds. Sensitive and specific diagnostic assays are essential for the assessment of pigs suspected of being infected and for prevention of spread of the virus within and between herds. Currently, the IDEXX HerdChek® PRRS ELISA is widely used for the detection of antibodies to either North American Type 2 or European-like Type 1 PRRSV. Concerns with suspect false positive IDEXX ELISA results in otherwise seronegative herds, have necessitated the use of a variety of follow-up serological assays to confirm the true status of individual animals. However, the sensitivity of common follow-up serological assays, such as the indirect fluorescent antibody (IFA) and virus neutralization assays are affected by viral antigenic variation and may not detect a serological response against antigenically diverse PRRSV isolates, such as Type 1 isolates. In this study, we evaluated the feasibility of developing an ELISA based diagnostic assay using the cysteine protease (CP) domain region and the conserved ES2 epitope of non-structural protein (Nsp2) as antigens. The CP regions of Type 1 and Type 2 PRRSV were expressed as recombinant proteins. Three hundred and fifty three serum samples from 32 individual pigs experimentally infected with Type 1 or Type 2 PRRSV were tested using CP-based ELISAs. Antibody specific to the CP domain can be detected as early as 14 dpi, and the antibody response lasted to 202 dpi. Receiver operating characteristic analysis based on the 81 known positive and 118 known negative samples showed good specificity (96.6%) and sensitivity (98.2%) of Nsp2 CP-based ELISA. The capability of the Nsp2 CP-based ELISA for detecting serum antibody response from pigs infected with various genetically different field strains was determined. Nine hundred and seventy-nine serum samples submitted to the SDSU diagnostic laboratory were tested. The Nsp2 CP-based ELISA possesses 91.6% agreement with the IDEXX ELISA. In further testing of 202 IDEXX suspect false positive samples, our Nsp2 CP-based ELISA resolved 93.56% of the samples as negative. To differentiate Type 1 and Type 2 PRRSV, we developed an epitope-based ELISA using a conserved epitope, ES2 in the CP region of Type 1 PRRSV. The results showed that the ES2 epitope-based ELISAs are specific for identifying Type 1 PRRSV with 94.4% specificity and 94.5% sensitivity. This project addresses the “proof of concept” phase for new diagnostic assay development and more detailed “full validation” studies will be pursued based on the preliminary data generated from this project. For detailed information about this project, please contact Dr. Ying Fang, Center for Infectious Disease Research and Vaccinology, Veterinary Science Department, South Dakota State University, Brookings, SD 57007; e-mail:; Phone: 605-688-6647"".