Senecavirus A (SVA) is a member of the Picornaviridae family and is associated with vesicular disease and neonatal mortality in swine. The primary concern of SVA associated disease is the development of vesicular lesions which are clinically indistinguishable from foot-and mouth disease, a highly contagious and economically devastating foreign animal disease. Currently, there is no vaccine and the diagnosis of SVA relies on the detection of viral RNA. Also, commercially available serological tests for herd surveillance or identification of new outbreaks are limited. The overall objective of this study was to develop improved diagnostic capabilities for SVA to fulfill industry needs for the differentiation between SVA and other picornaviruses in circulation. The specific objectives addressed include: 1) Development of monoclonal antibody (mAb) reagents for diagnostic assays including immunohistochemical (IHC) antigen detection and a blocking ELISA (bELISA); 2) Development and validation of a consistent and reproducible highly specific bELISA for the detection of SVA antibodies in serum. For our first objective, we addressed the need for SVA antigen and antibody reagents for use in various diagnostic tests. We initially identified conserved epitopes on the major capsid regions of SVA-VP2 that allowed for differentiation of SVA from other picornaviruses. We successfully cloned, expressed and purified the VP2 protein for use as antigen and for the immunization of mice for mAb development and the development of a bELISA for the detection of swine antibodies after disease outbreak. After a two-month immunization regimen, antibody producing hybridoma cells were produced from the fusion of mouse spleen cells and a mouse tumor cell line. Following screening of hybridoma cells, antibody ascites fluid was produced from selected hybridoma lines and purified. These mAbs were then characterized and evaluated for their ability to specifically recognize available strains of SVA by fluorescent antibody staining and IHC. In addition, these mAbs were utilized in development of a bELISA. The bELISA was optimized for providing a high degree of specificity then validated against a panel of serum of known SVA serostatus. The bELISA demonstrated a diagnostic specificity of 93.6% and a sensitivity of 88.2%. The bELISA shows enhanced specificity over our initial indirect ELISA format to differentiate between SVA, FMD and other picornaviruses that may share some common epitopes causing testing uncertainty. The bELISA as optimized, has the ability to detect a broad antibody response as soon as 5 days post infection including a robust IgM response which is produced early during infection. The significance of this test is that is it able to simultaneously detect the appearance of all antibody isotypes, thus decreasing the time of detection of a serological response. The bELISA is now validated for use to provide an additional testing platform for herd surveillance and confirmation of disease. In addition, resultant mAbs have also allowed for the development of an IHC test for routine in-situ detection of viral protein antigen within tissues, and a more sensitive serum virus neutralizing assay (SVN) capable of detecting infection beyond 6 weeks post infection. Taken together, both tests and antibody reagents will provide producers much needed, sensitive and specific assays to serologically monitor and differentiate incidences of vesicular disease outbreak.

Contact information: Steve Lawson, Veterinary & Biomedical Sciences, Animal Disease Research & Diagnostic Laboratory, South Dakota State University, Brookings, SD 57007-1396; Phone 605-688-5171; email