A novel porcine deltacoronavirus (PDCoV) was reported in China in 2012 and identified in the U.S. in early 2014. Since then, PDCoV has been identified in a number of U.S. states and linked with apparent clinical disease including acute diarrhea and vomiting in the absence of other identifiable pathogens. Since PDCoV was just recently linked with clinical disease, few specific antibody-based reagents were available to assist in diagnosis of PDCoV and limited serological capabilities were available to detect an antibody response to this virus. Therefore, the overall objective of this project was to develop and validate selected diagnostic reagents and assays for porcine deltacoronavirus (PDCoV) antigen and antibody detection. Specific objectives include:
1. Development of specific expressed protein and antibody reagents for diagnostic assay development and confirmation of virus isolation attempts, including critical reagents for immunohistochemistry (IHC), fluorescent antibody (FA) staining and serological and antigen capture assays.
2. Development and validation of an initial generation of serological assays for detection of antibody responses to PDCoV. These assays include indirect ELISA, fluorescent microsphere immunoassays (FMIA) and indirect immunofluorescence assays (IFA).
The nucleoprotein of PDCoV was expressed as a recombinant protein and purified for use as an antigen to immunize rabbits for specific antisera production. The resulting antisera was evaluated for use in fluorescent antibody staining methods to detect PDCoV infected cells following virus isolation attempts and for immunohistochemistry staining of intestinal tissues of infected pigs. These rabbit polyclonal antibodies appear to be specific for PDCoV and do not cross react with related viruses that cause similar disease syndromes, such as porcine epidemic diarrhea virus (PEDV) or transmissible gastroenteritis virus (TGEV). Therefore, they provide another valuable tool, in addition to PCR, for diagnostic laboratories and researchers to identify PDCoV and differentiate it from other related viruses. These reagents have been widely distributed to assist with research studies and diagnosis of PDCoV.
The same expressed nucleoprotein was also used as an antigen to develop serological tests to detect the antibody response to PDCoV in pigs following infection. Several different types of tests were developed to determine the best assays for different applications. Serum samples from swine herds with recent documentation of PDCoV infection and samples from expected naïve herds were used for initial assay optimization. ELISA and FMIA tests were developed since these test formats allow for high-volume testing in diagnostic laboratories. The tests were optimized in a checkerboard fashion to reduce signal to noise ratios using samples of known status. Statistical analysis was performed to establish assay cutoff values and assess diagnostic sensitivities and specificities. At least 260 known negative serum samples and 180 known positive samples were evaluated on each assay. The ELISA showed test sensitivity of approximately 95% and specificity of approximately 94%. The FMIA showed a sensitivity of 95% and specificity of 98%. Both ELISA and FMIA detected seroconversion of challenged pigs between 8-14 DPI.
Once PDCoV was adapted to grow in laboratory cell culture systems, additional serology assays using cell culture adapted virus were developed. An IFA test to detect a general antibody response and a virus neutralization test to quantify neutralizing antibody responses were developed. All of these tests continue to be further optimized and validated. These new tests should allow producers to determine if animals have recently been infected with PDCoV. Since many aspects of PDCoV infection and transmission are still not fully understood, the reagents and assays developed in this project should provide valuable tools to help understand this disease and to aid in the control and surveillance of porcine deltacoronavirus outbreaks.
Eric A. Nelson
Veterinary & Biomedical Sciences Department
Animal Disease Research & Diagnostic Laboratory
South Dakota State University
Brookings, SD 57007-1396