Effect of Porcine Epidemic Diarrhea Virus Infectious Doses on Infection Outcomes in Naïve Conventional Neonatal and Weaned Pigs

Thomas, Joseph T.; Chen, Qi; Gauger, Phillip C.; Giménez-Lirola, Luis G.; Sinha, Avanti; Harmon, Karen M.; Madson, Darin M.; Burrough, Eric R.; Magstadt, Drew R.; Salzbrenner, Holly M.; Welch, Michael W.; Yoon, Kyoung-Jin; Zimmerman, Jeffrey J.; Zhang, Jianqiang

A U.S. PEDV prototype strain whole virus-based ELISA was developed and validated at the ISU VDL for detection of PEDV-specific IgG antibody. Each batch of one liter of PEDV propagated in Vero cells (infectious titers ranging from 10 5 -10 6 TCID50/ml) were subjected to one freeze-thaw and then centrifuged at 4,000 × g for 15 min to remove cell debris. The virus was then pelleted by ultracentrifugation at 140,992 × g for 3 h. The virus pellet was resuspended in sterile PBS (1× pH 7.4) at a ratio of 1:100 of the original volume and stored at -80°C until use. Polystyrene 96-well microtitration plates (Thermo Fisher Scientific) were coated with the viral antigen solution (100μl/well) and incubated at 4°C overnight. Plates were washed 5 times, blocked (300μl/well) with PBS containing 1% bovine serum albumin (Jackson ImmunoResearch Inc., West Grove, Pennsylvania), and incubated at 25°C for 2 h. Plates were then dried at 37°C for 4 h and stored at 4°C in a sealed bag with desiccant packs until use. Serum samples were 1:50 diluted and added to the coated plates (100μl/well). Plates were incubated at 25°C for 1 h and then washed 5 times with PBS (1× pH 7.4). Subsequently 100μl of peroxidase-conjugated goat anti-pig IgG (Fc) antibody (Bethyl Laboratories Inc., Montgomery, Texas, USA) at 1:25,000 dilution was added to each well and the plates were incubated at 25°C for 1 h. After a washing step, 100μl of tetramethylbenzidine-hydrogen peroxide substrate solution (TMB, Dako North America Inc., Carpinteria, California, USA) was added to each well. The plates were incubated for 5 min at room temperature and the reaction was stopped by adding 50μl of stop solution (1 M sulfuric acid) to each well. Reactions were measured as optical density (OD) at 450 nm using an ELISA plate reader operated with commercial software (Biotek 1 Instruments Inc., Winooski, Vermont, USA). The antibody response in serum samples was represented as sample-to-positive (S/P) ratios calculated as: S/P ratio = (sample OD-blank well control mean OD) / (positive control mean OD-blank well control mean OD). After multiple optimizations, the S/P ratios of >0.8 were considered antibody positive, 0.6-0.8 as suspect, and <0.6 as negative [27, 28] .