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

Serum samples (N = 147) collected at 0, 7, 14, 21, and 28 DPI from the weaned pig study were tested for antibodies by the PEDV IFA assay following the previously described procedures [24] with modifications. For IFA plate preparation, confluent Vero cells grown in 96-well plates were inoculated with 100 μl/well of the U.S. PEDV prototype strain USA/IN19338/2013 at 1000 TCID 50 /ml and incubated at 37°C, with 5% CO 2 . Twenty four hours later, the inocula were removed and cells were fixed with cold 80% acetone for 10 min. Plates were air dried, sealed and stored at -80°C until use. Serum samples were serially two-fold diluted from 1:40 to 1:5120 in PBS, and 100μl of each diluted serum was added to each well of the IFA plates. After one hour incubation at 37°C, the sera were removed and plates rinsed twice with PBS. Then 60μl/well of 1:50 diluted goat anti-swine IgG (γ) antibody conjugated with FITC (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Maryland) was added to each well and incubated at 37°C for 1 h. Plates were then rinsed twice with distilled water and cell staining was examined under a fluorescent microscope. A positive signal at a serum dilution of 1:40 or higher was considered to be IFA antibody positive.