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) from the weaned pig study were tested for PEDV neutralizing antibodies. Serum samples were first inactivated at 56°C for 30 min, then 2-fold serially diluted from 1:4 to 1:512 dilutions in 96-well plates with a volume of 75μl per well after dilution. Subsequently 75μl of 4 × 10 3 TCID 50 /ml of PEDV strain USA/IN19338/2013 was mixed with the equal volume of diluted sera and incubated for 1 h at 37°C with 5% CO 2 . Then 100μl of the serum-virus mixture (containing 200 TCID 50 of virus) was transferred to 96-well plates with a Vero cell monolayer, and the plates were incubated for 1 h at 37°C. The cells were washed twice with post-inoculation media and incubated with 100μl/well of such media for 48 h at 37°C with 5% CO 2 . Then cells were fixed with cold 80% acetone and stained with PEDV N protein-specific monoclonal antibody SD6-29 conjugated to FITC (Medgene, Brookings, South Dakota) at a 1:100 dilution for 40 min. The staining was examined under a fluorescent microscope. The reciprocal of the highest serum dilution resulting in >90% reduction of staining as compared to the negative serum control was defined as the VN titer of the serum sample. A VN titer of 8 was considered positive.