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

The virus stock and serial dilutions were back titrated in Vero cells to determine their infectious titers in the unit of median tissue culture infective dose per milliliter (TCID 50 /ml) and plaque forming unit per milliliter (PFU/ml). To determine TCID 50 /ml, each sample was serially 10-fold diluted in post-inoculation media and inoculated into Vero cells grown in 96-well plates, 100μl per well, triplicate wells per dilution. The plates were incubated at 37°C with 5% CO 2 for 5 days. Viral cytopathic effects (CPE) were recorded daily. After 5-day inoculation, the plates were subjected to immunofluorescence staining using a monoclonal antibody conjugate SD6-29 against the PEDV nucleocapsid protein (SD-1F-1, Medgene Labs, Brookings, South Dakota, USA). The virus titers were determined according to the method described by Reed and Muench [23] and expressed as TCID 50 /ml. To determine PFU/ml, each sample was serially 10-fold diluted in post-inoculation media and inoculated into Vero cells grown in 6-well plates, 500μl per well, duplicate wells per dilution. After incubation at 37°C for 1 h, 4ml of overlay medium (post-inoculation media supplemented with 0.75% carboxymethyl cellulose) was added to each well. The plates were then incubated undisturbed at 37°C with 5% CO 2 for 4 days. The media were discarded and the plates were stained with crystal violet in 10% buffered formalin. The bottoms of the plates were then illuminated. Wells containing 20-200 plaques were counted and titers expressed as PFU/ml.