2.3  TCID50 Assay
The kinetics of PDCoV multiplication in IPEC-J2 cells was determined by measuring the TCID50 using a microtitration infectivity assay. Briefly, 100 μL/well of 10-fold serial dilutions of PDCoV were inoculated onto IPEC-J2 cells grown in 96-well microplates (Corning, NY, USA). After adsorption at 37 °C for 1.5 h, the inocula were removed, and serum-free DMEM/F12 medium containing 5 μg/mL of trypsin was added to the wells. After an additional 48-h cultivation, virus titers were measured by observing the presence of visible CPEs in the corresponding wells, and calculated using the Reed–Muench method.27