2.4.3. Growth Kinetics, Viral Titration and Plaque Assay
Confluent monolayers of Vero cells were seeded onto six-well plates (5 × 105 cells/well) and infected with each virus at a MOI of 1 and 0.001 at 37 °C for 1 h in triplicates. The cells were then washed twice with Dulbecco’s phosphate-buffered saline (DPBS) and maintained in PI medium. The supernatants at indicated time points were collected and proceeded for viral quantification on Vero cells in 96-well plates using the standard 50% tissue-culture infectious dose (TCID50) assay. In brief, Vero cells in 96-well plates were washed twice with DPBS and then incubated with a 10-fold serially diluted culture supernatant acquired from the aforementioned six-well plate at 37 °C for 1 h. After absorption, the inoculum was removed and replaced with fresh PI-medium following one wash step. The titers were determined at 72 h post-infection using the Reed–Muench method [21].
Plaque assays were performed as previously described [19] to characterize plaque morphologies. Briefly, after absorption of PEDVs at an MOI of 0.0001, confluent monolayers of Vero cells in six-well plates were washed twice with DPBS and then covered with an overlay of pre-warmed PI medium containing 1% agarose. After solidification of the overlays, the plates were incubated at 37 °C for 72 h to produce distinct plaques. The cells were then fixed in 3.16% neutral-buffered formalin for 1 h before removing the semisolid overlays. The plates were stained with 1% crystal violet in 20% ethanol and distilled water for 1 min. Viral plaques were inspected after washing off the crystal violet solution, rinsing the plates with water, and air-drying at room temperature (RT).