2.4.1. Immunofluorescence Assay and Syncytia Analysis
Immunofluorescence assay (IFA) was performed to detect PEDV antigens as previously described with modifications [18]. Briefly, Vero cells in 96-well plates (1.75 × 104 cells/well) were infected with the designated P1viruses at a multiplicity of infection (MOI) of 0.005. At 18 h post-infection, cells were fixed with 80% ice-cold acetone, air-dried, and then incubated with an in-house anti-PEDV S antibody, P4B [20], at a dilution of 1:1000 at room temperature (RT) for 1 h (h). After being washed three times with phosphate-buffered saline (PBS), the FITC-conjugated monoclonal goat anti-mouse-IG antibody (BD Pharmingen, San Jose, CA, USA) was applied at a dilution of 1:500 at RT for 1 h. Following the final wash step, the cells were counterstained with mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Abcam, Cambridge, MA, USA) in the dark for 1 min. Images were visualized and captured using ZOE fluorescent cell imager (Bio-Rad, Hercules, CA, USA). Syncytia analysis were performed concurrently along with IFA by calculating the number of nuclei per syncytium.