Picornavirus Infection Cell Staining

For cell staining, cells were plated at 1 × 10⁴ cells/well in 96-well plates and incubated overnight. The cells were then infected with PeV-A3 at multiplicity of infection (MOI) = 5. After 8- and 24-h infection, the cells were fixed with 1:1 Methanol/Ethanol solution (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Cells were incubated with blocking buffer [10% skim milk in phosphate-buffered saline (PBS)] and incubated with anti-PeV VP0 polyclonal antibody at 4°C overnight (LTK BioLaboratories, Taoyuan, Taiwan) (10 (link), 29 (link)). Then, the IRDye^® 800CW goat anti-rabbit IgG secondary antibody was added into the cells (Li-COR, #926-32211, Lincoln, NE, USA), followed by 2-h incubation. Images of immunofluorescence assay were captured, and the fluorescence intensity of PeV-A3-infected cells were quantified using Odyssey image system (Li-COR, Lincoln, NE, USA).