PEDVs were labeled with a lipophilic cationic dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD; Thermo Fisher, USA). A final concentration of 250 μM DiD dye was used to label the purified viruses, and it was first incubated at room temperature for 90 min with a soft vortex to ensure sufficient partition of DiD dye into the viral envelope (23 (link), 24 (link), 42 (link)). Next, excess dyes were removed using a NAP-10 filtration column (GE Healthcare). Finally, DiD-labeled PEDVs were filtered through a 0.22-μm pore filter (Millipore), aliquoted, and stored at −80°C.
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