Plaque assays were performed by infecting confluent PK-15 cells in six-well plates with the indicated viruses. The cells were washed three times with 1 × PBS and infected with an appropriate dilution of PRV HN1201 or rPRV HN1201-EGFP-Luc containing the same gene copy number for 1 h at 37 °C and 5% CO2. The viral inoculum was removed, and the cells were washed with 1 × PBS three times. Then, the cell monolayers were overlaid with DMEM (Gibco, Grand Island, NY, USA) containing 1% methylcellulose (M8070, Solarbio) and 2% FBS (10099-141C, Gibco) at 37 °C and 5% CO2 for 36 h. Next, the medium was removed, and the cell monolayer was fixed with 4% paraformaldehyde (P1110, Solarbio) for 1 h and stained with 1% crystal violet (C8470, Solarbio); plaques were then counted. The number of rPRV HN1201-EGFP-Luc plaques was normalized to the number of PRV HN1201 plaques to detect the effect of gI/gE gene knockout on PRV packaging. The plaque diameters were measured from scanned images by using ImageJ.

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