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MDCK cells were cultured in 6-well plates (4 × 105 cells/well) for 24 h. Antiviral effects were evaluated using two drug treatment approaches: “During infection” and “Pretreatment of the virus”, as mentioned above [17]. After treatment, cells were washed with PBS to remove the free virus and then cultured in 3 mL of serum-free MEM (2×) containing 1× TPCK-trypsin (1.6% AGAR for 72 h, as mentioned above). The cells were then fixed with 4% paraformaldehyde for 20 min, and then incubated with a 0.5% (w/v) crystal violet dye solution for 1 h at 37 °C. The effect of PPa on viral plaque formation was determined by counting the number of plaques.
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