FIG 1.

PDCoV entry into IPI-2I cells requires an acidic endosomal pH. (A) CCK-8-based cell viability assay for chloroquine as described in Materials and Methods. (B) Chloroquine inhibited PDCoV entry but not binding. IPI-2I cells were pretreated with subtoxic doses at 37°C for 1 h and infected with PDCoV (MOI of 5) at 4°C for 1 h (binding step) and then shifted to 37°C for 1 h (entry step). Cells were lysed to determine viral RNA copy numbers by RT-qPCR. (C and D) RT-qPCR, Western blotting, and viral titer detection analysis for viral infection inhibition by chloroquine. Cells were pretreated with increasing subtoxic doses of chloroquine or DMSO at 37°C for 1 h and then inoculated with PDCoV (MOI of 5) at 37°C for 6 h. Cells were lysed to determine viral RNA copy numbers via RT-qPCR, N protein levels via Western blotting (C), and viral titer (D) via TCID₅₀ assay. (E) IFA for viral infection inhibition by chloroquine. Cells were pretreated with subtoxic doses of these drugs at 37°C for 1 h and then inoculated with PDCoV (MOI of 5) at 37°C for 6 h. Cells were fixed and subjected to IFA. Relative fluorescence intensity was quantified by Image‐Pro Plus software as shown on the right. (F) CCK-8-based cell viability assay for NH₄Cl as described in Materials and Methods. (G to J) RT-qPCR, Western blot analysis, viral titer detection, and IFA for viral entry and infection inhibition by NH₄Cl. Cells were pretreated with increasing subtoxic doses of NH₄Cl or H₂O, followed by RT-qPCR (G), Western blotting (H), viral titer detection (I), and IFA (J) as described in corresponding panels B, C, D, and E. (K to N) Effect of chloroquine and NH₄Cl on the entry of VSV-GFP and SeV into IPI-2I cells. The cells were pretreated with increasing subtoxic doses of chloroquine (K and M) or NH₄Cl (L and N) and then were infected with VSV-GFP (K and L) and SeV (M and N) at 4°C for 1 h (binding step) and then shifted to 37°C for 1 h (entry step). Cells were lysed to determine viral RNA copy numbers by RT-qPCR. Target protein expression was quantitatively estimated by ImageJ software and presented as the density value relative to that of the β-actin. The presented results represent the means and standard deviations of data from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.