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. 2015 May 13;89(15):8077–8081. doi: 10.1128/JVI.00356-15

FIG 2.

FIG 2

Recombinant PEDV encoding S of the PEDV CV777 strain with an artificial furin cleavage site at the S2′ site (PEDV-SFCS) mediates trypsin-independent cell-cell fusion. (a) Cells were infected with GFP-expressing recombinant PEDVs carrying wild-type CV777 S (PEDV-SWT) or mutated CV777 S (PEDV-SFCS) in the presence of trypsin (15 μg/ml) or soybean trypsin inhibitor (SBTI; 40 μg/ml), as indicated. At 2 h postinfection, cells were washed and further cultured in the presence of soybean trypsin inhibitor (SBTI; 40 μg/ml) or trypsin (15 μg/ml), as indicated. Cells were fixed at 12 h p.i. Nuclei were stained with DAPI (blue), and images of PEDV-infected cells (GFP positive, green) were acquired. Experiments were repeated two times, and representative images are shown. (b) Vero cells were infected with PEDV-SWT and PEDV-SFCS in the presence of trypsin for 2 h and further cultured in the presence of soybean trypsin inhibitor (as indicated above). At 12 h p.i., the numbers of nuclei per focus of infection were counted. Statistical significance was assessed by unpaired one-tailed Student’s t test; **, P < 0.01. (c) Vero cells were infected with recombinant PEDV-SWT and PEDV-SV888R (MOI 0.01) in the absence or presence of trypsin. The infectivity in the culture medium was monitored by taking small samples from the medium at various time points postinfection and titrating them on Vero cells in the presence of trypsin. TCID50, 50% tissue culture infective dose. (d) Vero cells were mock infected or infected with PEDV-SWT and PEDV-SFCS in the presence of the trypsin inhibitor for 2 h (MOI, 10) and further cultured in the absence of trypsin, as described for panel a. At 24 h p.i., the cell culture supernatants of these cultures were pelleted through a 20% sucrose cushion. Pellets were dissolved in Laemmli sample buffer and subjected to Western blotting. Virion-incorporated SWT and SFCS proteins were detected by their C-terminally appended FLAG tag using an anti-Flag MAb (Sigma). Sizes of marker proteins are indicated at the left in kilodaltons.