FIG 4.

The paracellular route for CDV to enter the airway epithelial cells is governed by nectin-4. Ferret-adapted recombinant CDV-5804PeH and its mutants (SLAM-blind and nectin-4 blind viruses) were used to inoculate canine and ferret ALI cultures. After EGTA pretreatment for 10 min, the dog (A to D) and ferret (E to H) ALI cultures were inoculated with the recombinant viruses (MOI = 0.5) from the apical side as described above. On day 3 postinoculation, the ALI cultures were fixed by 3% PFA and the ciliated cells (β-actin, red) and signals for viral replication (eGFP, green) were visualized using Nikon fluorescence microscope (canine ALI, panels A to C; ferret ALI, panels E to G), and the numbers of virus foci (green) were calculated and statistically analyzed (canine ALI, panel D; ferret ALI, panel H). Bar, 100 μm. Statistical analysis: one-way ANOVA with Tukey’s post hoc test; ***, P < 0.001.