Plaque assay and immunostaining.

Confluent monolayers of MDCK cells (10⁶ cells/well in a 6-well-plate format) were infected for 1 h at room temperature, and after virus adsorption, cells were overlaid with agar and incubated at 33°C or 37°C. At 3 dpi, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After the overlays were removed, cells were permeabilized (0.5% Triton X-100 in 1× PBS) for 15 min at room temperature, and immunostaining was performed as previously described (58, 59), using the NP MAb HB-65 (ATCC HB-65, clone HL16-L10-4R5) and vector kits (Vectastain ABC kit and DAB HRP substrate kit; Vector) according to the manufacturer's specifications.