Virus titers (plaque forming units (PFU)/ml) were determined by plaque assay in Vero cells. Monolayers of Vero cells in TC6-well plates were inoculated with 10-fold serial dilutions of virus. After 1 hour adsorption, the inoculum was aspirated, the monolayer was overlaid with optiMEM diluted 1:1 with 2% methylcellulose, and the plates were incubated for 7 days at 35°C. Plaques were immunostained with ferret polyclonal antisera directed to hMPV diluted 1:500 in PBS containing 5% powdered milk (w/v) (PBS-milk). The cells were then incubated with horseradish peroxidase-conjugated goat anti-ferret antibody (Dako) diluted 1:1000, followed by incubation with 3-amino-9-ethylcarbazole (AEC) (Dako) to visualize plaques. Ferret polyclonal antisera were generated by collecting blood 4 weeks after infection of 8-10 week old ferrets with 6.0 log 10 wthMPV/NL/1/00 administered intranasally.