Measurement of viral load

Whole lung tissue was harvested in PBS at the indicated times, homogenized, and stored at − 70 °C. Virus titers were measured with a standard plaque assay by infecting MDCK cell monolayers with serial 5-fold dilutions of lung suspension in duplicate. Eighteen to twenty-four hours after infection, monolayers were washed and fixed with 80% acetone in water. Infected cell clusters were detected with a biotin-labeled mouse anti-influenza A monoclonal antibody (Chemicon), followed by staining with streptavidin-AP, and visualized with Sigma Fast BCIP/NBT substrate (Sigma). The number of viral-foci units (VFUs) was counted, and the data were shown as the VFU/lung.