Plaque assays were preformed as previously described [26] . Briefly, lungs from HK-x31 infected animals were collected and homogenized using a tissue lyser (Qiagen, Hilden, Germany). Monolayers of Madin-Darby kidney cells were incubated with 10fold serial dilutions of 10% homogenate in dilution media (16MEM, 1 mg/mL TPCK-treated trypsin) for 1 hour at 37uC. Cells were washed with 16 sterile PBS and overlaid with MEM containing 1.2% Avicel microcrystalline cellulose (FMC BioPoly-mer, Philadelphia, PA), 0.04 M HEPES, 0.02 mM L-glutamine, 0.15% NaHCO 3 (w/v), and 1 mg/mL TPCK-treated trypsin. After 72 hours, the overlay was removed, and the cells were washed with 16 sterile PBS, fixed by incubation with cold methanol/acteone (60:40%), and stained with crystal violet.