Lung Viral Plaque Assay Protocol

Plaque assays were performed as previously described (31 (link)). Briefly, whole lungs isolated from infected mice were isolated, weighed, and homogenized using a Tissue Lyser (Qiagen, Hilden, Germany). Serial dilutions of 10% homogenate were made in dilution media (1×MEM, 1 μg/mL TPCK-treated trypsin) and incubated for 1 hour atop confluent monolayers of Madin-Darby canine kidney cells (MDCKs) grown in 12 well plates for 1 hour at 37°C. Following infection, cell layers were washed with PBS and overlaid with MEM containing 1.2% Avicel microcrystalline cellulose (FMC BioPolymer, Philadelphia, PA), 0.04 M HEPES, 0.02 mM L-glutamine, 0.15% NaHCO₃ (w/v), and 1 μg/mL TPCK-treated trypsin. After 72 hours at 37°C, the overlay was removed, and the cells were washed with PBS, fixed by incubation with cold methanol/acetone (60:40%), and stained with crystal violet. Plaques were counted and plaque-forming units per mg of lung tissue determined.