Plaque Assay for Virus Quantification

The left lobe of the lung was homogenized in 1 mL serum-free DMEM with a disposable tissue grinder. Plaque assays were performed as described.^{13 (link)} In brief, Vero C1008, Clone E6 (ATCC CRL-1586) cells grown in DMEM (GIBCO 11995-040) with fetal bovine serum (FBS) were seeded into at a concentration of 2 × 10⁵ cells/well the night before the assay. Serial dilutions were added to the wells. The plate was incubated at 37°C, 5% CO₂ for 2 hours, shaking the plates every 15 minutes. After 2 hours, the media was replaced with a liquid overlay of DMEM, 2.5% FBS containing 1.2% Avicel PH-101 (Sigma Aldrich, St. Louis, MO) and incubated at 37°C, 5% CO₂. After 3 days, the overlay was removed, wells were fixed with 10% formaldehyde, and stained with 0.1% crystal violet to visualize plaques. Plaques were counted, and PFUs were calculated according to the following equation: Average # of plaques/dilution factor × volume diluted virus added to the well.