Quantifying Viral Titers in Lung Tissue

Lungs were weighed and quick-frozen in 10% MEM-10, and plaque assays were performed as described previously (62 (link)). Briefly, thawed lung tissue was dissociated using a GentleMACs Machine (Miltenyi Biotec). Cell suspensions were pelleted to remove cellular debris, and clarified supernatants were serially diluted and inoculated in triplicate on 80% confluent HEp-2 cell monolayers. After rocking for 1 h at room temperature, monolayers were overlaid with 0.75% methyl cellulose in MEM-10 and incubated at 37°C. Cells were fixed with 10% buffered formalin and stained with hematoxylin and eosin on day 4. Plaques were counted and expressed as Log₁₀ PFU/g of lung tissue. The limit of detection was 1.8 Log₁₀ PFU/g.