Lungs were harvested at day 5 pi and homogenized in 1 mL DMEM using GentleMACS tissue homogenizer (Miltenyi Biotec, Gaithersburg, MD) as described (55 (link)). Homogenates were centrifuged at 500 xG at 4°C for 8 min, supernatant was 10-fold diluted in DMEM (Hyclone) and overlaid onto 90% confluent Vero E6 cells in 24-well plates. After 2h of absorption, cells were overlaid with 2% methylcellulose (Sigma Aldrich) and incubated at 37°C for 6 days. Following incubation, methylcellulose was aspirated, wells were washed with PBS, fixed with acetone: methanol (60:40, Sigma-Aldrich), and air-dried overnight. Wells were washed 3x with KPL wash buffer and blocked with blotto overnight at 4°C. The next day, Blotto was removed and a mAb cocktail against RSV F and G proteins (clones 131-2A, 131-2G) was diluted in blotto was added overnight at 4°C. Wells were washed 3x with KPL wash buffer and goat anti-mouse-AP (ThermoFisher) was added overnight at 4°C. Wells were washed 3x with KPL wash buffer and virus plaques were developed with 1-Step™ NBT/BCIP substrate solution (ThermoFisher) for 5 min, rinsed with diH2O, and enumerated using a dissection microscope.
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