Agarose

Whole lung samples were thawed on ice. CA/09 lung samples were weighed and per 0.1 g a volume of 1 ml of Dulbecco modified Eagle medium (DMEM) supplemented with penicillin-streptomycin (P/S) was added. For SW/NC/152702/15 lung samples, whole lungs were suspended in 1 ml of DMEM + P/S. The tissue was macerated through a 0.70 μm nylon filter (Corning Cell Strainer, Sigma Aldrich, St. Louis, MO, USA) until thoroughly homogenized. Lung filtrate was then enumerated for viral lung titers. Ten-fold serial dilutions of lung homogenate were overlaid onto MDCK cells seeded at 5 × 10⁵ cells per well of a six-well plate. Samples were incubated for 1 h at RT with intermittent shaking every 15 min. Medium was removed, and the cells were washed twice with DMEM + P/S. Wash medium was replaced with 2 ml of L15 medium with 2 µg/ml TPCK-trypsin plus 0.8% agarose (Cambrex, East Rutherford, NJ, USA) and incubated at 37°C with 5% CO₂ for 72 h and 48 h for CA/09 and SW/NC/152702/15, respectively. agarose was removed and discarded. MDCK cells were fixed with 10% buffered formalin for 10 min and stained with 1% crystal violet for 15 min. The plates were thoroughly washed in distilled water to remove excess crystal violet, and the plaques were counted and recorded to determine the PFU per ml lung homogenate.

Plaque assays were performed to determine the viral burden in snap-frozen nasal washes, as previously described in (Bissel et al., 2019 (link)). Supernatants were gently thawed on ice, forced through a cell strainer (70 μm) and syringe plunger in phosphate-buffered saline, then spun down (2500 rpm, 5 minutes, 4°C) to collect the supernatant. Supernatants were diluted in Iscove’s modified Dulbecco’s minimum essential medium. Madin-Darby canine kidney (MDCK) cells were plated (5 × 10⁵) in each well of a six-well plate. Samples were diluted in Iscove’s modified Dulbecco’s minimum essential medium (final dilution factors of 10⁰ to 10⁻⁶) and overlaid onto the cells in 100 μL of Dulbecco’s modified Eagle’s medium supplemented with penicillin-streptomycin followed by 1 hour incubation. Samples were removed, cells were washed twice, and medium was replaced with 2 ml of L15 medium plus 0.8% agarose (Cambrex, East Rutherford, NJ, USA) and incubated for 72 hours at 37°C with 5% carbon dioxide. agarose was removed and discarded. The cells were fixed with 10% buffered formalin and then stained with 1% crystal violet for 15 minutes. After thorough washing in distilled water to remove excess crystal violet, the plates were dried, the number of plaques was counted, and the number of PFU per milliliter was calculated.

Plaque assays were performed to determine the viral burden in lung tissue (52 (link)). Lung tissue supernatants were obtained from frozen lung pieces that were gently thawed on ice, forced through a cell strainer (70 μm) and syringe plunger in PBS, and then spun down (2500 rpm, 5 min, 4°C) to collect the supernatant. Lung supernatants were diluted in Iscove’s modified Dulbecco’s minimum essential medium.
Madin-Darby canine kidney cells were plated (5 × 10⁵) in each well of a six-well plate. Samples were diluted (final dilution factors of 10⁰ to 10⁻⁶) and overlaid onto the cells in 100 μl of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin-streptomycin and incubated for 1 hour. Samples were removed, cells were washed twice, and the medium was replaced with 2 ml of L15 medium plus 0.8% agarose (Cambrex, East Rutherford, NJ, USA) and incubated for 72 hours at 37°C with 5% carbon dioxide. agarose was removed and discarded. The cells were fixed with 10% buffered formalin and then stained with 1% crystal violet for 15 min. After thorough washing in distilled water to remove excess crystal violet, the plates were dried, the number of plaques was counted, and the number of plaque-forming units (PFU) per milliliter was calculated.