2.3. Viral Titration and Measurement of Cytokines in Bronchoalveolar Lavage (BAL) Fluids

For viral titration and cytokine measurements, bronchoalveolar lavage (BAL) fluids from infected mice were collected at the indicated time points by washing with 1 ml of PBS [16].

The viral titers were determined by a standard PR8 plaque assay on Madin–Darby canine kidney (MDCK) cells, as previously reported [17]. Briefly, MDCK cells (1 × 10⁶ cells/well) were cultured in a 6-well plate and incubated in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Welgene, Daegu, Korea) overnight at 37 °C. After confluency, culture media were removed, and serially-diluted BAL fluids (200 μL) were added to the wells. Cells were then incubated at 37 °C for 1 h with shaking every 20 min, after which they were washed twice with PBS. Afterwards, cells were incubated with 2 ml of a 0.6% agarose medium per well at 37 °C for 72 h. The number of plaques was counted.

The levels of IL-6, IL-12p40, and IL-33 in BAL fluids were measured by an enzyme-linked immunosorbent assay (ELISA; BD Bioscience, San Jose, CA, USA), according to the manufacturer’s protocol.