Multicycle Virus Growth Kinetics in MDCK Cells

Multicycle virus growth kinetics were carried out in confluent MDCK cells (12-well plate format, 5 × 10⁵ cells/well, triplicates) infected with the indicated viruses at multiplicity of infection (MOI) of 0.001. After 1 h of virus adsorption at room temperature, cells were washed and overlaid with DMEM containing 0.3% BA and TPCK-treated trypsin and incubated at 33 °C or 37 °C. At the indicated times post infection (24, 48, 72, and 96 h), virus titers in the tissue culture supernatants were determined by immuno-focus assay (fluorescent focus-forming units, FFU/mL) using a mouse monoclonal antibody (MAb) against IAV NP (MAb HB-65; ATCC H16-L10-4R5) and a fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Dako), as previously described [11 (link),30 (link),69 (link)]. The mean value and standard deviation (SD) were calculated using Microsoft Excel software. Cell monolayers were imaged to evaluate mCherry expression, and the presence of NLuc was quantified using Nano-Glo luciferase substrate (Promega, Madison, WI, USA) following the manufacturer’s specifications.