Infectious Virus Titer Determination by Plaque Assay

Infectious virus titers were determined by plaque assays as described elsewhere (47 (link), 48 (link), 91 (link)). Briefly, supernatants were serially diluted 10-fold in MEM with 1 μg/ml TPCK-trypsin and were inoculated onto 90% confluent MDCK cell monolayers in 12-well tissue culture plates (Corning Costar, Cambridge, MA). The virus was adsorbed for 1 h at 37°C under 5% CO₂ before the addition of 3 ml of overlay. The overlay medium contained 1 part liquid medium containing 10× MEM supplemented with 200 mm l-glutamine (Gibco), HEPES solution (Gibco), 7.5% NaCHO₃ (Gibco), penicillin-streptomycin-amphotericin B solution (Gibco), and 1 part 2.4% Avicel (FMC BioPolymer, Philadelphia, PA) in water or 1 part 1% agarose in water. Samples from A/WSN/33 or A/CA/04/2009 wells were incubated at 37°C under 5% CO₂ for 3 days. B/Yamagata/16/1988 was incubated at 37°C under 5% CO₂ for 5 days to allow for better plaque formation. The overlays were removed, the plates were washed twice with PBS, and the cell monolayers were fixed with acetone-methanol (80:20) for 20 min at RT. Following fixation, the plates were stained with crystal violet as described previously, and viral titers were determined (92 (link), 93 (link)).