2.1 Cells, viruses, and aprotinin
Madin-Darby canine kidney (MDCK) cells were cultured in Growth Medium: 1× Minimum Essential Medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA), 3% L-glutamine (Gibco), 0.75% sodium bicarbonate (Gibco), 1% MEM vitamin solution (Sigma-Aldrich, St. Louis, MO, USA), 50 μg/ml gentamicin (Gibco), and 1% antibiotic-antimycotic antibiotics solution (Gibco).
Seven IAVs and one IBV were used in this study: A/Puerto Rico/8/1934 (A/PR/8/34; H1N1); A/California/04/2009 (A/CA/04/09; H1N1); A/Philippines/2/1982 (A/PH/2/82; H3N2); A/Brisbane/10/2007 (A/Bris/10/07; H3N2); A/Aquatic Bird/Korea/CN2/2009 (A/AB/Kor/CN2/09; H5N2); A/Aquatic Bird/Korea/CN5/2009 (A/AB/Kor/CN5/09; H6N5); A/Chicken/Korea/01310/2001 (A/Ck/Kor/01310/01; H9N2); and B/Seoul/32/2011 (B/Seoul/32/11). The IAVs were grown in 10-day-old embryonated chicken eggs for 48 h at 35 °C. The allantoic fluid was harvested, and aliquots were stored at ‒70 °C until use. The IBV was propagated in MDCK cells in Infection Medium: 1× MEM supplemented with 0.3% bovine serum albumin (Sigma-Aldrich) (instead of FBS) and 1.0 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (Worthington Biochemical Corporation, Lakewood, NJ, USA). After incubation for 72 h, the supernatant was harvested, and aliquots were stored at ‒70 °C until use.
Aprotinin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was dissolved in distilled water to make stock solutions, and aliquots were stored at ‒20 °C until use.