L 1 tosylamido 2 phenylethyl chloromethyl ketone tpck trypsin

Influenza virus A/PR/8/1934 (PR8) (H1N1) was propagated in the allantoic cavity of embryonated chicken eggs at 37°C for 48 hrs. Influenza virus A/Philippines/2/1982/X-79 (H3N2): PR8 (H1N1), a 2:6 recombinant influenza virus containing HA and NA from the influenza A/Philippines/1982 and all the other segments from influenza virus PR8, and the mouse-adapted influenza A/Netherlands/602/2009 (pH1N1) were kindly provided by Drs. F. Krammer and P. Palese (Mount Sinai School of Medicine, New York, NY). All viruses were titrated on MDCK cells in the presence of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-trypsin (Sigma-Aldrich), as previously described [26] (link). Resulting virus stocks were aliquoted and stored at −80°C until use. The LD₅₀ of the influenza viruses was calculated in female BALB/c mice by the method of Reed and Muench [27] . All experiments were reviewed and approved by the institutional biosafety program at Weill Medical College of Cornell University.

IAV stocks (H1N1 A/Puerto Rico/8/34, pandemic H1N1 A/Luxembourg/46/2009, seasonal H3N2 A/Luxembourg/01/2005, H7N9 A/Anhui/01/2013) were grown on MDCK cells using serum free virus growth medium supplemented with 2 µg/ml L-1-tosylamido-2-phenylethyl chloromethylketone-(TPCK) trypsin (Sigma-Aldrich). Half maximal tissue culture infectious dose (TCID50) determinations of IAV were done on MDCK cells, incubating them in quadruplicates for 3 days at 37°C and 5% CO2 with 3-fold serial dilutions of virus-containing supernatant. The cytopathic effect was scored and TCID50 was calculated by the ID-50 5.0 program (http://www.ncbi.nlm.nih.gov/CBBresearch/Spouge/html_ncbi/html/index/software.html#1). Adenovirus (Type 5, ATCC reference strain) was propagated and titered by TCID50 determination on A549 cells, measles virus stocks (Schwarz vaccine strain, GSK, Belgium) on VeroSlam cells.

The viruses and cell lines used in the study are described in Table 1. Cells were maintained in minimal essential medium (MEM) (Corning^®, Mediatech, Inc., Manassas, VA, USA) and supplemented with 10% fetal bovine serum (FBS) (Seradigma^®, VWR International, LLC, Radnor, PA, USA), 2 mM L-glutamine (Corning^®), 1% Antimycotic-Antibiotic 100× (Gibco™, Grand Island, NY, USA), and 50 µm/mL of gentamicin (Corning^®). PEDV was amplified in FBS-free media supplemented with 2 µg/mL of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) trypsin (Sigma-Aldrich^®, St. Louis, MO, USA). Cells were incubated at 37 °C and in a 5% CO₂ environment. Virus stocks were titrated using the Reed and Muench method [12 (link)], and the titers were calculated and expressed in a median tissue culture infectious dose per milliliter (TCID₅₀/mL).