Tpck treated trypsin

Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco’s minimum essential medium supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories Inc., Dartmouth, MA, USA) and penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA) (100 U/mL) were used in this study for propagating cell and viral cultures. The IDV strain used for the study was influenza D/bovine/California/0363/2019 virus. The virus was propagated on MDCK cells. Following infection, fresh DMEM with 1 μg/mL tolyl-sulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma, Saint Louis, MO, USA) was added and incubated at 33 °C in 5% CO₂ for 5 days. The supernatant was spun at 500× g for 10 min at 4 °C to remove the cellular debris. The virus titers were determined using MDCK cells according to the method of Reed and Muench [18 (link)]. Virus loads in nasopharyngeal swabs were also titrated using MDCK cells. DMEM supplemented with penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA) (200 U/mL) and TPCK-treated trypsin (Sigma, Saint Louis, MO, USA) (1.0 μg/mL) was used as the virus growth and maintenance medium.

Recombinant influenza virus PR8-NS1(1-73)-GFP was rescued using the A/Puerto Rico/8/34 based reverse genetics system40 (link). To generate recombinant PR8-NS1(1-73)-GFP virus, 1 μg of each pHW-plasmid (pHW191-PB2, pHW192-PB1, pHW193-PA, pHW194-HA, pHW195-NP, pHW196-NA, pHW197-M and pHW-NS1(1-73)Dmd-GFP-NEP) was transfected in a HEK293T/MDCK coculture using calcium phosphate precipitation in Optimem. After 36 h, TPCK-treated trypsin (Sigma) was added to a final concentration of 2 μg/ml. After 72 h, the medium was collected. The virus in the medium was amplified on MDCK.PIV5V cells in serum-free cell culture medium in the presence of 2 μg/ml TPCK-treated trypsin (Sigma).

MDCK cells were seeded at 5 × 10⁴ cells per well in Dulbecco’s modified eagle medium (DMEM; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (Gibco, Logan, UT, USA) in 24-well plates under 5% CO₂ at 37 °C for overnight. When the cells reached approximately 80% confluences, the media were removed. The cells were infected with mock influenza B virus Victoria lineage (B/Thailand/CU-B5522/2011), or Yamagata lineage (B/Massachusetts/2/2012) at the multiplicity of infection (MOI) of 0.01. After incubation with each viral suspension in overlay medium (DMEM supplemented with 0.2 µg/mL TPCK-treated trypsin (Sigma-Aldrich, St. Louis, MO, USA)) for 1 h, the viral suspensions were removed. The cells were washed with phosphate buffer saline (PBS; Merck Millipore, Darmstadt, Germany), and cultured with fresh infection medium (DMEM supplemented with 0.2% (w/v) bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA)), and 0.2 µg/mL TPCK-treated trypsin under 5% CO₂ at 37 °C for 24 h.

Completely differentiated ALI-PREC cultures were inoculated with 250 μl of a 1:1 dilution of PHEV 67N (1:128 HA titer) and infection medium for ALI-PRECs containing DMEM/F-12 supplemented with 2% Ultroser G, 1× MEM nonessential amino acids, 1× HEPES, Pen-Strep, and 2 μg/ml N-tosyl-l-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Millipore-Sigma) or mock inoculated with infection medium and incubated for 6 h at 37°C and 5% CO₂. Then, the inoculum was removed, the cell cultures were washed once with DMEM/F-12, fresh infection medium was added into the plate wells, and plates were incubated for 24, 36, and 48 h postinoculation (hpi) at 37°C with 5% CO₂. ALI-PREC cultures were monitored daily under the microscope for the presence of cytopathic changes, and subnatants were collected at different time points over the course of the infection for assessment of virus replication.

Influenza viruses, A/Michigan/45/2015 (Flu A/H1N1), A/Kansas/14/2017 (Flu A/H3N2), B/Phuket/3073/2013 (Flu B/Phuket), and B/Colorado/06/2017 (FluB/Colorado), were propagated in MDCK cells using EMEM supplemented with 0.1% bovine serum albumin (Hyclone; Logan, UT, USA), 1% l-glutamine, 1 mM Sodium Pyruvate, 1% non-essential amino acids, 1 × penicillin–streptomycin, 25 mM HEPES (ThermoFisher Scientific, Waltham, MA USA), 1% l-glutamine, and 1 mg/ml TPCK-treated trypsin (Millipore Sigma; Oakville, ON, Canada). SARS-CoV-2 (hCoV-19/Canada/ON_ON-VIDO-01-2/2020; Accession ID: EPI_ISL_425177), human Rhinovirus 49 (Strain 8213, ATCC VR-1644), and RSV (Strain long, ATCC VR-26) were amplified in Vero cells, HeLa cells, or Hep2 cells, respectively, using EMEM supplemented with 2% FBS, 1 mM Sodium Pyruvate, 1% non-essential amino acids, 1 × penicillin–streptomycin, and 1% l-glutamine. Human Coronavirus 229E (229E, ATCC VR-740) and Human Coronavirus OC43 (OC43, ATCC VR-1558) were amplified in MRC7 cells using DMEM supplemented with 2% FBS, 1 mM Sodium Pyruvate, 1% non-essential amino acids, 1 × penicillin–streptomycin, and 1% l-glutamine. Virus stocks were inactivated by gamma irradiation; SARS-CoV-2 was irradiated using 3Mrads and the remaining viruses were irradiated with 2Mrads. All viruses were safety tested prior to use in containment level 2.

Five million cells or 50 mg of tissue sample was used as starting material and lyophilized. After lyophilization, the samples were resuspended in 1 mL of 500 μg/mL TPCK-treated trypsin (MilliporeSigma) solution and incubated at 37°C overnight. The trypsin-digested samples were then loaded onto the columns before being washed with 6 mL of 5% acetic acid. Peptides were eluted with 2 mL of 20% 1-propanol, then 2 mL of 40% 1-propanol followed by 2 mL of 100% 1-propanol. The lyophilized peptides were resuspended in 200 μL of 50 mM ammonium bicarbonate, to which 3 μL of PNGase F (New England Biolabs) was added for a 4 hour incubation at 37°C. The PNGase F–treated samples were loaded onto the columns before being washed with 6 mL of 5% of acetic acid. Flow-through and wash fraction containing the released N-glycans were collected, pooled, and lyophilized and were ready for permethylation. If O-glycans were to be analyzed, the PNGase F–treated glycopeptides were eluted from the column with 2 mL of 20% 1-propanol, then 2 mL of 40% 1-propanol, and then 2 mL of 100% 1-propanol. Fractions were pooled and lyophilized and were ready for O-glycan preparation. For more details please refer to Supplemental Methods.

MDCK cells were seeded in plain Dulbecco’s modified Eagle medium (Gibco DMEM, Thermo Fisher Scientific) containing 10% FBS in 96-well plates overnight. The cells were washed 3 times with Virus Growth Medium (VGM) (DMEM with 2% BSA and 2 μg/mL TPCK-treated trypsin, MilliporeSigma) and 100 μL of 1 multiplicity of infection (MOI) of A/Shanghai/2/2013 (H7N9)-PR8-IDCDC-RG32A virus in VGM added to the cells and incubated for 3 hours at 37°C in 5% CO₂. The cells were then washed with VGM again and replenished with VGM containing 3-fold serial dilutions of mAbs or zanamivir (GlaxoSmithKline), starting at the highest concentration of mAbs 10 μg/mL or equimolar. The plates were incubated for 21 hours at 37°C in 5% CO₂, and the supernatants were collected for performing the HA assay. For HA assay, we used turkey red blood cells (Rockland Immunochemicals) that were washed and diluted to 0.5% in PBS. A volume of 50 μL of the supernatants was incubated with 50 μL of the 0.5% turkey red blood cells in V-bottom plates for 1 hour at 4°C. The IC₁₀₀ values were defined as the lowest antibody concentration added to infected MDCK cells that corresponded to absence of virus in supernatant according to HA of red blood cells.