*2.4. Experiments with Ferrets*

Three groups of three seronegative (800- to 1000-*g*) male ferrets (Triple F Farms Inc., Gillett, PA, USA) were housed consecutively in the system for 7 to 12 days. The ventilation system was set at 200 L/min, with 160 holes of the particle separator for all experiments. Ferrets housed in cage one were infected intra-nasally with 250 μL (125 μL per nostril) containing 4.5 log TCID50/mL of the A/California/7/2009 (H1N1) influenza A virus. Nasal wash was collected every day by instillation of 5 mL Phosphate-Buffered Saline (PBS) into the intranasal cavity.

System ventilation was stopped before opening the cages' sealed doors. Animals were manipulated in the following order: first the ferret from cage three, followed by the ferret from cage two, and then the ferret from cage one. Viral titer from the nasal wash was determined by plaque assay on ST6GalI-MDCK cells.

Air samples were collected every day using NIOSH two-stage bioaerosol cyclone samplers and SKC BioSamplers. Air samplers were connected to sampling ports located in cage two (between the perforated grates of cages one and two) as well as in cage three (between the particle separator and the perforated grate). Air sampling with NIOSH two-stage bioaerosol cyclone samplers was performed at 2 L/min for 24 h. At this flow rate, the cut-off separations of the NIOSH two-stage bioaerosol cyclone sampler were: 4 μm for first stage, 1.7 μm for second stage, and the remaining particles were collected on the backup filter. Air sampling started when ferrets were placed in cages after the infection of the ferret from cage one, and was stopped before shutting down the ventilation system for the daily nasal wash. Samples were eluted from NIOSH two-stage bioaerosol cyclone samplers by vortexing for 1 min in MEM (minimal essential medium; 5 mL in first stage, 500 μL in second stage, 5 ml in backup filter). Air sampling with SKC BioSamplers was performed at 11–14 L/min (determined by critical opening of the instrument) for 20 min and was set before shutting down the ventilation system for daily animal care. SKC BioSamplers were filled with 20 mL of MEM (minimal essential medium) without bovine serum albumin (BSA). After air sampling, 150 μL of BSA was added to the remaining liquid of the SKC BioSampler. Air samples were kept frozen at −80 ◦C until further quantitation. The virus concentration in NIOSH two-stage bioaerosol cyclone air samples was measured using qPCR [16]. The virus concentration in BioSampler air samples was measured using plaque assays on ST6GalI-MDCK cells and embryonated chicken eggs [17].

Animal procedures were approved by the Institutional Animal Care Committee of Université Laval according to the guidelines of the Canadian Council on Animal Care (protocol 2015031).
