Viral RNA was extracted from ferret nasal washes by processing 200 μl through the Purelink Pro 96 Viral RNA/DNA Purification Kit (Thermofisher 12280-096A). The viral load in each sample was measured by RT-qPCR using primers/probe specific for the open reading frame of segment 7 (M1/M2) of influenza A virus: forward primer 5’- GACCRATCCTGTCACCTCTGAC-3’, reverse primer 5’- AGGGCATTYTGGACAAAKCGTCTA-3’, and probe 5’-(FAM)- TGCAGTCCTCGCTCACTGGGCACG-(BHQ1)-3’. Each reaction contained 5.4 μL of nuclease-free water, 0.5 μL of each primer at 40 μM, 0.1 μL of ROX dye, 0.5 μL SuperScript III RT/Platinum Taq enzyme mix, 0.5 μL of 10 μM probe, 12.5 μL of 2x PCR buffer master mix, and 5 μL of extracted viral RNA. The PCR master mix was thawed and stored at 4°C, 24 hours before reaction set-up. The RT-qPCR was performed on a 7500 Fast real-time PCR system (Applied Biosystems) with a machine protocol of 50°C- 30min, 95°C-2min followed by 45 cycles of 95°C-15sec, 55°C-30sec. To relate genome copy number to Ct value, we used a standard curve based on serial dilutions of a plasmid control, run in triplicate on the same plate. H3N2 samples were compared to a plasmid containing the M segment of A/Perth/16/2009. H1N1 samples were compared to a plasmid containing the M segment of A/California/07/2009.

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