Fast7500 equipment

Total RNA was isolated from LCM tissues, using the miRNeasy FFPE Kit (no. 217504, Qiagen), as described in the above section.
Relative mRNA expression levels of IFNα, IL-6, TLR-3, IL-8, RIG-I, IFNγ, TNFα, CCL2, CCL3 and the housekeeping gene β-actin, at each different lung compartment, were assessed by two-step RRT-PCR. Primer sequences and source are illustrated in the Additional file 1. Amplicon sizes of the target genes range between 90 and 120 base pairs. Briefly, RRT-PCR was performed using a Power SYBR green kit (Applied Biosystems) and Fast 7500 equipment (Applied Biosystems). RNA extraction was performed on the ferret lung tissue samples with an RNeasy Mini Kit (Qiagen), using the RNA stabilization and on-column DNase digestion protocols (Qiagen). Reverse transcription was performed using an ImProm-II reverse transcription system (Promega, Madison, WI, USA), at 0.5 µg RNA. PCR reactions were performed in 10 µL reaction volumes in quadruplicates; 45 amplification cycles were used, and the annealing temperature was 60 °C. The expression levels were normalized using the house-keeping gene β-actin using the relative standard curve method and taking into account primer efficiency. The results are expressed as arbitrary units.

Viral RNA quantification was performed in nasal swab samples using the NucleoSpin RNA isolation kit (MACHEREY-NAGELGmbH&CoKG, Düren, Germany) following the manufacturer’s instructions. Subsequently, a TaqMan RT-qPCR designed to detect influenza viruses (IVs) using the PCR primers and hydrolysis probe already described [45 (link)] was run in a Fast7500 equipment (Applied Biosystems, CA, USA) with the conditions already set and described [14 (link)].

Viral RNA was extracted with the NucleoSpin^® RNA Virus Kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions, using a 5 mm square sample of the proximal mediastinic right caudal lobe of each ferret. The IAV M gene was quantified by TaqMan one-step quantitative real time RT-PCR (rRT-PCR) using primers, a probe, and the amplification conditions described in [31 (link)] using Fast7500 equipment (Applied Biosystems, Foster City, CA, USA). Individual samples were run in duplicate and each assay contained known positive and negative amplification controls that were also run in duplicate. Threshold cycle (Ct) values ≤40 were considered positive for IAV.
The IAV antigen was also quantified in the lung by immunohistochemistry (IHC). Fixed paraffin-embedded tissues were stained with a primary antibody against the influenza A nucleoprotein and IAV antigen immune staining was quantified as previously described [32 (link)].

Relative mRNA expression levels of selectin P-ligand (SELPLG), and the housekeeping gene β-actin in the vascular compartment, were assessed by two-step RRT-PCR, as described in the above section. Briefly, RRT-PCR was performed using a Power SYBR green kit (Applied Biosystems) and Fast 7500 equipment (Applied Biosystems). PCR reactions were performed as described in the above section and primer sequences and the GenBank accession number are illustrated in Additional file 1. The expression levels were normalized using the house-keeping gene β-actin using the relative standard curve method and taking into account primer efficiency. The results are expressed as arbitrary units. Primer sequences for the SELPLG gene was designed as described previously [26 (link)]. The ferret-specific SELPLG gene is available in the NCBI nucleotide database [27 ]. The amplification product was detected by electrophoresis to validate the size of the product, in accordance with the primer design, and the products were purified using a QIAquick PCR Purification Kit (Qiagen). Sequencing reactions were performed with ABI Prism BigDye Terminator Cycle Sequencing v.3.1 Ready Reaction (Applied Biosystems), and analysed using an ABI PRISM model 3730 automated sequencer (Applied Biosystems). The amplified sequence correlated with the ferret specific target sequences.