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)].
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)].
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