Lung viral titer assay.

Infected mice were euthanized and the whole lungs were removed, resuspended in 2 ml of 1× PBS containing protease inhibitors (Complete Ultra tablets, Roche), and homogenized using a Precellys 24 homogenizer (Bertin Technologies). Samples were centrifuged to remove tissue debris and supernatants were transferred to a new tube. Viral titers were determined by plaque assay in MDCK cells followed by immunostaining. For the plaque assays, MDCK cells were seeded on 6-well plates 12 to 18 h prior to the assay. The virus suspensions were serially diluted in 1× PBS and 1 ml of the virus dilution was used to infect each well of MDCK cells for 1 h at 37°C. After the incubation, the virus was removed, the cells washed twice with 1× PBS and 3 ml of Avicell mix (1.2% Avicell in 1× MEM with bovine serum albumin [BSA] and trypsin) was added to each well. Plates were incubated at 37°C for 48 h (influenza A viruses) or at 34°C for 72 h (influenza B virus). After incubation, cells were fixed with 3 ml of 4% paraformaldehyde (PFA). For the immunostaining, the cells were blocked for 20 min with 5% milk in 1× PBS and the primary (test antibodies) and secondary antibody (anti-human IgG-HRP) were added sequentially. After the final wash, 500 μl of TrueBlue was added to visualize and count the plaques.