(A, B) Groups of sex-matched WT (B6), Ifnar1-/- and B2m-/- mice were infected intranasally with 600 PFU of FM-MA virus. Single-cell suspensions were prepared from uninfected lungs and those infected with the virus for 5 days. Cells were stained with antibodies against H-2Kb or H-2Db and CD326 (EpCAM—epithelial cell marker), and analyzed by flow cytometry. Surface expression of H-2Kb or H-2Db was determined on EpCAM+ lung epithelial cells. The following mAb were used in this experiment: anti-LFA-1, anti-EpCAM, anti-H-2Kb, and anti-H-2Db. One representative image from each group is shown. This experiment was performed three times with similar results. (C, D) Ly49G, I1, O, R, and V expression was detected on lung NK cells of uninfected WT and NKCKD mice using anti-NKp46, anti-TCRβ, and a combination of 4D11, 4E5 and 14B11 mAb. NKG2D, NKp46, CD11b, and CD27 expression was detected on lung NK cells, defined using anti-CD49b (DX5) and anti-TCRβ. The gray line represents staining with an isotype antibody. This experiment was performed three times with similar results. (E) Groups of age and sex-matched WT, Ly49QKO, and NKCKD mice were infected with FM-MA virus (1050 PFU) and monitored for 2 weeks. Data are pooled from two independent experiments (n = 19 in each group). (F) Groups of age and sex-matched NKCKD mice with or without NK depletion by anti-asialoGM1 were infected and monitored as above. A group of uninfected, NK-depleted mice was included as a control (n = 10 in each group). The percentage of surviving mice is shown. *p < 0.05, **p < 0.01 and ***p < 0.001. Statistical analysis was performed with the log rank test.

https://doi.org/10.1371/journal.ppat.1006210.g001