Fig. 1: MHCII is required for sOH/04 infection in the absence of sialic acid.

a Representative (n = 3 different animals) right cranial lung samples from mock and sOH/04-infected pigs were deparaffinized, rehydrated and stained for SLA-DR (green) and HA (red) detection. Nuclei were stained using DAPI (blue). Scale bar represents 30 μm. b Neuraminidase treatment efficiently removed sialic acid from the surface of PAMs. Macrophages were incubated with 100 mU/mL of neuraminidase, and sialic acid was stained at 24 h post-treatment. Scale bar represents 30 μm. Imaging was performed three independent times. c SLA-DR expression in desialylated and non-desialylated PAMs was evaluated at 24 h post-neuraminidase treatment by immunofluorescence using an αSLA-DR specific antibody. Scale bar represents 30 μm. Representative images were selected after three independent experiments. PAMs were left untreated or desialylated and further infected ex vivo for 1 h at an MOI of 0.01 of either sOH/04 (blue, d) or hVIC/11 (orange, e) and viral titers in the supernatant were determined at 0, 12, 24, 48, and 72 hpi. Data are presented as the mean ± SEM of three independent experiments. f MHCII availability is critical for sOH/04 infection of desialylated PAMs. 24 h after deacetylation, PAMs were incubated with concentrations ranging 0–10 μg/mL of an αSLA-DR specific antibody or with 10 μg/mL of an anti-GFP control antibody (g) for 1 h before infection. PAMs were infected at an MOI of 0.1 for 1 h, and then cells were supplemented with fresh media containing the desired antibody concentration, and viral titers were measured at 48 hpi. Data are presented as the mean ± SEM of three independent experiments. L.D. limit of detection.