Figure 6.

Hemoglobin has antiviral function and reduces influenza virus replication

(A) MDCK cells were incubated with the BALF of WT and CD47^−/− mice isolated at the indicated time points. After 30 min cells were infected with 1000 PFU of influenza virus and 3 dpi the antiviral activity of BALF samples was determined based on plaque count (n = 8).

(B) MDCK cells were incubated with 1 μg/mL hemoglobin (HB) for 30 min. Afterward, cells were infected and 3 dpi the antiviral activity of HB was determined based on plaque count. For post infection treatment, HB was added 2 h after infection.

(C) MDCK cells were incubated prior or after infection with 1 μg/mL HB as described. 48 h after infection cells were harvested and analyzed for IFN-β and Mx1 gene expression by qRT-PCR (n = 6). Non infected (n.i.) cells served as controls.

(D) Mice were i.n. infected with IAV. 3 dpi IFN-β concentrations were assessed in lung homogenates (n = 4). One representative experiment of two is shown.

(E) IAV-infected mice were intranasally treated with 50 μg HB protein on day 0 and 3 post infection. IFN-β expression by ELISA was analyzed 6 dpi from lung homogenates (n = 3). One representative experiment of two is shown.

(F) Virus titers were assessed by qRT-PCR from lung homogenates of HB treated and untreated mice 6 dpi. One representative experiment of two is shown.

(G) A549-AT cells were incubated with indicated HB concentrations for 30 min. Afterward, cells were infected and 24 h later the antiviral activity of HB was determined by in cell ELISA.

(H) For post infection treatment, HB was added after SARS CoV-2 infection of A549-AT cells. Two independent experiments are shown unless otherwise noted.

Data shown are mean ± SEM. One-way ANOVA with Tukey’s multiple-comparisons post-test was performed in (A–C, G, and H). Two-way ANOVA with Sidak’s multiple-comparisons post-test was performed in (D). Student’s t test was performed in (E and F). ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001.