Fig. 2.

Activation of endothelial cells is virus dose dependent. (A) Noninfected (mock) (M) and infected (MOIs, 0.1, 1, and 10) HUVECs were harvested at 6, 24, 48, and 72 h p.i., followed by protein separation by SDS-PAGE and staining for the proteins ICAM1 and CCHFV NP and the loading control calnexin. CCHFV NP is observed at increasing levels depending on the initial MOI, with the highest intensity at 72 h p.i. ICAM1 levels are increased upon exposure to LPS (L) (100 μg/ml) at 48 and 72 h p.i. and also upon infection with CCHFV at an MOI of 10 at 48 h p.i. At 72 h p.i., the ICAM1 levels increase in a virus dose-dependent manner. (B) Noninfected HUVECs (mock) and HUVECs infected with VSV at an MOI of 0.1 were harvested at 48 h p.i., followed by protein separation by SDS-PAGE and staining for the proteins ICAM1 and VSV-G and the loading control calnexin. VSV-G proteins are observed only in infected cells. ICAM1 protein expression is not induced by VSV in HUVECs at 48 h p.i. (C) HUVECs were incubated for 48 h with TNF-α (10 ng/ml), mock treated with cell medium, or infected with CCHFV at MOIs of 1, 10, and 20, followed by fixing and staining for ICAM1 on the cell surface. All results are normalized against CD105, and ICAM1 is shown as a percentage of CD105 expression. Each treatment was performed in triplicate for two independent experiments. Endothelial cells respond in a virus dose-dependent manner by upregulating ICAM1 on the cell surface. Statistical significance is shown (ns, not significant; ***, P < 0.001), and the error bars indicate standard deviations. The statistics were calculated by comparing stimulated cells to mock-infected cells, and the tests used were ANOVA followed by Dunnett's multiple-comparison test. (D) HUVECs were treated with TNF-α (10 ng/ml) or cell medium or infected with CCHFV at MOIs of 1, 10, and 20 or with UV-inactivated virus at an MOI of 20 and incubated for 48 h p.i., followed by addition of BCECF-AM-stained leukocytes for 1 h. Nonadherent leukocytes were washed away, followed by lysis of ECs and adherent leukocytes in lysis buffer. The intensity of fluorescence of BCECF-AM from lysed leukocytes in the lysates was then measured. The level of adhesion was calculated by normalizing against leukocytes adhered to nontreated ECs, and the results are shown as the fold change to leukocytes adhered to ECs pretreated with only medium. With increasing levels of virus, there is an increasing level of leukocyte adherence to infected endothelial cells. The results are means ± standard errors of the mean (SEM). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. The statistics were calculated by comparing results from treated cells to those from the UV-inactivated virus control utilizing ANOVA followed by Dunnett's multiple-comparision test.