Figure 5

Impaired amount of virus progeny after stimulation of BV-2 cells with IFN-γ or IFN-γ and LPS. BV-2 cells were stimulated with LPS (10 µg/ml), IFN-γ, (10 ng/ml), LPS + IFN-γ or IL-4 (10 ng/ml) for 24 h in medium with 2% FBS. Polarization of cells was analyzed by Griess assay (a), Western blot (b) and by detection of the percentage of MHCII⁺ cells by FACS analysis (c). The amount of virus progeny (pfu/ml) was analyzed by standard plaque assay in the presence of stimulating factors and in normal growth medium in cells and supernatants (d). Statistical analysis was performed related to infection medium. To analyze the recovery of viral replication, normal growth medium was applied after infection of pre-stimulated cells. Standard plaque assay was performed to determine viral progeny in cells and supernatant after 24, 48, 72 and 96 hpi (e). To analyze the relative survival of the cells in the presence of stimulating factors a MTT assay was performed (f). The cellular density of stimulated cells relative to unstimulated (w/o) cells was detected via optical density measurement after 24 and 48 h of cultivation (g). Two-sided t test with unequal variances was used for statistics *p < 0.05, **p < 0.01, ***p < 0.001. Active replication was defined as virus titer above the virus titer of the infection medium (red line). All experiments were performed in triplicate and repeated in an independent experiment.