European genotype PRRSV does not need to be infectious to downregulate PAM phagocytic capacity. (A–B) Flow cytometric analysis of the role of PRRSV infectivity in the inhibitory effect of European genotype PRRSV on macrophage phagocytosis: The effect of Marc-145-grown infectious (Black circle) and BEI-inactivated (Grey circle) concentrated PRRSV (LV) on phagocytosis of beads by viable macrophages, expressed as the percentage of viable cells that have beads associated with them (A) or the MFI per viable cell (B), which is a measure for the amount of beads per cell. PAM were inoculated with infectious or inactivated PRRSV (LV) at different moi as indicated and further incubated at 37°C for 1 h, after which a phagocytosis assay was performed. Mock infected cells were included as a control. Also, a phagocytosis assay was performed at 4°C (dotted line) after mock infection, to assess the percentage of macrophages that have beads bound to their surface and their MFI. Data represent the mean ± SEM of 4 independent experiments. *p < 0.05; ** p < 0.01; *** p < 0.001; (C) Confocal microscopical representation of the inhibitory effect of European genotype PRRSV on macrophage phagocytosis: the effect of Marc-145-grown, concentrated PRRSV (LV) on phagocytosis of beads by viable macrophages (performed as described above). An immunofluorescence staining was performed following the phagocytosis assay. Mock infected cells were included as a control and a phagocytosis assay was performed at 4°C after mock infection. For each moi, 4 single confocal z-sections throughout the middle of the cell are given, representative of each situation. Yellow-green fluorescent beads were used, nuclei are stained with Hoechst 33342 (blue), PRRSV virions are shown in green and cortical actin is visualized using TexasRed-X phalloidin. Upper panel: PAM phagocytosis of beads; Lower panel: internalized PRRSV particles.