Fig. 4

Comparative virulence between USUV strains in primary human astrocytes. Primary human astrocytes were infected with the six lineages of USUV at a MOI of 0.1. a Left panel. Bright light images of CT- and USUV-infected astrocytes showing at 7 dpi an atypical CPE which appeared specifically with the EU2 strain. This CPE is characterized by dark clustered cells that detach from the cell mat. Right panel: Supernatants from infected cells were collected at 2 and 7 dpi and subjected to TCID50 measurement on Vero cells. EU2 strain replicates longer in astrocytes. Results are expressed as mean ± SEM of 3 independent experiments. b Apoptosis (4 dpi) and cellular proliferation (7 dpi) measured by MTT and BrDU are affected by USUV infection. c Left panel. Mock and EU2 USUV-infected cells were fixed at 2 dpi and labeled with the pan-flavivirus antibody (in green) or double-stranded RNA antibody (in green) by indirect immunofluorescence. Nuclei are labeled with Hoechst (in blue) immunofluorescence (× 20 and × 40). The white square represents a zoom on infected cells. Scale bar = 50 μm. Right panel: Quantification of infected cells at 2 dpi (using pan-flavivirus antibody) showing more infected cells with the EU2 strain. For all experiments, n = 3 independent triplicates. *p < 0.05 and **p < 0.01