FIG 8.

PEAV is dynamically transported in Rab5, Rab7, and Rab9 vesicles. (A) Vero cells were inoculated with Alexa Fluor 647 NHS ester-labeled PEAV (MOI = 20) (1 h, 4°C), washed, incubated (30 min, 37°C), and fixed (4% PFA, 15 min, RT). The cells were immunodetected using an anti-PEAV N protein antibody (green) and observed using confocal microscopy. (B) Labeled PEAV was prepared with negative staining and observed under a transmission electron microscope. Scale bar = 100 nm. (C) Vero cells were transfected with WT Rab plasmid (2.5 μg, 24 h, 37°C), and then inoculated with Alexa Fluor 647 NHS ester-labeled PEAV (MOI = 10) (11 h, 4°C), washed, and incubated in maintenance medium at 37°C for 15 min (Rab5) or 30 min (Rab7 and Rab9). Single-virus tracking images were acquired using a live-cell workstation with the light scanning module of a Leica TCS SP8 STED 3×confocal microscope. Scale bar = 5 μm.