Fig. 3

IHNV infects and disrupts blood vessels.

A: scheme of the imaged regions (corresponding panel indicated with red letters), overlaid on a composite transmission (grayscale) and GFP fluorescence (green) picture from a 4dpf fli1:GFP larva viewed laterally (images taken with a stereomicroscope; to get a more readable GFP signal, short exposure of the head has been combined with longer exposure of the rest of the body). B–K, confocal images (maximal projections from multiple Z-stacks) of WIHC stainings of fli1:GFP larvae fixed 24 hpi after IHNV inoculation at 72 hpf (except C, uninfected control larva); latero-dorsal views, anterior to right, dorsal to top. Colours in B–F and I: Red: IHNV P protein; green: GFP; blue: nuclei. B: eye, showing disruption of the inner optic circle, with infected cells located close to the damaged site (arrow; compare with C). D and E: brain region of two infected larvae, showing variable pattern. Notice in D the infection and loss of GFP expression of dorsal longitudinal vein (arrow). E shows infection of a cell of the posterior cerebral vein; to demonstrate colocalization of GFP and viral protein, single Z-plane images (cropped to the boxed area in E) are shown in F–H with overlaid signals in F, GFP signal in G, and P-IHNV staining in H. I: view of intersomitic vessels, showing infection and loss of GFP of ventral part of these vessels (arrows indicate where endothelial cells are expected to be found) while the dorsal part appears intact. Partial colocalization of GFP and P-IHNV in the ventral half of one vessel is shown in J and K (GFP and P-IHNV signal channels, respectively). B–H, 40x objective; scale bar, 50 μm. I–K 63x objective; scale bar, 25 µm. hb: hindbrain; l: lens; mb: midbrain; n: neuropile; r: retina.