A to F: WNV antigens in different regions of the mouse CNS. Mice were inoculated with 103 FFU of IS-98-ST1 WNV upon different routes (i.c., i.p., i.n., i.d.); at Day 7 of infection, mice were euthanazied, brains were cut in 14 μm thick cryostat sections, and processed for immunofluorescence using anti-WNV serum (obtained from i.p.-inoculated resistant mice) as primary antibody. A: hippocampus (pyramidal layer), i.c. inoculation. B: frontal cortex, i.c. inoculation. C: spinal cord, i.p. inoculation. D: olfactory bulb, i.n. inoculation. Magnification: × 350. E: Average levels of infection of the different brain structures was estimated on 10 different sections for each of the 3 animals per group (I.C.: intracerebral, I.P.: intraperitoneal, I.D.: intradermal; I.N.: intranasal) according to the scale: +++: more than 10 positive cells per microscopic field; ++: between 3 and 9 positive cells; +: 1 or 2 positive cells; -: no positive cell. F: Immunodetection of WNV antigens (green) and Glial Fibrillary Acidic Protein (red) in cryostat section of WNV-infected mouse brain, day 7 of infection, i.c Magnification: × 700. G, H: WNV infection in primary neural cultures from C57BL/6 mouse brain cortex. Primary cultures were performed as described in text and infected with IS-98-ST1 WNV. G: Detection of WNV antigens (using anti-WNV mouse immune serum and a FITC-conjugated secondary antibody, green staining) and neuronal specific enolase (using a rabbit polyclonal antiserum and an anti-rabbit polyclonal antibody made in goat conjugated with Texas Red, red staining) by immunofluorescence at 24 h p.i. (m.o.i. 12.5). Magnification: × 700. H: Kinetics of infection and variation of cell number at various times post-infection for different m.o.i; three cultures for each m.o.i. were fixed and processed for WNV antigen detection by immunofluorescence, whereas cell nuclei were visualized with DAPI. Cell nuclei of adherent cells were counted in 8 different different fields for the three cultures (histogram) whereas the percentage of infected cell was estimated by counting WNV antigen positive cells and cell nuclei; the percentage of infected cells is indicated as values (%) in white squares.