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Additional file 7 Four microglia phenotypes identified in the homeostatic and infected brain have differential immune profiles. Histograms showing the expression of selected markers on/in P2RY12hiCD86+ (dark purple), P2RY12loCD86- (light green), P2RY12loCD86+ (light purple) and P2RY12hiCD86- (dark green) microglia at dpi 0 (blue) and dpi 7 (orange).

Additional file 2 Overlapping expression of 'infiltrating macrophage' markers on resident and infiltrating myeloid populations in WNV-infected brains. a-e Histograms and FACs plots showing the expression of CD44 (a), LFA1 (CD11a) (b), VLA4 (CD49d) (c), Ly6C (d) and CCR2 (e) on microglia from homeostatic (blue) and infected (green) brains relative to infiltrating microglia-like (orange) and Ly6Chi macrophages (dark pink) in WNV dpi 7 brains.

Additional file 12 IL-23 is under the detection threshold in WNV infected brains. Quantity of IL-23 (p19/p40) in mock and WNV-infected mice at dpi 7, as determined using an ELISA.

Additional file 8 ‘Microglia-specific’ marker TMEM119 is expressed by infiltrating macrophages populations in the brain but not in the bone marrow or blood. a, b Histograms showing the expression of TMEM119 on microglia (a) in naïve and WNV-infected mice and on microglia, Ly6Chi macrophages and microglia-like macrophages (b) in WNV dpi 7 brains. Fluorescence minus one (FMO) and single stains (ss) for TMEM119 are shown each plot. c-f tSNE plot clustered on live cells from naïve and WNV dpi 7 brains. c, d Annotation of populations found in the mock-infected (c) and infected (d) brain tSNE. e, f Relative expression TMEM119 on tSNE plots representing naïve (e) and WNV-dpi 7 (f) brains. g Heatmap showing the expression TMEM119 on neutrophils, microglia, microglia-like macrophages and Ly6Chi macrophages from four brain samples (1-4). h Expression of TMEM119 on peripheral bone marrow and blood Ly6Chi monocytes and Ly6Chi macrophages in WNV dpi 7 brains. i-k tSNE plot clustered on live, SSC-Alo and Ly6G+/- bone marrow, live, SSC-Alo and Ly6G+/- blood cells and live brain cells from WNV dpi 7 brains. Data is representative of at least two independent experiments, with a minimum 7 animals per group.

Additional file 3 Microglia are easily identifiable in the homoeostatic brain. a-d Quality control gates, including time (a), single cells (b), non-debris (c) and live cell (d) gates were applied before analysing cells. Neutrophils were also excluded by their expression of Ly6G (e). f Gating strategy one identifies a ‘resting’ microglia population (CD45lo CD11b+) (R1: blue) and an ‘activated’ microglia population (CD45int CD11b+) (R2: red). h, j Gating strategy two, does not use ‘microglia-specific markers’ and identifies microglia as CX3CR1+ CD45lo-int CD11b+ Ly6C-/lo. Gating strategy three is a revised gating strategy which identifies microglia as CX3CR1+ CD45lo-int CD11b+ P2RY12+ Ly6C-/lo (h, I, l). n, o Gating strategy four uses a limited number of markers and identifies microglia as CD45lo-int P2RY12+CD11b+CX3CR1+. g, k, m, p ‘Microglia’ populations gated using strategies 1 (g), 2 (k), 3 (m), and 4 (p), overlaid onto a tSNE plot, clustered on live cells from mock-infected brains. q, r Number (q) and frequency (r) of ‘microglia’ gated using strategies 1-4. Data is presented as mean ± SEM, from two independent experiments with at least six mice per group. **P

Additional file 5 Immune profiles of resident microglia and infiltrating myeloid cells in WNE at dpi 7.

Additional file 9 ‘Microglia-specific’ marker P2RY12 is expressed by infiltrating macrophages populations in the brain but not in the bone marrow or blood. a, b Histograms showing the expression of P2RY12 on microglia (a) in naïve and WNV-infected mice and on microglia, Ly6Chi macrophages and microglia-like macrophages (b) in WNV dpi 7 brains. Fluorescence minus one (FMO) and single stains (ss) for P2RY12 are shown each plot. c-f tSNE plot clustered on live cells from naïve and WNV dpi 7 brains. c, d Annotation of populations found in the mock-infected (c) and infected (d) brain tSNE. e, f Relative expression P2RY12 on tSNE plots representing naïve (e) and WNV-dpi 7 (f) brains. g Heatmap showing the expression P2RY12 on neutrophils, microglia, microglia-like macrophages and Ly6Chi macrophages from four brain samples (1-4). h Expression of P2RY12 on peripheral bone marrow and blood Ly6Chi monocytes and Ly6Chi macrophages in WNV dpi 7 brains. i-k tSNE plot clustered on live, SSC-Alo and Ly6G+/- bone marrow, live, SSC-Alo and Ly6G+/- blood cells and live brain cells from WNV dpi 7 brains. Data is representative of at least two independent experiments, with a minimum 7 animals per group.