Extended Data Figure 3: Despite synaptic terminal loss, no changes to synaptic terminal size, axons, or astrocyte or antibody association with terminals during WNV infection.

a, Immunostaining for the presynaptic marker, synaptophysin, at 7 dpi comparing mock (n = 7) with WNV-NS5-E218A-infected (n = 5) mice. Quantification of synaptophysin⁺ puncta size was performed within the hippocampal CA3. Scale bar, 10 μm. b, Immunostaining for the presynaptic marker, synapsin1, within the hippocampal CA3 in uninfected controls (n = 3) and footpad-infected WNV-NY-1999 (n = 4) at 8 dpi. Quantification was performed on the numbers of synapsin1⁺ puncta per mm² with *P < 0.05 considered significant. c, Immunostaining within the hippocampal CA3 for SMI-31, which detect phosphorylated neurofilament and marks axons at 25 dpi (n = 5–6 mice per group). Quantification of the area of SMI-31 per mm² (not significant by Student’s t-test). d, Immunostaining within the hippocampal CA3 for the presynaptic marker, synaptophysin, co-labelled with the astrocyte marker, S100β at 7 dpi (n = 3 mice per group). Quantification of the percentage of total S100β⁺ area and synaptophysin⁺ area colocalized with S100β (not significant by Student’s t-test). e, Electron microscopy was performed on hippocampal CA3 sections from day 7 after mock (left panel) or WNV-NS5-E218A (right panels) infection, with immune-DAB enhancement of IBA1. Note the presence of many phagosomes and cytoplasmic inclusions within the WNV-E218A-infected microglia. Electron micrographs shown are representative of n = 3 mice per group. Scale bars, 1 μm. f, Immunostaining for the presynaptic marker, VGlut1, and endogenous murine IgG (mIgG) at 7 days after mock (n = 4) or WNV-NS5-E218A (n = 4) infection. Quantification was performed on the total per cent of mIgG staining area as well as the per cent of VGlut1⁺ staining area colocalized with mIgG. g, Immunostaining for the postsynaptic marker, Homer1, and endogenous mIgG at 25 days after mock (n = 4) or WNV-NS5-E218A-infection, which were divided into WNV-infected mice which made fewer than 8 errors on day 2 of the Barnes maze (WNV good learners, n = 5) and WNV-infected mice which made greater than 9.5 errors on day 2 of the Barnes maze testing (WNV poor learners, n = 3). Quantification was performed on the total per cent of mIgG staining area as well as the percent of Homer1⁺ staining area colocalized with mIgG. Significance was determined by Student’s two-tailed t-test with P < 0.05 considered as significant. NS, not significant. h, Immunostaining and quantification of number of VGlut1 hippocampal CA3 presynaptic terminals at 7 dpi in wild-type and μMT^−/− mice. (*P < 0.05, NS, not significant, by Student’s two-tailed t-test). Scale bars, 10 μm.