Extended Data Figure 2: At 25–52 days post-WNV-NS5-E218A infection, mice do not show any appreciable loss in brain volume, neuron or astrocyte numbers, or macrophage infiltration.

a, Immunostaining for the neuronal marker, NeuN, with TUNEL staining for apoptotic cells within the hippocampus at 52 dpi. Quantification of the number of TUNEL⁺ neurons and total TUNEL⁺ cells is shown in mock (n = 3) and WNV-NS5-E218A (n = 6). Scale bar, 20 μm. b, Immunostaining and quantification of the number of NeuN⁺ neurons per mm² within the CA1, CA3, dentate gyrus and entorhinal cortex at 25 days after mock (n = 4) or WNV-NS5-E218A infection. WNV-infected animals were subdivided into good (n = 5) and poor (n = 3) learners. Scale bar, 100 μm. c, Post-mortem mouse brains were imaged by MRI at 52 dpi to determine tissue volume of the hippocampus (outlined in red) and total brain (n = 5 mice per group). Scale bar, 1 mm. Not significant by student’s two-tailed t-test (P < 0.05 considered significant). d, Immunostaining for the reactive astrocyte marker, GFAP, shows that WNV-NS5-E218A-infected mice do not exhibit greater hippocampal astrocyte activation than mock-infected controls at 52 dpi. NS, not significant by student’s two-tailed t-test. e, Haematoxylin and eosin (H&E) staining was performed at 52 dpi in WNV-NS5-E218A-recovered and mock-recovered mice. Occasional microglial nodules (arrowhead) surrounded by lymphocytes were observed within the hippocampus. CA1 pyr, CA1 pyramidal layer. f, Flow cytometric analysis of whole brain from mock and WNV-NS5-E218A-infected mice at 8 and 25 dpi was performed to determine numbers of microglia (CD45^low, CD11b^low), macrophages (CD45^high, CD11b^high), and lymphocytes (CD45^high, CD11b^negative). Note the decrease in macrophage population from 7 to 25 dpi.