Extended Data Figure 1: Murine intracranial infection with attenuated WNV-NS5-E218A induces similar viral loads and inflammatory response as wild-type WNV-NY99, but greater overall survival.

a, Plaque assay for infectious virus (measured in plaque-forming units per g of tissue) performed on dissected brain tissue at various days post-infection with either footpad infection with 10² pfu of WNV-NY99 or intracranial infection with 10⁴ pfu of WNV-NS5-E218A. Each point represents an individual mouse. b, Survival curves of mice infected at 8-weeks-old by the footpad with WNV-NY99 or intracranially with WNV-NY99 or WNV-NS5-E218A. c, Flow cytometric analysis of dissected cortex, hippocampus and cerebellum at 6 dpi with WNV-NY99 and WNV-NS5-E218A with plots for CD45 and CD11b. d, Quantification of flow cytometry data from c. Shown are numbers of leukocytes (CD45^high), lymphocytes (CD45^high, CD11b^low), and activated macrophages and microglia (CD45^high, CD11b^high) compared to mock-infected controls (n = 4 mice per group). e, Immunostaining and counts for TUNEL staining for apoptotic cells with co-staining for the neuronal marker, NeuN, during peak infection (7 dpi) of WNV-NS5-E218A (n = 5) compared to mock-infected controls (n = 4). DG, dentate gyrus, CTX, entorhinal, perirhinal, and visual cortex. f, Some mice were tested at 22 dpi on a three-day version of the Barnes maze, and evaluated for latency to find target hole (*P < 0.05 by repeated measures two-way ANOVA). g, Prior to Barnes maze testing, mice were tested on open field for total lines crossed in 2 min at 21 dpi. h, qPCR for positive strand (non-replicating strand) and negative strand (replicating) WNV envelope protein message remaining in hippocampal tissue at 7, 25 and 52 dpi (n = 13, 4, and 14 mice per group for 7, 25, and 52 dpi, respectively), measured in copies per Gapdh. i, qPCR for positive strand WNV envelope protein at 52 dpi in WNV good learners (fewer than 8 errors on day 2 of Barnes maze, n = 5) and WNV poor learners (greater than 9.5 errors on day 2 of Barnes maze, n = 9). j, qPCR for negative strand WNV envelope protein at 52 dpi in WNV good learners (fewer than 8 errors on day 2 of Barnes maze, n = 5) and WNV poor learners (greater than 9.5 errors on day 2 of Barnes maze, n = 9). Result was not significant by student’s two-tailed t-test.