Fig 2: Metabolomic profiles in cerebrospinal fluid samples from control, encephalitis and meningitis TBEV patients. (A) 2D PLS-DA plots with 95% confidence ellipses showing the effects of encephalitis and meningitis on the CSF metabolome. The metabolites contributing to the separation of samples are labelled with arrows indicating their direction and magnitude. Cross-validated 2D PLS-DA score plot comparing TBEV patients with (B) encephalitis (with follow-up) and control, or TBEV patients with (C) meningitis, follow-up, and control groups on the CSF metabolome. Volcano plots comparing a two-fold change in concentration of CSF metabolites in (D) encephalitis vs. meningitis, or (E) control vs. encephalitis or (F) meningitis patients. Significantly altered metabolites are highlighted in red (increased) or blue (decreased). Data were compared using a two-sided Mann-Whitney U test followed by Benjamini-Hochberg multiple comparison test with FDR < 0.05 and fold-change > 1. (G) Heatmap showing CSF metabolites of significantly higher (red) or lower (blue) abundance in TBEV-patients with encephalitis vs. meningitis. Metabolite alterations are represented by colour intensity. Borders are colour-coded according to statistical significance (p < 0.05)

Fig 3: Changes in the cerebrospinal fluid metabolomes in encephalitis and meningitis TBEV. (A) Area under the curve (AUC) in the top panel and box plot representation in the lower panel, showing cut-off values of 4 CSF biomarkers with significant up-regulation (S-Adenosylmethionine: AUC = 0.818, p = 0.0016348; 1-Methylnicotinamide: AUC = 0.78, p = 0.0006835; Phosphoenolpyruvic acid: AUC = 0.759, p = 0.0031707; Fructose 1,6-bisphosphate: AUC = 0.755, p = 0.012209) identified in encephalitis vs. meningitis. Statistical significance was determined using a one-way ANOVA followed by Bonferroni multiple comparison test (*p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001); ns, not significant. (B) KEGG metabolic pathway enrichment analysis and (C) scatter plot of pathway impact versus pathway significance, where each node represents a significant metabolic pathway contributing to the differences between encephalitis and meningitis TBEV-patient groups. The white to red node colour scale indicates the p-value, and node size reflects the pathway impact score. Red circles indicate the most highly affected metabolic pathways in TBEV patients, with the highest number of metabolite hits (9) from the pathway analysis (Impact score = 0.65877). (D) Network diagrams of metabolites in the CSF from encephalitis and meningitis patients. (E) Magnification of a new cluster of the PPP visualized from (D) in Cytoscape