Fig. 1

RSA59 infection upregulates NOS2 expression in WT brains at the acute phase (day 5/6 p.i.) and its deficiency results in drop in survival at the acute-adaptive transition phase (day 9/10 p.i.) NOS2 transcripts were determined in the brain of WT mice (n = 3 to 4 per group per time point) infected at 20,000 PFUs at days 6, 10, 15 and 30 p.i. by qRT-PCR. Results were normalised to GAPDH, and values calculated using the ΔCT method were compared and expressed as Mean ± SEM (A). NOS2 expression was determined by flow cytometry in the brain harvested from WT mice infected at 10,000 PFUs at day 5/6 p.i. (B) shows representative flow cytometry dot plots and histograms showing percentages of NOS2 + cells, gated from live cell populations that were gated from singlets, and their absolute numbers and NOS2 MFI are graphically represented in (C) and (D), respectively. Wildtype (WT) (n = 11 to 14) and NOS2-/- (n = 17 to 18) mice were infected with 20,000 PFUs of RSA59 and monitored daily for weight change (E), development of clinical disease (F) and survival (G). Clinical scores were assigned by an arbitrary scale of 0–4 as described in Materials and Methods. N gene (viral nucleocapsid gene) transcripts levels in the CNS at day 9/10 p.i. (H) were determined by qRT-PCR and values calculated using the ΔCT method were represented graphically as mRNA expression normalised to GAPDH. (n = 3 to 5). Plaque assay was performed to determine viral titers CNS of mice (n = 4 to 7) infected at 10,000 PFUs day 9/10 p.i. (I). Statistical analysis was performed using Ordinary One- way ANOVA test for (A); unpaired student’s t test with Welch’s correction for (C) and (D); Two- Way ANOVA with Tukey’s multiple comparison test for (E), (F), (H), and (I); and Log-rank (Mantel-Cox) test for survival proportions (G). Results were expressed as Mean ± SEM. *Asterisk represents statistical significance between infected WT and infected NOS2-/- groups. p < 0.05 was considered as significant. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Each dot in the dot plots represents one biological sample