Macrophages produce NO, which is pathogenic during WNV infection. (A-E) Analysis of sections from sham-infected and West Nile virus (WNV)-infected mice at day 7 p.i. showed significant upregulation of nitric oxide synthase (NOS)-2 message in the WNV-infected brains. (C, F) Furthermore, immunohistochemical staining for NOS-2 protein (red) and lectin (green) showed that most cells expressing NOS-2 in the WNV-infected brain parenchyma at day 7 p.i. were lectin-positive. (G) Using the dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM), which, in the presence of NO becomes fluorescent, flow cytometry showed that more than 70% of macrophages (CD45hi/CD11b+/CD11c−/Ly6C+) expressed NO compared with approximately 25% of resident microglia (CD45lo/int/CD11b+/CD11c−/Ly6C−), and treatment of mice with aminoguanidine resulted in a significant reduction in macrophages expressing NO. (H) Inhibition of NO with aminoguanidine did not increase survival when used over the entire infectious period, but it had a demonstrably protective effect given at day 6 p.i. (P < 0.05). (I) A significant reduction in DAF-FM-positive macrophages was seen from day 7 p.i. in the brains of WNV-infected mice that had been treated with anti-VLA-4 neutralizing antibodies on day 6 p.i. In situ hybridization was performed on sagittal brain sections from at least six mice per group. DAF-FM data are representative of three experiments with at least three mice per group. Aminoguanidine therapy was performed four times with at least ten mice per group. Values shown are means ± SD. Statistical analysis was conducted using one-way ANOVA with Tukey-Kramer post hoc test. * P < 0.05; **P < 0.001; ***P < 0.001. Statistical analysis of survival was determined using the log-rank (Mantel-Cox) test. *P < 0.05 was considered significant.