DNA microarray technology was used to determine the initial response of human macrophages exposed to Ebola virions. Total RNA isolated from primary human macrophages of three donors (D1, D2, D3) at 1 h and 6 h after in vitro exposure to purified Ebola virions was compared to RNA from mock-exposed cells derived from the same donors. The time points were chosen as virus gene expression does not occur within one hour of cell-virion contact, whereas it will have commenced five hours later while ZEBOV replication is still absent (see below). A total of 12 HG-U95Av2 GeneChip microarrays were analyzed to determine differences in cellular gene expression levels between Ebola virion-exposed and mock-exposed macrophages from donors D1-D3 and also compared to the responses of LPS-treated cells. Genes detected as absent (p#0.05) on all 12 arrays were removed from a total of 12,606 probe sets prior to ratio analysis resulting in a reduced number of 8,861 probe sets. The log 2 ratio was plotted as a function of the average log 2 intensity of Ebola virion-exposed versus mock-exposed samples to test for hybridization quality and for the extent of variation among biological replicates (cells from the three donors). The MA plots for both the 1 h and 6 h time points demonstrate a low variability among donors and good overall reproducibility with the mean log 2 being zero over the EBOV-Induced Changes in Macrophage Gene Expression www.plosntds.org entire signal intensity range ( Figure S1 ). The low overall variability among donors was corroborated quantitatively by an analysis of variance of the signal strength. A single factor analysis of variance (ANOVA) indicated no significant difference among the 12 arrays (p = 0.83), confirming that the expression of the majority of cellular genes was not affected by exposure to Ebola virions. Genes responsive to Ebola virion exposure were identified by data reduction, i.e. by exclusion of all genes that were identified as 'no change' (NC, p#0.05) for all conditions tested. The resulting 2,025 probe sets (genes) were characterized by at least one significant change in one donor at either 1 h or 6 h post infection. ANOVA indicated that differences in expression patterns among these probe sets were statistically significant (p = 3610 26 ).