immunization with the adjuvant alone likely had no effect on immune responses after RVFV challenge. The expression of all 84 genes remained unaltered in the spleen of recNP immunized mice compared to normal mice, whereas nine genes were significantly upregulated and 20 downregulated in adjuvant control mice spleens, compared to 11 upregulated and 20 downregulated in PBS control mice spleens. Comparing tissues, only 10 genes were similarly up- or downregulated in liver and spleen of adjuvant control mice, whereas 45 genes yielded contrasting results between tissues. Only 13 genes were similarly expressed in liver and spleen of PBS control mice, compared to 44 genes which yielded contrasting results between tissues (Table S1), indicating that there was a tissue specific modulation of host gene expression in response to RVFV infection. Selected genes that were significantly up- or downregulated in any of the experimental groups and indicating a clear difference or interesting similarity between immunized and control mice were grouped according to known effects on specific immune functions and shown in Figure 4 (a–b), Figure 5 (a–f) and Figure 6 (a–d). There was a significant upregulation of several genes in the liver that have pro-apoptotic effects [34–42] in non-immunized adjuvant and PBS control mice, whereas these genes were normally expressed in recNP-immunized mice (Figure 4a, Figure 5a). One gene in particular, encoding the early growth response 1 protein (Egr-1) which is a transcription factor involved in proliferation, differentiation and activation of cell death pathways [43], that was upregulated in non-immunized mice was downregulated in recNP-immunized mice. Only one gene encoding the transcription factor RelB, part of the NF-KB family of proteins and responsible for counter-regulating the effects of NF-KB, was upregulated in liver of immunized and non- immunized mice (Figure 5a) [40]. Several genes with anti- apoptotic effects [44–49] were downregulated in the liver of non-immunized control mice but normal in recNP-immunized mice (Figure 5b). Most notable of these were the genes encoding the prolactin receptor (Prlr), an anti-inflammatory protein known to promote proliferation, protect against apoptosis and enhance cell survival, and the WW domain containing E3 ubiquitin protein ligase 1 (Wwp1), an anti-apoptotic protein playing a role in proliferation [49]. There was also evidence of severe liver inflammation [43,50–58] in non-immunized adjuvant and PBS control mice, but not in recNP immunized mice (Figure 5c). Despite this the gene encoding the anti-inflammatory cytokine IL- 10 was upregulated in all mice. The expression of osteopontin (gene Spp1), important for wound healing, was upregulated significantly in liver of non-immunized control mice, compared to normal expression in immunized mice (Table S1) [58,59]. The gene expressing the Cyclin-dependent kinase inhibitor P21 (Cdkn1a), a protein with pro- or anti-apoptotic effects and normally upregulated in response to liver injury, was upregulated in liver of all mice but more pronounced in non-immunized mice (Table S1) [60]. The gene expressing the inducible nitric oxide synthase (Nos2), an effector of the innate immune system targeting viral proteases and inhibiting viral replication, was normal in liver of recNP-immunized mice but upregulated in non-immunized mice. Nitric oxide, a product of the Nos2 gene, is a radical molecule that can become toxic under oxidative stress conditions (Table S1) [61]. Both arms of the adaptive immune response, humoral (Th2) and cellular (Th1) [45,50,52,62–74], were activated in liver of non- immunized control mice, but normal in immunized mice, at 72 hours. The gene encoding the Phosphatidylinositol 3-kinase catalytic delta polypeptide (Pik3cd), involved in the regulation of B-cells and antibody production, was also upregulated in liver of Protective Anti-RVFV NP Responses in Mice Figure 4. (a–b). Heat maps showing fold changes in liver and spleen at 72 hours after RVFV infection. Expression of genes in RecNP immunized, adjuvant control and PBS control mice are organized according to function. Livers are indicated in panel a, and spleens in panel b. The genes shown in orange are upregulated, those in blue are downregulated and those in black or darker shades of orange and blue have fold-change values between 22 and 2 and/or have p-values.0.05. doi:10.1371/journal.pone.0025027.g004 recNP-immunized mice indicating re-activation of humoral immunity in mice recognizing one of the viral proteins as one it has encountered before (Table S1) [75]. The genes encoding the Dedicator of cyto-kinesis 2 (Dock2) protein and interleukin-12b, which are involved in the development and induction of NKT cells, were upregulated in liver of non-immunized control mice. Some important genes were, however, downregulated in liver of non-immunized control mice (Figure 5 d and Table S1). The gene encoding the Cd1d1 antigen, which is important for the presentation of antigens to, and activation of NKT cells, was downregulated with statistical significance in non-immunized control mice (Figure 5 d) [65,76,77]. The expression of the gene encoding interleukin-7, necessary for B- and T-cell and NK cell PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e25027