Protective Anti-RVFV NP Responses in Mice Figure 6. (a–d). Fold changes in expression of genes in the spleen of experimental groups at 72 hours after RVFV infection. RecNP- immunized mice (n = 3) are indicated by solid black bars, adjuvant control mice (n = 3) by grey bars and PBS control mice (n = 3) by white bars. The horizontal dotted lines indicate the cut-off values for upregulation (+2) or downregulation (22). The asterisk (*) indicates where the P-value is smaller than or equal to 0.05 (statistically significant results). Standard deviation from the mean fold changes are indicated by the error bars. doi:10.1371/journal.pone.0025027.g006 however, downregulated in the spleens of adjuvant and PBS control mice (Figure 6c–d). The most notable of these were Cd40 and Cd40 ligand which are responsible for inducing effective CD8+ cytotoxic T-lymphocyte (CTL) responses and activation of B-cells for antibody production [51], and Cd8a and Cd8b1 which are responsible for CTL activation (Figure 6c) [87]. Expression of Complement receptor 2 (Cr2), which plays an important role in B- cell activation and maturation, was also downregulated in spleen of non-immunized control mice (Figure 6d). Discussion The immune evasion mechanisms known for RVFV are directed against the type I interferon response [24,28,88] and programmed cell death [29]. However, it was recently shown that mice displaying an earlier and stronger type I interferon response were less susceptible to RVFV infection than mice with a delayed and partial response which suggests some additional evasive or regulatory effects on innate or adaptive immune mechanisms enabling its replication [33]. Immunization of mice with recNP combined with Alhydrogel protected against disease and significantly reduced viral replication [17]. A study by Lorenzo et al (2008) implicated a cellular (Th-1) response to the RVFV NP as a protective mechanism [12], however, our present data shows that immunization with recNP/ Alhydrogel induces Th-2 humoral immunity. The discrepant outcome of the two studies might be explained by the fact that Lorenzo et al (2008) used DNA vaccination [12], whereas in our study a subunit antigen combined with adjuvant known to favour Th-2 immunity, was used. Immunized mice launched an earlier and stronger type I interferon response compared to non- immunized mice which showed activation of several genes with pro-apoptotic and pro-inflammatory effects, and suppression of anti-apoptotic genes during acute phase of RVFV infection in the liver. This possibly contributed to hepatic damage, which is the main pathological feature of RVF [89]. The expression of several genes involved in the activation and function of NK cells and B- and T-lymphocytes were suppressed in infected mice, indicating additional immune evasion tactics of RVFV. The induction of expression of IFNb has been shown to occur in vitro at 3–6 hours p.i [33], while in the livers and spleens of immunized mice in this study it occurred at 3 hours p.i. Taking into account that after host infection, the virus is likely conveyed to the lymph nodes where it first replicates before it can spread to the liver and other organs [2], our results show the ability of RVFV to rapidly spread in the infected host. The expression of the same gene was, however, not upregulated in the tissues of adjuvant and PBS control mice early after infection. Type I interferon could not PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e25027