be upregulated as a direct result of recNP immunization since it is not memory dependent. The fact, however, that the protective anti-recNP responses shown here was largely humoral, but without neutralizing effects, suggests that some other form of antibody dependent mechanism, such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), might be responsible. It appears that the RVFV nucle- ocapsid protein might be released from infected cells indepen- dently from other viral proteins [13], and/or processed by the infected host cell proteasome into peptides that are displayed on their surface by MHC-1. Consequently these cells can be coated with anti-NP IgG antibodies complexed with presented NP or peptides, and become a target for ADCC or CDC. It has been shown for influenza virus, with highly variable envelope glycoprotein antigens but a conserved internal nucleoprotein, that previous infection causes heterosubtypic immunity and that this is due to antibodies against the NP involving CD8+ cells in an antibody dependent manner [90]. The resultant lysis of RVFV infected cells, that would otherwise have evaded the host innate immune response and produced progeny virus because of the inhibitory action of NSs on type I interferon might have resulted in the activation of the type I interferon response in neighbouring uninfected cells. The lysed cells would release dsRNA, a by- product of viral replication and activator of type I interferon, helping close-by uninfected cells to remain uninfected and thus decrease virus spread [91]. It has been shown that an early type I interferon response is protective against RVFV infection [18,33] and it is thus highly likely that the early expression of IFNb in immunized mice contributed to effective viral clearance and protection. The excessive expression of IFNb, however, in control mice later after infection (72–120 h) was not able to curb the replication of the virus, as evidenced by high viral titers, and probably contributed to the pathology in the liver and spleen. It has been suggested that the role of IFNc in RVFV pathogenesis is negligible [28] yet it has been shown in vivo that IFNc does act as an antiviral against RVFV [27]. In our study expression of IFNc was upregulated in all mice after infection, and there was only a decrease in viral replication in immunized mice indicating that IFNc did not play a significant role in protection. Also, the massive expression of IFNc in control mice late after infection did not contain viral replication in the liver and likely rather contributed to liver pathology. The improper expression of IL-10 during a viral infection might contribute to immune escape events since IL-10 is an anti- inflammatory and immunosuppressive cytokine that inhibits the actions of Th-1 and NK cells, decreases antigen presentation and limits the production of various important cytokines (i.e. IL-12, IL- 18 and TNF-a) [73]. It has been shown that IL-10 is upregulated after West Nile virus (WNV) infection, and that IL-10 deficient mice are less susceptible to WNV infection than mice expressing the gene normally [92]. Dengue virus has also been shown to replicate less efficiently when IL-10 expression is suppressed [93]. Some viruses even express IL-10 homologs to enable them to modulate the host immune system and escape viral clearance [94– 96]. There was an early upregulation of IL-10 expression in liver of adjuvant control mice when an anti-inflammatory response would seem unnecessary. The early detection of replicating RVFV in liver of adjuvant control mice might be contributed to immune escape because of this early activation of IL-10. During the later stages of infection when high level virus replication was noted in non-immunized mice, the host upregulated expression of IL-10 to counteract severe inflammation of tissues. A consequence of this, however, is the suppression of various immune responses as described above which possibly contributed to inefficient clearance Protective Anti-RVFV NP Responses in Mice of virus in control mice or immune escape. The cytokines IL-10 and IFNc are counter-regulatory of each other with IFNc being responsible for pathogen clearance and IL-10 minimizing pathology [73], and therefore their relative expression levels can be an indication of host immune response bias towards virus clearance or minimizing immunopathology. At the acute phase of infection and height of viral replication in non-immunized mice the IL-10 to IFNc ratio was massively favoured towards IL-10, indicating that these host responses were biased towards decreasing inflammation and pathology rather than clearing the virus, which most likely lead to ineffective viral clearance and high virus titers still being detectable at 120 hours p.i. It was interesting to note replication of virus to a much higher titer in spleen of recNP immunized mice compared to liver. This, however, did not result in any form of clinical disease in immunized mice which is further evidence that early disease progression is proportional to viral replication in the host liver and not necessarily other organs. The activation of genes with pro-apoptotic and pro-inflamma- tory effects and the suppression of genes with anti-apoptotic effects in the liver of control mice as a result of uncontrolled viral replication, most probably contributed to severe hepatic disease. In the spleen activation of apoptosis and inflammation was not as pronounced as in the liver of non-immunized mice at 72 hours p.i. The liver is, however, the main target organ for viral replication, the primary site for lesions and rapid severe hepatic damage is probably responsible for the early clinical signs of RVF [89]. The overexpression of CD40, a member of the TNF receptor superfamily and potent activator of nuclear factor kappa beta, in the liver of non-immunized mice is of particular importance to apoptosis and inflammation. Mice deficient in the expression of CD40 have been shown to have improved survival during bacterial sepsis as a result of decreased induction of IL-6, IL-10, IL-12 and IFNc expression [97]. Contrary to upregulation in liver the expression of CD40, as well as its ligand CD40L (CD154), was downregulated in spleen of non-immunized mice during acute infection. Apart from its role in apoptosis of hepatocytes, CD40 plays a very important role in mediating B- and T-cell responses by controlling cytokine secretion, proliferation and differentiation of B- and T-cells [51]. In addition to downregulation of CD40, various other genes involved in B- and T-cell immunity were also downregulated in the spleens of non-immunized mice. These include NOS2, CD3e, IL7, IL18, CD8a,CD1D1, CD74, CR2 and IL4. The spleen is a very important organ in the host immune system and these results indicate that infection with RVFV has a marked effect on the regulation of the host immune response on a molecular level. It is possible that this broad downregulation of humoral and cellular immunity could have resulted in uncon- trolled replication of RVFV, since the virus was still detectable at high titers 2 days later in non-immunized mice. The expression of a TNF receptor superfamily ligand (Tnfsf14) was also upregulated in liver of control mice, but normal in their spleens. This protein is able to block TNFa-mediated apoptosis but not FAS-mediated apoptosis, and is a co-stimulatory factor that enhances T-cell- mediated immunity, leading to severe inflammation [41,98]. The upregulation of the genes expressing the cell survival factor osteopontin (Spp1) and the Cyclin-dependent kinase inhibitor P21 (Cdkn1a), which is upregulated in response to tissue injury [99,100], is evidence of the host’s attempt to counteract the damaging effects of the infection. P21 interacts, amongst others, with the growth arrest and DNA damage-inducible gene 45 (Gadd45) [101], which was also upregulated in control mice. Gadd45 has been implicated in DNA repair, apoptosis, regulation of signal transduction and cell cycle control [102]. The NSs protein of RVFV has been shown to interact with some specific PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e25027