regions of host cell DNA, causing defects in host chromosome structure and segregation [103]. Therefore it might be that these DNA damage inducible proteins are upregulated in an attempt to arrest the cell cycle of affected cells and prevent apoptosis. The fact that P21 was upregulated in healthy recNP-immunized mice is probably as a result of the very low level of viral replication in their livers. Although the expression of some genes were similarly regulated during acute infection in the livers and spleens of non-immunized mice, a number of genes were quite distinctly only up- or downregulated in either liver or spleen, with some even being upregulated in one tissue and downregulated in the other. This distinct expression pattern between tissues is very likely a result of the different cellular composition of the liver and spleen. The liver consists mostly of hepatocytes, whereas the spleen consists of a red pulp where red blood cells are filtered and monocytes are stored, and a white pulp which consists of B- and T-lymphocytes. It seems like the specific pattern of genes regulated during acute infection in the liver actually contributed to liver pathology by enhancing apoptosis and inflammation without being able to decrease viral replication to less virulent levels. On the other hand in the spleen the expression of genes involved in activation and modulation of B- and T-cell immunity were altered during acute infection, probably contributing to persistence of the virus in the host up to 120 hours. It was very interesting to note that, despite viral replication to a relatively high level in spleen of recNP immunized mice at 72 hours p.i., none of the 84 genes analyzed were up- or downregulated in their spleens. The mechanism by which recNP immunized mice can, 72 hours after infection, maintain expres- sion levels of genes involved in immune activation similar to expression levels in spleens of uninfected mice, despite viral replication close to that in non-immunized mice where several genes were altered, needs to be further investigated. It is, however, fair to assume that this normal expression of genes involved in immune activation at 72 hours p.i. contributed to efficient viral clearance in immunized mice spleens, as is evidenced by the absence of replicating virus at the next sampling point (120 h). Further it is also fair to assume that the downregulation of B- and T-cell immunity in non-immunized mice at 72 hours p.i. contributed to the virus still replicating in their spleens at 120 hours p.i. Therefore, although not the main target organ of RVFV and not as important for disease progression as the liver, the spleen seems to be very important in regulating immune responses and decreasing the replication of the virus with dysregulation resulting in immune evasion. Further evidence of the dysregulation of host immune responses by RVFV infection is the fact that up- or downregulation of several genes (i.e. IL-18, IL- 12b, CD40 and SOCS5) indicate bias towards both Th1 and Th2 immunity in the same host. In conclusion, the expression of type I IFN was upregulated in the liver and spleen of immunized mice shortly after RVFV challenge, compared to a delayed upregulation of the same gene in non-immunized mice. In the acute phase of liver infection, however, there was a massive upregulation of type I and II interferon in the presence of high viral titers in non-immunized mice associated with downregulation of several genes involved in the activation of B- and T-cells in the spleen, compared to normal expression in immunized mice. Furthermore various genes with pro-apoptotic and pro-inflammatory effects were strongly upregu- lated, and anti-apoptotic genes downregulated in liver of non- immunized mice. Host gene responses identified in this study may be useful targets for the development of therapeutic interventions, e.g. suppressing inflammatory and apoptotic effects of RVFV infection in the liver, Protective Anti-RVFV NP Responses in Mice or limiting the suppression of B- and T-cell activation in the spleen, and further aid the evaluation of subunit candidate vaccines. Materials and Methods Ethics statement This study was carried out in strict accordance with the recommendations of the South African National Standards for the Care and Use of Animals for Scientific Purposes (SANS 10386:2008) and the Guidelines for the Use and Care of Animals in Experimental Education and other Scientific Purposes of the University of the Witwatersrand, South Africa. The protocol was approved by the University of the Witwatersrand Animal Ethics Screening Committee (clearance certificate number AESC 2008/ 16/4). Blood collection after immunization was done under Ketamine/Xylazine anaesthesia, organ tissue collection after viral challenge was done post-mortem after euthanasia with carbon dioxide (CO2) asphyxiation, and all efforts were made to minimize suffering. Bacterial expression of recombinant RVFV N protein Bacterial expression and purification of the recombinant N protein was carried out as described previously [10]. Mouse immunization Four-week old female BALB/cOlaHsd (Harlan Laboratories, U.K LTD) mice were housed in groups of six in standard plastic mouse cages with wood shaving bedding in a specific pathogen free (SPF) facility during the immunization part of the experiment. Group sizes were calculated so as to have three mice per group per time point for each collection to enable statistical relevance. The immunized group consisted of 60 mice each immunized with a 200 ml inoculum containing 70 mg RVFV recNP in combination with Alhydrogel (Sigma, U.S.A). The adjuvant control group consisted of 18 mice inoculated with Alhydrogel in PBS. The placebo control group consisted of 18 mice which were inoculated with PBS buffer. The normal control group consisted of 18 mice that did not receive any inoculation during the immunization experiment, but were included as an age related control group for the gene expression experiment. All mice, except the age-related normal control group, were inoculated subcutaneously (s.c.) and received identical booster inoculums on day 14 after the initial inoculation. Three mice from the recNP-immunized group were anaesthetized by intramuscular inoculation with a combination of Ketamine (35 mg/kg body mass) and Xylazine (5 mg/kg body mass) and bled by cardiac puncture on each of the following days post-immunization to monitor development of immune responses: day 0, 3, 5, 7, 10 and 12 after the first immunization, and day 0, 3, 5, 7, 12, 18, 21 and 27 after the booster immunization. Monitoring of immune response Anti-recNP humoral responses were monitored by the indirect ELISA which was done as described previously [17]. A mouse serum with high levels of both IgG1 and IgG2A subclass antibodies, generated by immunization with RVFV recNP combined with SaponinQ adjuvant (Sigma, U.S.A) in a previous study [17], was used as a positive control. The positive control serum yielded similar optical density values at 405 nm for IgG (1.094), IgG1 (1.01) and IgG2A (0.94) subclass antibodies at the same dilution used for experimental sera (1:400). Optical density (OD) was determined at 405 nm for each IgG subclass and means calculated for duplicate measurements of each sample or control. The mean OD values of samples for different IgG subclass PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e25027