Type I interferons (IFNs) are essential elements during host antiviral defense [55] . Both recombinant RVFV strains inoculated i.p. were able to kill Ifnar1-deficient 129S2/SvPas mice within 2 days whereas wild-type 129S2/SvPas mice survived infection, indicating that a functional IFN-a/b pathway is critical for the protection of mice from fatal infection with these attenuated viruses. We showed that the recombinant viruses could replicate in known target tissues and cells of RVFV. It is not clear whether in the absence of the IFN-a/b receptor, the reporter RVFV can replicate in tissues and cells that are not normally susceptible to infection with a fully virulent RVFV in wild-type mice. Hence, we infected wild-type 129S2/SvPas with the virulent RVFV ZH548 strain and observed infected macrophages in the spleen and pancreaticoduodenal lymph node. However, we failed to identify the peritonitis seen repeatedly in recombinant RVFV-infected Ifnar1 2/2 mice. This suggests that, in Ifnar1 2/2 mice, cells of the macrophage lineage displayed an increase susceptibility to RVFV compared to wild-type mice. The high susceptibility of cells of the macrophage/dendritic lineage to viral infection in the absence of a functional type I IFN system has been previously observed. An increased infection of cells of the macrophage/dendritic lineage was observed in Ifnar1 2/2 mice infected with either the Sindbis virus [56] , or the mouse hepatitis virus [57] . Similarly, macrophages showed the greatest increase in susceptibility among the different splenocyte populations in West Nile virus-infected Ifnar1 2/2 mice compared to wild-type mice [58] . More generally, an increase in the replication of viruses in tissues and cells normally susceptible to virus infection has been previously observed in Ifnar1 2/2 mice. Coxsackievirus replicated dramatically in the liver of Ifnar1-deficient compared with wild-type mice [59] . Finally, previous investigation of poliovirus replication sites in infected Ifnar1 2/2 mice expressing the human poliovirus receptor showed that nontarget tissues became potentially permissive for virus infection when IFNa/b signaling was disrupted [60] . Therefore, the fact that Ifnar1 2/2 mice inoculated with NSsdeficient RVFV strains develop acute hepatitis and eventually die, as wild-type mice infected with a virulent RVFV strain, does not mean that the exact mechanisms of the cellular pathogenesis are the same in Ifnar1 2/2 and wild-type mice. Thus, although Ifnar1 2/2 mice have proven to be a tractable system in which to study the progression of RVFV infection in vivo, the immunocompromised nature of this mutant strain remains a limitation in translating these results directly to wild-type mice. Additional work needs to be done to develop similar whole-organ imaging and flow cytometry analysis in immune-competent mice. Further studies involving the use of fully virulent RVFV -i.e. carrying the NSs gene -which express a reporter gene, might allow us to give a comprehensive picture of the dynamics of natural infection in mammals. Our work provides the basis for the use of bioluminescent and fluorescent RVFV to study the effects of specific mutations in the viral genome and of host genetic factors on the tissue tropism and replication kinetics in living mice.  Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells. To date, quantitative real-time PCR (qRT-PCR) is the most reliable and easy to perform technique to measure the expression level of a selected gene of interest (GOI) by quantifying mRNA transcripts. qRT-PCR is fast and the sensitivity of the method allows precise quantification of minimal differences in expression across a wide dynamic range even when working with limited amounts of starting material. However, several variables associated with the different steps of qRT-PCR experimental procedures can lead to considerable inter-sample variation and possibly to erroneous results: the different amount and quality of starting material; RNA integrity; efficiency in cDNA synthesis and PCR amplification; and differences between tissues or cells in overall transcriptional activity [1] . Among the strategies proposed to control for technical and sample variation in qRT-PCR experiments [2] , the use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors generated in the quantification of gene expression. In this normalization strategy, reference genes are used as internal controls and are submitted to the same experimental protocol of the GOI. The expression level measured for the target gene is then normalized according to the values of the internal controls. It is clear, therefore, that an ideal reference gene should be stably expressed within the samples to be compared irrespective of experimental conditions or external factors, otherwise the detection of small changes become unfeasible and unreliable. A number of studies have well assessed that genes classically thought to be stable for their ubiquitous expression and involvement in cell homeostasis (e.g., GAPDH, ACTB, 18S rRNA) are not always the best reference genes, as they show different behaviour across various cell types and tissues [3, 4] . Accordingly, a proper evaluation of several candidate genes should be performed before any gene expression study [2] .