Rift Valley Fever Virus Propagates in Human Villous Trophoblast Cell Lines and Induces Cytokine mRNA Responses Known to Provoke Miscarriage.

The mosquito-borne Rift Valley fever (RVF) is a prioritised disease that has been listed by the World Health Organization for urgent research and development of counteraction. Rift Valley fever virus (RVFV) can cause a cytopathogenic effect in the infected cell and induce hyperimmune responses that contribute to pathogenesis. In livestock, the consequences of RVFV infection vary from mild symptoms to abortion. In humans, 1-3% of patients with RVFV infection develop severe disease, manifested as, for example, haemorrhagic fever, encephalitis or blindness. RVFV infection has also been associated with miscarriage in humans. During pregnancy, there should be a balance between pro-inflammatory and anti-inflammatory mediators to create a protective environment for the placenta and foetus. Many viruses are capable of penetrating that protective environment and infecting the foetal-maternal unit, possibly via the trophoblasts in the placenta, with potentially severe consequences. Whether it is the viral infection per se, the immune response, or both that contribute to the pathogenesis of miscarriage remains unknown. To investigate how RVFV could contribute to pathogenesis during pregnancy, we infected two human trophoblast cell lines, A3 and Jar, representing normal and transformed human villous trophoblasts, respectively. They were infected with two RVFV variants (wild-type RVFV and RVFV with a deleted NSs protein), and the infection kinetics and 15 different cytokines were analysed. The trophoblast cell lines were infected by both RVFV variants and infection caused upregulation of messenger RNA (mRNA) expression for interferon (IFN) types I-III and inflammatory cytokines, combined with cell line-specific mRNA expression of transforming growth factor (TGF)-ß1 and interleukin (IL)-10. When comparing the two RVFV variants, we found that infection with RVFV lacking NSs function caused a hyper-IFN response and inflammatory response, while the wild-type RVFV suppressed the IFN I and inflammatory response. The induction of certain cytokines by RVFV infection could potentially lead to teratogenic effects that disrupt foetal and placental developmental pathways, leading to birth defects and other pregnancy complications, such as miscarriage.

Production of small ruminant morbillivirus, rift valley fever virus and lumpy skin disease virus in CelCradle™ -500A bioreactors.

BACKGROUND: Animal vaccination is an important way to stop the spread of diseases causing immense damage to livestock and economic losses and the potential transmission to humans. Therefore effective method for vaccine production using simple and inexpensive bioprocessing solutions is very essential. Conventional culture systems currently in use, tend to be uneconomic in terms of labor and time involved. Besides, they offer a limited surface area for growth of cells. In this study, the CelCradle™-500A was evaluated as an alternative to replace conventional culture systems in use such as Cell factories for the production of viral vaccines against small ruminant morbillivirus (PPR), rift valley fever virus (RVF) and lumpy skin disease virus (LSD). RESULTS: Two types of cells Vero and primary Lamb Testis cells were used to produce these viruses. The study was done in 2 phases as a) optimization of cell growth and b) virus cultivation. Vero cells could be grown to significantly higher cell densities of 3.04 × 109 using the CelCradle™-500A with a shorter doubling time as compared to 9.45 × 108 cells in Cell factories. This represents a 19 fold increase in cell numbers as compared to seeding vs only 3.7 fold in Cell factories. LT cells achieved modestly higher cell densities of 6.7 × 108 as compared to 6.3 × 108 in Cell factories. The fold change in densities for these cells was 3 fold in the CelCradle™-500A vs 2.5 fold in Cell factories. The titers in the conventional system and the bioreactor were not significantly different. However, the Cell-specific virus yield for rift valley fever virus and lumpy skin disease virus are higher (25 virions/cell for rift valley fever virus, and 21.9 virions/cell for lumpy skin disease virus versus 19.9 virions/cell for rift valley fever virus and 10 virions/cell for lumpy skin disease virus). CONCLUSIONS: This work represents a novel study for primary lamb testis cell culture in CellCradle™-500A bioreactors. In addition, on account of the high cell densities obtained and the linear scalability the titers could be further optimized using other culture process such us perfusion.

In Vitro Evaluation of Anti-Rift Valley Fever Virus, Antioxidant and Anti-Inflammatory Activity of South African Medicinal Plant Extracts.

Rift valley fever virus (RVFV) is a mosquito-borne virus endemic to sub-Saharan African countries, and the first sporadic outbreaks outside Africa were reported in the Asia-Pacific region. There are no approved therapeutic agents available for RVFV; however, finding an effective antiviral agent against RVFV is important. This study aimed to evaluate the antiviral, antioxidant and anti-inflammatory activity of medicinal plant extracts. Twenty medicinal plants were screened for their anti-RVFV activity using the cytopathic effect (CPE) reduction method. The cytotoxicity assessment of the extracts was done before antiviral screening using the MTT assay. Antioxidant and reactive oxygen/nitrogen species' (ROS/RNS) inhibitory activity by the extracts was investigated using non-cell-based and cell-based assays. Out of twenty plant extracts tested, eight showed significant potency against RVFV indicated by a decrease in tissue culture infectious dose (TCID50) < 105. The cytotoxicity of extracts showed inhibitory concentrations values (IC50) > 200 µg/mL for most of the extracts. The antioxidant activity and anti-inflammatory results revealed that extracts scavenged free radicals exhibiting an IC50 range of 4.12-20.41 µg/mL and suppressed the production of pro-inflammatory mediators by 60-80% in Vero cells. This study demonstrated the ability of the extracts to lower RVFV viral load and their potency to reduce free radicals.

Rift Valley Fever Virus: Propagation, Quantification, and Storage.

Rift Valley fever virus (RVFV) is an arthropod-borne, zoonotic disease endemic to sub-Saharan Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever have had up to 100% mortality rates in fetal and neonatal sheep. Upon infection of ruminant and human hosts alike, RVFV infection causes an at times severe hepatitis and pathology in many other organs. The enveloped virion contains a tripartite, predominantly negative-sense, single-stranded RNA genome, which codes for the proteins the virus needs to replicate both in mammalian hosts and insect vectors. Endemic countries often use attenuated RVFV strains for vaccination of livestock but there are no commercially licensed vaccines for humans or livestock in non-endemic areas. In the laboratory, RVFV can be readily propagated and manipulated in vitro using cell culture systems. Presented in this article are techniques routinely used in RVFV research that have proven successful in our laboratories. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Propagation of Rift Valley fever virus in mammalian cells Basic Protocol 2: Quantification of Rift Valley fever virus by plaque assay Basic Protocol 3: Quantification of Rift Valley fever virus by 50% tissue culture infectious dose (TCID50 ) assay Basic Protocol 4: Quantification of Rift Valley fever virus by focus-forming assay Basic Protocol 5: Storage and disinfection Alternate Protocol 1: Propagation of Rift Valley fever virus in MRC-5 cells Alternate Protocol 2: Propagation of RVFV in mosquito-derived cells Alternate Protocol 3: TCID50 detection using fluorescence visualization Support Protocol 1: Calculation of the amount of virus needed to infect a flask at a chosen multiplicity of infection Support Protocol 2: Calculation of the virus titer by plaque assay or focus-forming assay Support Protocol 3: Calculation of the TCID50 titer by the method of Reed and Muench Support Protocol 4: Calculation of the antibody volume for the focus-forming assay.

Host-based processes as therapeutic targets for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is an enveloped, segmented, negative sense RNA virus that replicates within the host's cytoplasm. To facilitate its replication, RVFV must utilize host cell processes and as such, these processes may serve as potential therapeutic targets. This review summarizes key host cell processes impacted by RVFV infection. Specifically the influence of RVFV on host transcriptional regulation, post-transcriptional regulation, protein half-life and availability, host signal transduction, trafficking and secretory pathways, cytoskeletal modulation, and mitochondrial processes and oxidative stress are discussed. Therapeutics targeted towards host processes that are essential for RVFV to thrive as well as their efficacy and importance to viral pathogenesis are highlighted.

Rift Valley fever virus: strategies for maintenance, survival and vertical transmission in mosquitoes.

Rift Valley fever virus (RVFV) is a mosquito-borne arbovirus causing severe disease in humans and ruminants. Spread of RVFV out of Africa has raised concerns that it could emerge in Europe or the USA. Virus persistence is dependent on successful infection of, replication in, and transmission to susceptible vertebrate and invertebrate hosts, modulated by virus-host and vector-virus interactions. The principal accepted theory for the long-term maintenance of RVFV involves vertical transmission (VT) of virus to mosquito progeny, with the virus surviving long inter-epizootic periods within the egg. This VT hypothesis, however, is yet to be comprehensively proven. Here, evidence for and against the VT of RVFV is reviewed along with the identification of factors limiting its detection in natural and experimental data. The observations of VT for other arboviruses in the genera Alphavirus, Flavivirus and Orthobunyavirus are discussed within the context of RVFV. The review concludes that VT of RVFV is likely but that current data are insufficient to irrefutably prove this hypothesis.

Rapamycin modulation of p70 S6 kinase signaling inhibits Rift Valley fever virus pathogenesis.

Despite over 60 years of research on antiviral drugs, very few are FDA approved to treat acute viral infections. Rift Valley fever virus (RVFV), an arthropod borne virus that causes hemorrhagic fever in severe cases, currently lacks effective treatments. Existing as obligate intracellular parasites, viruses have evolved to manipulate host cell signaling pathways to meet their replication needs. Specifically, translation modulation is often necessary for viruses to establish infection in their host. Here we demonstrated phosphorylation of p70 S6 kinase, S6 ribosomal protein, and eIF4G following RVFV infection in vitro through western blot analysis and in a mouse model of infection through reverse phase protein microarrays (RPPA). Inhibition of p70 S6 kinase through rapamycin treatment reduced viral titers in vitro and increased survival and mitigated clinical disease in RVFV challenged mice. Additionally, the phosphorylation of p70 S6 kinase was decreased following rapamycin treatment in vivo. Collectively these data demonstrate modulating p70 S6 kinase can be an effective antiviral strategy.

Experimental Infection of Calves by Two Genetically-Distinct Strains of Rift Valley Fever Virus.

Recent outbreaks of Rift Valley fever in ruminant livestock, characterized by mass abortion and high mortality rates in neonates, have raised international interest in improving vaccine control strategies. Previously, we developed a reliable challenge model for sheep that improves the evaluation of existing and novel vaccines in sheep. This sheep model demonstrated differences in the pathogenesis of Rift Valley fever virus (RVFV) infection between two genetically-distinct wild-type strains of the virus, Saudi Arabia 2001 (SA01) and Kenya 2006 (Ken06). Here, we evaluated the pathogenicity of these two RVFV strains in mixed breed beef calves. There was a transient increase in rectal temperatures with both virus strains, but this clinical sign was less consistent than previously reported with sheep. Three of the five Ken06-infected animals had an early-onset viremia, one day post-infection (dpi), with viremia lasting at least three days. The same number of SA01-infected animals developed viremia at 2 dpi, but it only persisted through 3 dpi in one animal. The average virus titer for the SA01-infected calves was 1.6 logs less than for the Ken06-infected calves. Calves, inoculated with either strain, seroconverted by 5 dpi and showed time-dependent increases in their virus-neutralizing antibody titers. Consistent with the results obtained in the previous sheep study, elevated liver enzyme levels, more severe liver pathology and higher virus titers occurred with the Ken06 strain as compared to the SA01 strain. These results demonstrate the establishment of a virulent challenge model for vaccine evaluation in calves.

High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection.

Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection.

Cytokine response in mouse bone marrow derived macrophages after infection with pathogenic and non-pathogenic Rift Valley fever virus.

Rift Valley fever virus (RVFV) is the most pathogenic member of the genus Phlebovirus within the family Bunyaviridae, and can cause severe disease in humans and livestock. Until recently, limited information has been published on the cellular host response elicited by RVFV, particularly in macrophages and dendritic cells, which play critical roles in stimulating adaptive and innate immune responses to viral infection. In an effort to define the initial response of host immunomodulatory cells to infection, primary mouse bone marrow derived macrophages (BMDM) were infected with the pathogenic RVFV strain ZH501, or attenuated strains MP-12 or MP-12 based Clone13 type (rMP12-C13 type), and cytokine secretion profiles examined. The secretion of T helper (Th)1-associated antiviral cytokines, chemokines and various interleukins increased rapidly after infection with the attenuated rMP12-C13 type RVFV, which lacks a functional NSs virulence gene. In comparison, infection with live-attenuated MP-12 encoding a functional NSs gene appeared to cause a delayed immune response, while pathogenic ZH501 ablates the immune response almost entirely. These data demonstrate that NSs can inhibit components of the BMDM antiviral response and supports previous work indicating that NSs can specifically regulate the type I interferon response in macrophages. Furthermore, our data demonstrate that genetic differences between ZH501 and MP-12 reduce the ability of MP-12 to inhibit antiviral signalling and subsequently reduce virulence in BMDM, demonstrating that viral components other than NSs play a critical role in regulating the host response to RVFV infection.

Role of the cytosolic tails of Rift Valley fever virus envelope glycoproteins in viral morphogenesis.

The correct folding, heterodimerization and trafficking of Gn/Gc envelope glycoproteins of Rift Valley fever virus, RVFV (Bunyaviridae and Phlebovirus genus) are essential for Golgi assembly and budding of viral particles. The Gn and Gc carboxy-terminus contain a Golgi targeting and an ER-retrieval signal, respectively. We generated RVFV-like particles with mutations in the cytosolic tails of Gn or Gc and identified regions important for release of infectious particles. The role of specific amino-acids in these regions was further investigated by creating recombinant mutant viruses by reverse-genetics. Residues outside the suspected Golgi targeting motif, i.e. the di-lysine K29-K30 motif and the N43, R44 and I46 residues of the Gn cytosolic domain, appeared important for Golgi localization and RNP packaging. Concerning the Gc tail, replacement of K2 or K3 in the di-lysine motif, had a drastic impact on Gn trafficking and induced an important organelle redistribution and cell remodeling, greatly affecting particle formation and release.

Single-dose immunization with virus replicon particles confers rapid robust protection against Rift Valley fever virus challenge.

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The potential for RVFV introduction outside the area of endemicity highlights the need for fast-acting, safe, and efficacious vaccines. Here, we demonstrate a robust system for the reverse genetics generation of a RVF virus replicon particle (VRP(RVF)) vaccine candidate. Using a mouse model, we show that VRP(RVF) immunization provides the optimal balance of safety and single-dose robust efficacy. VRP(RVF) can actively synthesize viral RNA and proteins but lacks structural glycoprotein genes, preventing spread within immunized individuals and reducing the risk of vaccine-induced pathogenicity. VRP(RVF) proved to be completely safe following intracranial inoculation of suckling mice, a stringent test of vaccine safety. Single-dose subcutaneous immunization with VRP(RVF), although it is highly attenuated, completely protected mice against a virulent RVFV challenge dose which was 100,000-fold greater than the 50% lethal dose (LD(50)). Robust protection from lethal challenge was observed by 24 h postvaccination, with 100% protection induced in as little as 96 h. We show that a single subcutaneous VRP(RVF) immunization initiated a systemic antiviral state followed by an enhanced adaptive response. These data contrast sharply with the much-reduced survivability and immune responses observed among animals immunized with nonreplicating viral particles, indicating that replication, even if confined to the initially infected cells, contributes substantially to protective efficacy at early and late time points postimmunization. These data demonstrate that replicon vaccines successfully bridge the gap between safety and efficacy and provide insights into the kinetics of antiviral protection from RVFV infection.

Creation of a recombinant Rift Valley fever virus with a two-segmented genome.

Rift Valley fever virus (RVFV; family Bunyaviridae) is a clinically important, mosquito-borne pathogen of both livestock and humans, which is found mainly in sub-Saharan Africa and the Arabian Peninsula. RVFV has a trisegmented single-stranded RNA (ssRNA) genome. The L and M segments are negative sense and encode the L protein (viral polymerase) on the L segment and the virion glycoproteins Gn and Gc as well as two other proteins, NSm and 78K, on the M segment. The S segment uses an ambisense coding strategy to express the nucleocapsid protein, N, and the nonstructural protein, NSs. Both the NSs and NSm proteins are dispensable for virus growth in tissue culture. Using reverse genetics, we generated a recombinant virus, designated r2segMP12, containing a two-segmented genome in which the NSs coding sequence was replaced with that for the Gn and Gc precursor. Thus, r2segMP12 lacks an M segment, and although it was attenuated in comparison to the three-segmented parental virus in both mammalian and insect cell cultures, it was genetically stable over multiple passages. We further show that the virus can stably maintain an M-like RNA segment encoding the enhanced green fluorescent protein gene. The implications of these findings for RVFV genome packaging and the potential to develop multivalent live-attenuated vaccines are discussed.

Novel suspension cell-based vaccine production systems for Rift Valley fever virus-like particles.

Rift Valley fever virus (RVFV) is an arthropod-borne pathogen that often results in severe morbidity and mortality in both humans and livestock. As its geographic range continues to expand, it presents a real threat to naïve populations around the world by accidental introduction (e.g., the result of increased travel) or intentional release (e.g., a bioterror event). While there is a clear need for a safe and efficacious vaccine against this emerging and re-emerging pathogen, no FDA-approved vaccine is currently available. This need was addressed by the establishment of novel mammalian and insect suspension cell line systems for the efficient production of RVF virus-like particle (VLP)-based vaccine candidates. A direct comparison of the production of RVF VLPs in these systems was performed. Optimization and characterization resulted in a production platform suitable for scale-up. Furthermore, RVF VLP-based vaccines were tested in a lethal challenge model and showed full protection, demonstrating that RVF VLPs present promising RVFV vaccine candidates.

Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation.

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-beta mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD) or alpha-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR)-mediated eukaryotic initiation factor (eIF)2alpha phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2alpha accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2alpha phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2alpha phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts.

RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses.

Rift Valley fever virus (RVFV, Bunyaviridae, Phlebovirus) is a mosquito-transmitted arbovirus that causes human and animal diseases in sub-Saharan Africa and was introduced into the Arabian Peninsula in 2000. Here, we describe a method of reverse genetics to recover infectious RVFV from transfected plasmids based on the use of the cellular RNA polymerase I promoter to synthesize viral transcripts. We compared its efficiency with a system using T7 RNA polymerase and found that both are equally efficient for the rescue of RVFV generating titers of approx. 10(7) to 10(8) pfu/ml. We used the RNA polymerase I-based system to rescue both attenuated MP12 and virulent ZH548 strains as well as chimeric MP12-ZH548 viruses, and in addition RVFV expressing reporter proteins.

Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene.

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.

Immunogenicity of combination DNA vaccines for Rift Valley fever virus, tick-borne encephalitis virus, Hantaan virus, and Crimean Congo hemorrhagic fever virus.

DNA vaccines for Rift Valley fever virus (RVFV), Crimean Congo hemorrhagic fever virus (CCHFV), tick-borne encephalitis virus (TBEV), and Hantaan virus (HTNV), were tested in mice alone or in various combinations. The bunyavirus vaccines (RVFV, CCHFV, and HTNV) expressed Gn and Gc genes, and the flavivirus vaccine (TBEV) expressed the preM and E genes. All vaccines were delivered by gene gun. The TBEV DNA vaccine and the RVFV DNA vaccine elicited similar levels of antibodies and protected mice from challenge when delivered alone or in combination with other DNAs. Although in general, the HTNV and CCHFV DNA vaccines were not very immunogenic in mice, there were no major differences in performance when given alone or in combination with the other vaccines.

Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs.

Rift Valley fever virus (RVFV), a phlebovirus of the family Bunyaviridae, is a major public health threat in Egypt and sub-Saharan Africa. The viral and host cellular factors that contribute to RVFV virulence and pathogenicity are still poorly understood. All pathogenic RVFV strains direct the synthesis of a nonstructural phosphoprotein (NSs) that is encoded by the smallest (S) segment of the tripartite genome and has an undefined accessory function. In this report, we show that MP12 and clone 13, two attenuated RVFV strains with mutations in the NSs gene, were highly virulent in IFNAR(-/-) mice lacking the alpha/beta interferon (IFN-alpha/beta) receptor but remained attenuated in IFN-gamma receptor-deficient mice. Both attenuated strains proved to be excellent inducers of early IFN-alpha/beta production. In contrast, the virulent strain ZH548 failed to induce detectable amounts of IFN-alpha/beta and replicated extensively in both IFN-competent and IFN-deficient mice. Clone 13 has a defective NSs gene with a large in-frame deletion. This defect in the NSs gene results in expression of a truncated protein which is rapidly degraded. To investigate whether the presence of the wild-type NSs gene correlated with inhibition of IFN-alpha/beta production, we infected susceptible IFNAR(-/-) mice with S gene reassortant viruses. When the S segment of ZH548 was replaced by that of clone 13, the resulting reassortants became strong IFN inducers. When the defective S segment of clone 13 was exchanged with the wild-type S segment of ZH548, the reassortant virus lost the capacity to stimulate IFN-alpha/beta production. These results demonstrate that the ability of RVFV to inhibit IFN-alpha/beta production correlates with viral virulence and suggest that the accessory protein NSs is an IFN antagonist.