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Rift Valley fever virus (RVFV) is a mosquito-borne virus endemic to Africa and the Middle East. RVFV infection can cause encephalitis, which is associated with significant morbidity and mortality. Studies of RVFV encephalitis following percutaneous inoculation, as would occur following a mosquito bite, have historically been limited by a lack of consistent animal models. In this review, we describe new insights into the pathogenesis of RVFV and the opportunities provided by new mouse models. We underscore the need to consider viral strain and route of inoculation when interpreting data obtained using animal models. We discuss the trafficking of RVFV and the role of host genetics and immunity in modulating the pathogenesis of RVFV encephalitis. We also explore potential strategies to prevent and treat central nervous system disease caused by RVFV and discuss remaining knowledge gaps.

Rift Valley fever (RVF) is an important zoonotic viral disease affecting several species of domestic and wild ruminants, causing major economic losses and dozens of human deaths in various geographical areas of Africa, where it is endemic. Although it is not present in Europe, there is a risk of its introduction and spread linked to globalisation and climate change. At present, the only measure that could help to prevent the disease is vaccination of flocks in areas at risk of RVF. Available live attenuated vaccines are an effective means of controlling the disease, but their use is often questioned due to residual virulence, particularly in susceptible hosts such as pregnant sheep. On the other hand, no vaccine is currently licensed for use in humans. The development of safe and effective vaccines is therefore a major area of research. In previous studies, we selected under selective mutagenic pressure a highly attenuated RVFV 56/74 virus variant called 40Fp8. This virus showed an extremely attenuated phenotype in both wild-type and immunodeficient A129 (IFNARKO) mice, yet was still able to induce protective immunity after a single inoculation, thus supporting its use as a safe, live attenuated vaccine. To further investigate its safety, in this work we have analysed the attenuation level of 40Fp8 in immunosuppressed mice (A129) when administered by the intranasal route, and compared it with other attenuated RVF viruses that are the basis of vaccines in use or in development. Our results show that 40Fp8 has a much higher attenuated level than these other viruses and confirm its potential as a candidate for safe RVF vaccine development., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: INIA-CSIC (BB and AB) has filed an international patent application (code # WO2021/245313A1) for 40-FP8 based RVF vaccines. The authors declare that the research was conducted in the absence of any commercial, financial, personal, or professional relationships that could be construed as a potential conflict of interest., (Copyright: © 2024 Borrego et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rift Valley fever (RVF) is an emerging arboviral disease affecting both humans and livestock. In humans, RVF displays a spectrum of clinical manifestations, including encephalitis. To date, there are no FDA-approved vaccines or therapeutics for human use, although several are in preclinical development. Few small-animal models of RVF encephalitis exist, further complicating countermeasure assessment. Human mAbs RVFV-140, RVFV-268, and RVFV-379 are recombinant potently neutralizing antibodies that prevent infection by binding the RVFV surface glycoproteins. Previous studies showed that both RVFV-268 and RVFV-140 improve survival in a lethal mouse model of disease, and RVFV-268 has prevented vertical transmission in a pregnant rat model of infection. Despite these successes, evaluation of mAbs in the context of brain disease has been limited. This is the first study to our knowledge to assess neutralizing antibodies for prevention of RVF neurologic disease using a rat model. Administration of RVFV-140, RVFV-268, or RVFV-379 24 hours prior to aerosol exposure to the virulent ZH501 strain of RVFV resulted in substantially enhanced survival and lack of neurological signs of disease. These results using a stringent and highly lethal aerosol infection model support the potential use of human mAbs to prevent the development of RVF encephalitis.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by RVF virus (RVFV). RVFV infections in humans are usually asymptomatic or associated with mild febrile illness, although more severe cases of haemorrhagic disease and encephalitis with high mortality also occur. Currently, there are no licensed human vaccines available. The safety and efficacy of a genetically engineered four-segmented RVFV variant (hRVFV-4s) as a potential live-attenuated human vaccine has been tested successfully in mice, ruminants, and marmosets though the correlates of protection of this vaccine are still largely unknown. In the present study, we have assessed hRVFV-4s-induced humoral and cellular immunity in a mouse model of RVFV infection. Our results confirm that a single dose of hRVFV-4s is highly efficient in protecting naïve mice from developing severe disease following intraperitoneal challenge with a highly virulent RVFV strain and data show that virus neutralizing (VN) serum antibody titres in a prime-boost regimen are significantly higher compared to the single dose. Subsequently, VN antibodies from prime-boost-vaccinated recipients were shown to be protective when transferred to naïve mice. In addition, hRVFV-4s vaccination induced a significant virus-specific T cell response as shown by IFN-γ ELISpot assay, though these T cells did not provide significant protection upon passive transfer to naïve recipient mice. Collectively, this study highlights hRVFV-4s-induced VN antibodies as a major correlate of protection against lethal RVFV infection.

Introduction: This immunoinformatic study identified potential epitopes from the envelopment polyprotein (Gn/Gc) of Rift Valley fever virus (RVFV), a pathogenic virus causing severe fever in humans and livestock. Effective vaccination is crucial for controlling RVFV outbreaks. The identification of suitable epitopes is crucial for the development of safe and effective vaccines., Methodology: Protein sequences were obtained from the UniProt database, and evaluated through VaxiJen v2.0 to predict the B and T-cell epitopes within the RVFV glycoprotein. Gn/Gc protein sequences were analyzed with bioinformatics tools and algorithms. The predicted T-cell and B-cell epitopes were evaluated for antigenicity, allergenicity, and toxicity by the VaxiJen v2.0 system, AllerTop v2.0, and ToxinPred server, respectively., Results: We employed computational methods to screen the RVFV envelopment polyprotein encompassing N-terminal and C-terminal glycoprotein segments, to discover antigenic T- and B-cell epitopes. Our analysis unveiled multiple potential epitopes within the RVFV glycoprotein, specifically within the Gn/Gc protein sequences. Subsequently, we selected eleven cytotoxic T-lymphocytes (CTL) and four helper T-lymphocytes (HTL) for population coverage analysis, which collectively extended to cover 97.04% of the world's population, representing diverse ethnicities and regions. Notably, the CTL epitope VQADLTLMF exhibited binding affinity to numerous human leukocyte antigen (HLA) alleles. The identification of glycoprotein (Gn/Gc) epitopes through this immunoinformatic study bears significant implications for advancing the development of an effective RVFV vaccine., Conclusions: These findings provide valuable insights into the immunological aspects of the disease and may contribute towards the development of broad-spectrum antiviral therapies targeting other RNA viruses with similar polymerase enzymes., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2024 Tanjir Hosen, Saaimatul Huq, Mohammad Abdullah-Al-Shoeb, Shahidul Islam, Muhammad Abul Kalam Azad.)

Rift Valley fever (RVF) is a mosquito-borne zoonotic viral disease endemic to Africa and the Middle East. Live-attenuated RVF vaccines have been studied for both veterinary and human use due to their strong immunogenicity and cost-effective manufacturing. The live-attenuated MP-12 vaccine has been conditionally approved for veterinary use in the U.S.A., and next-generation live-attenuated RVF vaccine candidates are being actively researched. Assessing the virulence phenotype of vaccine seeds or lots is crucial for managing vaccine safety. Previously, preweaning 19-day-old outbred CD1 mice have been used to evaluate the MP-12 strain. This study aimed to characterize the relative virulence of three live-attenuated RVF vaccine strains in 19-day-old inbred C57BL/6 mice: the recombinant MP-12 (rMP-12), the RVax-1, and the ∆NSs-∆NSm-rZH501 strains. Although this mouse model did not show dose-dependent pathogenesis, mice that succumbed to the infection exhibited distinct brain pathology. Mice infected with ∆NSs-∆NSm-rZH501 showed an infiltration of inflammatory cells associated with infected neurons, and focal lesions formed around virus-infected cells. In contrast, mice infected with rMP-12 or RVax-1 showed a minimal association of inflammatory cells in the brain, yet the virus spread diffusely. The preweaning model is likely useful for evaluating host responses to attenuated RVFV strains, although further refinement may be necessary to quantitate the virulence among different RVFV strains or vaccine lots.

Rift Valley fever (RVF) caused by Rift Valley fever virus (RVFV) is a major health concern for both domesticated animals and humans in certain endemic areas of Africa. With changing environmental conditions and identification of vectors capable of transmitting the virus, there is high risk of RVFV spreading into other parts of the world. Furthermore, unavailability of effective vaccines in the event of an outbreak can be a major challenge as witnessed recently in case of SARS-CoV2 pandemic. Hence, identifying potential vaccines and testing their protective efficacy in preclinical models before clinical testing is the absolute need of the hour. Here, we describe methods used to quantify virus-specific T cell responses in mice that were immunized with RVFV strains or antigens., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Single-domain antibodies, referred to as VHH (variable heavy chains of heavy chain-only antibodies) or in their commercial name as nanobodies, are potent tools for the detection of target proteins in biological samples. They have the advantage of being highly stable, specific, and sensitive, with affinities reaching the nanomolar range. We utilized this tool to develop a rapid detection method that discriminates cells infected with Rift Valley fever virus (RVFV), based on the intracellular detection of the viral nonstructural NSm protein localized on the outer membrane of mitochondria. Here we describe how NSm-specific VHHs have been produced, cloned, and characterized, highlighting their value in RVFV research and diagnosis. This work may also raise interest in other potential applications such as antiviral therapy., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Type I interferons (IFN) are pro-inflammatory cytokines which can also exert anti-inflammatory effects via the regulation of interleukin (IL)-1 family members. Several studies showed that interferon receptor (IFNAR)-deficient mice develop severe liver damage upon treatment with artificial agonists such as acetaminophen or polyinosinic:polycytidylic acid. In order to investigate if these mechanisms also play a role in an acute viral infection, experiments with the Bunyaviridae family member Rift Valley fever virus (RVFV) were performed. Upon RVFV clone (cl)13 infection, IFNAR-deficient mice develop a severe liver injury as indicated by high activity of serum alanine aminotransferase (ALT) and histological analyses. Infected IFNAR -/- mice expressed high amounts of IL-36γ within the liver, which was not observed in infected wildtype (WT) animals. In line with this, treatment of WT mice with recombinant IL-36γ induced ALT activity. Furthermore, administration of an IL-36 receptor antagonist prior to infection prevented the formation of liver injury in IFNAR -/- mice, indicating that IL-36γ is causative for the observed liver damage. Mice deficient for adaptor molecules of certain pattern recognition receptors indicated that IL-36γ induction was dependent on mitochondrial antiviral-signaling protein and the retinoic acid-inducible gene-I-like receptor. Consequently, cell type-specific IFNAR knockouts revealed that type I IFN signaling in myeloid cells is critical in order to prevent IL-36γ expression and liver injury upon viral infection. Our data demonstrate an anti-inflammatory role of type I IFN in a model for virus-induced hepatitis by preventing the expression of the novel IL-1 family member IL-36γ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Anzaghe, Niles, Korotkova, Dominguez, Kronhart, Ortega Iannazzo, Bechmann, Bachmann, Mühl, Kochs and Waibler.)

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes severe and potentially fatal hemorrhagic fever in humans. Autophagy is a self-degradative process that can restrict viral replication at multiple infection steps. In this study, we evaluated the effects of RVFV-triggered autophagy on viral replication and immune responses. Our results showed that RVFV infection triggered autophagosome formation and induced complete autophagy. Impairing autophagy flux by depleting autophagy-related gene 5 ( ATG5 ), ATG7 , or sequestosome 1 ( SQSTM1 ) or treatment with autophagy inhibitors markedly reduced viral RNA synthesis and progeny virus production. Mechanistically, our findings demonstrated that the RVFV nucleoprotein (NP) C-terminal domain interacts with the autophagy receptor SQSTM1 and promotes the SQSTM1-microtubule-associated protein 1 light chain 3 B (LC3B) interaction and autophagy. Deletion of the NP C-terminal domain impaired the interaction between NP and SQSTM1 and its ability to trigger autophagy. Notably, RVFV-triggered autophagy promoted viral infection in macrophages but not in other tested cell types, including Huh7 hepatocytes and human umbilical vein endothelial cells, suggesting cell type specificity of this mechanism. It was further revealed that RVFV NP-triggered autophagy dampens antiviral innate immune responses in infected macrophages to promote viral replication. These results provide novel insights into the mechanisms of RVFV-triggered autophagy and indicate the potential of targeting the autophagy pathway to develop antivirals against RVFV. IMPORTANCE We showed that RVFV infection induced the complete autophagy process. Depletion of the core autophagy genes ATG5 , ATG7 , or SQSTM1 or pharmacologic inhibition of autophagy in macrophages strongly suppressed RVFV replication. We further revealed that the RVFV NP C-terminal domain interacted with SQSTM1 and enhanced the SQSTM1/LC3B interaction to promote autophagy. RVFV NP-triggered autophagy strongly inhibited virus-induced expression of interferon-stimulated genes in infected macrophages but not in other tested cell types. Our study provides novel insights into the mechanisms of RVFV-triggered autophagy and highlights the potential of targeting autophagy flux to develop antivirals against this virus.

Rift Valley fever (RVF) is a zoonotic disease caused by RVF Phlebovirus (RVFV). The RVFV MP-12 vaccine strain is known to exhibit residual virulence in the case of a deficient interferon type 1 response. The hypothesis of this study is that virus replication and severity of lesions induced by the MP-12 strain in immunocompromised mice depend on the specific function of the disturbed pathway. Therefore, 10 strains of mice with deficient innate immunity (B6-IFNAR tmAgt , C.129S7(B6)-Ifng tm1Ts /J, B6-TLR3 tm1Flv , B6-TLR7 tm1Aki , NOD/ShiLtJ), helper T-cell- (CD4 tm1Mak ), cytotoxic T-cell- (CD8 atm1Mak ), B-cell- (Igh-J tm1Dhu N?+N2), combined T- and B-cell- (NU/J) and combined T-, B-, natural killer (NK) cell- and macrophage-mediated immunity (NOD.Cg-Prkdc scid Il2rg tm1WjI /SzJ (NSG) mice) were subcutaneously infected with RVFV MP-12. B6-IFNAR tmAgt mice were the only strain to develop fatal disease due to RVFV-induced severe hepatocellular necrosis and apoptosis. Notably, no clinical disease and only mild multifocal hepatocellular necrosis and apoptosis were observed in NSG mice, while immunohistochemistry detected the RVFV antigen in the liver and the brain. No or low virus expression and no lesions were observed in the other mouse strains. Conclusively, the interferon type 1 response is essential for early control of RVFV replication and disease, whereas functional NK cells, macrophages and lymphocytes are essential for virus clearance.

Rift Valley fever virus (RVFV) is an arbovirus found throughout Africa. It causes disease that is typically mild and self-limiting; however, some infected individuals experience severe manifestations, including hepatitis, encephalitis, or even death. Reports of RVFV encephalitis are notable among immunosuppressed individuals, suggesting a role for adaptive immunity in preventing this severe complication. This phenomenon has been modeled in C57BL/6 mice depleted of CD4 T cells prior to infection with DelNSs RVFV (RVFV containing a deletion of nonstructural protein NSs), resulting in late-onset encephalitis accompanied by high levels of viral RNA in the brain in 30% of animals. In this study, we sought to define the specific type(s) of CD4 T cells that mediate protection from RVFV encephalitis. The viral epitopes targeted by CD4 and CD8 T cells were defined in C57BL/6 mice, and tetramers for both CD4 and CD8 T cells were generated. RVFV-specific CD8 T cells were expanded and of a cytotoxic and proliferating phenotype in the liver following infection. RVFV-specific CD4 T cells were identified in the liver and spleen following infection and phenotyped as largely Th1 or Tfh subtypes. Knockout mice lacking various aspects of pathways important in Th1 and Tfh development and function were used to demonstrate that T-bet, CD40, CD40L, and major histocompatibility complex class II (MHC-II) mediated protection from RVFV encephalitis, while gamma interferon (IFN-γ) and interleukin-12 (IL-12) were dispensable. Virus-specific antibody responses correlated with protection from encephalitis in all mouse strains, suggesting that Tfh/B cell interactions modulate clinical outcome in this model. IMPORTANCE The prevention of RVFV encephalitis requires intact adaptive immunity. In this study, we developed reagents to detect RVFV-specific T cells and provide evidence for Tfh cells and CD40/CD40L interactions as critical mediators of this protection.

Discovered in 1931, Rift Valley fever virus (RVFV) is an arbovirus that causes disease in humans and livestock. In humans, disease ranges from a self-limiting febrile illness to a more severe hepatitis or encephalitis. There are currently no licensed human therapeutics for RVFV disease. Given the recent advances in the use of monoclonal antibodies (MAbs) for treating infectious disease, a panel of anti-RVFV Gn glycoprotein MAbs was developed and characterized. RVFV MAbs spanned a range of neutralizing abilities and mapped to distinct epitopes along Gn. The contribution of Fc effector functions in providing MAb-mediated protection from RVFV was assessed. IgG2a version MAbs had increased capacity to induce effector functions and conferred better protection from RVFV challenge in a lethal mouse model than IgG1 version MAbs. Overall, this study shows that Fc-mediated functions are a critical component of humoral protection from RVFV. IMPORTANCE Rift Valley fever virus (RVFV) is a mosquito-borne virus found throughout Africa and into the Middle East. It has a substantial disease burden; in areas of endemicity, up to 60% of adults are seropositive. With a case fatality rate of up to 3% and the ability to cause hemorrhagic fever and encephalitis, RVFV poses a serious threat to human health. Despite the known human disease burden and the fact that it is a NIAID category A priority pathogen and a WHO priority disease for research and development, there are no vaccines or therapeutics available for RVF. In this study, we developed and characterized a panel of monoclonal antibodies against the RVFV surface glycoprotein, Gn. We then demonstrated therapeutic efficacy in the prevention of RVF in vivo in an otherwise lethal mouse model. Finally, we revealed a role for Fc-mediated function in augmenting the protection provided by these antibodies.

Rift Valley fever phlebovirus (RVFV) is a mosquito-transmitted phlebovirus (Family: Phenuiviridae, Order: Bunyavirales) causing severe neonatal mortality and abortion primarily in domestic ruminants. The susceptibility of young domestic swine to RVFV and this species' role in geographic expansion and establishment of viral endemicity is unclear. Six commercially bred Landrace-cross piglets were inoculated subcutaneously with 105 plaque-forming units of RVFV ZH501 strain and two piglets received a sham inoculum. All animals were monitored for clinical signs, viremia, viral shedding, and antibody response for 14 days. Piglets did not develop evidence of clinical disease, become febrile, or experience decreased weight gain during the study period. A brief lymphopenia followed by progressive lymphocytosis was observed following inoculation in all piglets. Four piglets developed a brief viremia for 2 days post-inoculation and three of these had detectable virus in oronasal secretions three days post-inoculation. Primary inoculated piglets seroconverted and those that developed detectable viremias had the highest titers assessed by serum neutralization (1:64-1:256). Two viremic piglets had a lymphoplasmacytic encephalitis with glial nodules; RVFV was not detected by immunohistochemistry in these sections. While young piglets do not appear to readily develop clinical disease following RVFV infection, results suggest swine could be subclinically infected with RVFV.

Phleboviruses (genus P hlebovirus , family Phenuiviridae ) are emerging pathogens of humans and animals. Sand-fly-transmitted phleboviruses are found in Europe, Africa, the Middle East, and the Americas, and are responsible for febrile illness and nervous system infections in humans. Rio Grande virus (RGV) is the only reported phlebovirus in the United States. Isolated in Texas from southern plains woodrats, RGV is not known to be pathogenic to humans or domestic animals, but serologic evidence suggests that sheep ( Ovis aries ) and horses ( Equus caballus ) in this region have been infected. Rift Valley fever virus (RVFV), a phlebovirus of Africa, is an important pathogen of wild and domestic ruminants, and can also infect humans with the potential to cause severe disease. The introduction of RVFV into North America could greatly impact U.S. livestock and human health, and the development of vaccines and countermeasures is a focus of both the CDC and USDA. We investigated the potential for serologic reagents used in RVFV diagnostic assays to also detect cells infected with RGV. Western blots and immunocytochemistry assays were used to compare the antibody detection of RGV, RVFV, and two other New World phlebovirus, Punta Toro virus (South and Central America) and Anhanga virus (Brazil). Antigenic cross-reactions were found using published RVFV diagnostic reagents. These findings will help to inform test interpretation to avoid false positive RVFV diagnoses that could lead to public health concerns and economically costly agriculture regulatory responses, including quarantine and trade restrictions.

Diagnostic performance of an indirect enzyme-linked immunosorbent assay (I-ELISA) based on a recombinant nucleocapsid protein (rNP) of the Rift Valley fever virus (RVFV) was validated for the detection of the IgG antibody in sheep ( n = 3367), goat ( n = 2632), and cattle ( n = 3819) sera. Validation data sets were dichotomized according to the results of a virus neutralization test in sera obtained from RVF-endemic (Burkina Faso, Democratic Republic of Congo, Mozambique, Senegal, Uganda, and Yemen) and RVF-free countries (France, Poland, and the USA). Cut-off values were defined using the two-graph receiver operating characteristic analysis. Estimates of the diagnostic specificity of the RVFV rNP I-ELISA in animals from RVF-endemic countries ranged from 98.6% (cattle) to 99.5% (sheep) while in those originating from RVF-free countries, they ranged from 97.7% (sheep) to 98.1% (goats). Estimates of the diagnostic sensitivity in ruminants from RVF-endemic countries ranged from 90.7% (cattle) to 100% (goats). The results of this large-scale international validation study demonstrate the high diagnostic accuracy of the RVFV rNP I-ELISA. Standard incubation and inactivation procedures evaluated did not have an adverse effect on the detectable levels of the anti-RVFV IgG in ruminant sera and thus, together with recombinant antigen-based I-ELISA, provide a simple, safe, and robust diagnostic platform that can be automated and carried out outside expensive bio-containment facilities. These advantages are particularly important for less-resourced countries where there is a need to accelerate and improve RVF surveillance and research on epidemiology as well as to advance disease control measures.

Rift Valley fever virus (RVFV), an arbovirus belonging to the Phlebovirus genus of the Phenuiviridae family, causes the zoonotic and mosquito-borne RVF. The virus, which primarily affects livestock (ruminants and camels) and humans, is at the origin of recent major outbreaks across the African continent (Mauritania, Libya, Sudan), and in the South-Western Indian Ocean (SWIO) islands (Mayotte). In order to be better prepared for upcoming outbreaks, to predict its introduction in RVFV unscathed countries, and to run efficient surveillance programmes, the priority is harmonising and improving the diagnostic capacity of endemic countries and/or countries considered to be at risk of RVF. A serological inter-laboratory proficiency test (PT) was implemented to assess the capacity of veterinary laboratories to detect antibodies against RVFV. A total of 18 laboratories in 13 countries in the Middle East, North Africa, South Africa, and the Indian Ocean participated in the initiative. Two commercial kits and two in-house serological assays for the detection of RVFV specific IgG antibodies were tested. Sixteen of the 18 participating laboratories (88.9%) used commercial kits, the analytical performance of test sensitivity and specificity based on the seroneutralisation test considered as the reference was 100%. The results obtained by the laboratories which used the in-house assay were correct in only one of the two criteria (either sensitivity or specificity). In conclusion, most of the laboratories performed well in detecting RVFV specific IgG antibodies and can therefore be considered to be prepared. Three laboratories in three countries need to improve their detection capacities. Our study demonstrates the importance of conducting regular proficiency tests to evaluate the level of preparedness of countries and of building a network of competent laboratories in terms of laboratory diagnosis to better face future emerging diseases in emergency conditions., Competing Interests: The authors have declared that no competing interests exist.

The DNA fragment encoding predicted main antigenic region, aa 14-245 on N protein of Rift Valley virus (RVFV) was cloned into the vector pET-28a (+) and p3xFLAG-CMV-10. The recombinant pET-28a-N1 protein was expressed in Escherichia coli BL21 (DE3) with 1 mM isopropyl- b -thio-galactopyranoside at 37°C for 5 hours, and purified by protein purifier. Three monoclonal antibodies (mAbs) named 3A5, 3A6, and 3A7 against N protein were obtained by fusing mouse myeloma cell line SP2/0 with spleen lymphocytes from pET-28a-N1 protein-immunized mice. Finally, the mAbs were characterized by enzyme-linked immunosorbent assays, indirect immunofluorescent assays, and Western blot. The results show that all the mAbs possess high specificity and react with both prokaryotic and eukaryotic N protein, which could provide important materials for the research on the function of N protein and the diagnostic methods of RVFV.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes an important disease in ruminants, with great economic losses. The infection can be also transmitted to humans; therefore, it is considered a major threat to both human and animal health. In a previous work, we described a novel RVFV variant selected in cell culture in the presence of the antiviral agent favipiravir that was highly attenuated in vivo. This variant displayed 24 amino acid substitutions in different viral proteins when compared to its parental viral strain, two of them located in the NSs protein that is known to be the major virulence factor of RVFV. By means of a reverse genetics system, in this work we have analyzed the effect that one of these substitutions, P82L, has in viral attenuation in vivo. Rescued viruses carrying this single amino acid change were clearly attenuated in BALB/c mice while their growth in an interferon (IFN)-competent cell line as well as the production of interferon beta (IFN-β) did not seem to be affected. However, the pattern of nuclear NSs accumulation was modified in cells infected with the mutant viruses. These results highlight the key role of the NSs protein in the modulation of viral infectivity.

γδ T cells are a numerically significant subset of immune cells in ruminants, where they may comprise up to 70 % of all peripheral blood mononuclear cells (PBMCs) in young animals and 25 % in adults. These cells can be activated through traditional TCR-dependent mechanisms, or alternatively in a TCR-independent manner by pattern recognition receptors and have been shown to uptake antigen, as well as process and present it to αβ T cells. We have identified a novel CD11b+ subset of γδ T cells in normal sheep peripheral blood. An increase in the frequency of these cells in sheep peripheral blood in response to immunization with an experimental recombinant subunit Rift Valley fever (RVF) vaccine was observed. However, injection of the vaccine adjuvant ISA-25VG alone without the recombinant RVF virus antigens demonstrated the same effect, pointing to an antigen-independent innate immune function of CD11b+ γδ T cells in response to the adjuvant. In vitro studies showed repeatable increases of CD11b-, CD14-, CD86-, CD40-, CD72-, and IFNγ- expressing γδ T cells in PBMCs after 24 h of incubation in the absence of a mitogen. Moreover, the majority of these myeloid-like γδ T cells were demonstrated to process exogenous antigen even in the absence of mitogen. ConA activation increased CD25- and MHCII- expression in γδ T cells, but not the myeloid associated receptors CD14 or CD11b or co-stimulatory molecules such as CD86 and CD40. Considering the role of CD11b and CD14 in the activation of innate immunity, we hypothesize that this subpopulation of sheep γδ T cells may function as innate antigen presenting and pro-inflammatory cells during immune responses. The results presented here also suggest that stress molecules and/or damage-associated molecular patterns may be involved in triggering antigen presenting and pro-inflammatory functions of γδ T cells, given their appearance in vitro in the absence of specific stimulation. Taken together, these data suggest that the early appearance of γδ T cells following adjuvant administration and their possible role in early activation of αβ T cell subsets may non-specifically contribute to augmented innate immunity and may promote strong initiation of the adaptive immune response to vaccines in general., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Rift Valley fever (RVF) is a zoonotic viral disease of domestic ruminants in Africa and the Arabian Peninsula caused by a mosquito-borne Phlebovirus. Outbreaks in livestock and humans occur after heavy rains favour breeding of vectors, and the virus is thought to survive dry seasons in the eggs of floodwater-breeding aedine mosquitoes. We recently found high seroconversion rates to RVF virus (RVFV) in cattle and goats, in the absence of outbreaks, in far northern KwaZulu-Natal (KZN), South Africa. Here, we report the prevalence of, and factors associated with, neutralizing antibodies to RVFV in 326 sera collected opportunistically from nyala (Tragelaphus angasii) and impala (Aepyceros melampus) culled during 2016-2018 in two nature reserves in the same area. The overall seroprevalence of RVFV, determined using the serum neutralization test, was 35.0% (114/326; 95%CI: 29.8%-40.4%) and tended to be higher in Ndumo Game Reserve (11/20; 55.0%; 95%CI: 31.5%-76.9%) than in Tembe Elephant Park (103/306; 33.6%; 95%CI: 28.4%-39.3%) (p = .087). The presence of antibodies in juveniles (6/21; 28.6%; 95%CI: 11.3%-52.2%) and sub-adults (13/65; 20.0%; 95%CI: 11.1%-37.8%) confirmed that infections had occurred at least until 2016, well after the 2008-2011 RVF outbreaks in South Africa. Odds of seropositivity was higher in adults than in sub-adults (OR = 3.98; 95%CI: 1.83-8.67; p = .001), in males than in females (OR = 2.66; 95%CI: 1.51-4.68; p = .001) and in animals collected ≤2 km from a swamp or floodplain compared with those collected further away (OR = 3.30; 95%CI: 1.70-6.38; p < .001). Under similar ecological conditions, domestic and wild ruminants may play a similar role in maintenance of RVFV circulation and either or both may serve as the mammalian host in a vector-host reservoir system. The study confirms the recent circulation of RVFV in the tropical coastal plain of northern KZN, providing the basis for investigation of factors affecting virus circulation and the role of wildlife in RVF epidemiology., (© 2020 Blackwell Verlag GmbH.)

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis showing complex epidemiological patterns that are poorly understood in South Africa. Large outbreaks occur in the central interior at long, irregular intervals, most recently in 2010-2011; however, the level of herd immunity of ruminant livestock, a key determinant of outbreaks, is unknown. During 2015-2016 a cross-sectional study on 234 randomly-selected farms investigated the prevalence, patterns of, and factors associated with, antibodies to RVF virus (RVFV) in livestock in an area heavily affected by that outbreak. A RVFV inhibition ELISA was used to screen 977 cattle, 1,549 sheep and 523 goats and information on potential risk factors was collected using a comprehensive questionnaire. The estimated RVFV seroprevalence, adjusted for survey design, was 42.9% in cattle, 28.0% in sheep and 9.3% in goats, showing a high degree of farm-level clustering. Seroprevalence increased with age and was higher on private vs. communal land, on farms with seasonal pans (temporary, shallow wetlands) and perennial rivers and in recently vaccinated animals. Seropositivity amongst unvaccinated animals born after the last outbreak indicates likely viral circulation during the post-epidemic period. The current level of herd immunity in livestock may be insufficient to prevent another large outbreak, should suitable conditions recur.

Rift Valley fever virus (RVFV) is a zoonotic arbovirus that causes severe disease in humans and ruminants. The infection is characterized by abortions in pregnant animals, high mortality in neonates as well as febrile illness in humans that develop in 1% of cases encephalitis or hemorrhagic fever. There is presently no specific antiviral treatment for RVFV infection available. In this study, two monoclonal antibodies (mAbs), raised against glycoprotein Gn, were applied in a therapeutic study. Treatment of RVFV infected mice with neutralizing mAb Gn3 alone at two different time points (30 minutes before or 30 minutes after virus challenge) showed only moderate efficacy of about 58.3% survival in both applications. However, a combination therapy together with non-neutralizing mAb Gn32 demonstrated complete protection (100% survival) when applied 30 minutes after the lethal challenge dose. The increase of mAb efficacy is probably based on cooperative neutralization effects. These data suggest that a combination therapy with mAbs Gn3 and Gn32 could be an effective treatment option against RVFV infection., Competing Interests: The authors have declared that no competing interests exist.

The Rift Valley fever virus (RVFV) is an arthropod-borne virus that can not only cause severe disease in domestic animals but also in humans. However, the licensed vaccines or available therapeutics for humans do not exist. Here, we report two Gn-specific neutralizing antibodies (NAbs), isolated from a rhesus monkey immunized with recombinant human adenoviruses type 4 expressing Rift Valley fever virus Gn and Gc protein (rHAdV4-GnGcopt). The two NAbs were both able to protect host cells from RVFV infection. The interactions between NAbs and Gn were then characterized to demonstrate that these two NAbs might preclude RVFV glycoprotein rearrangement, hindering the exposure of fusion loops in Gc to endosomal membranes after the virus invades the host cell. The target region for the two NAbs is located in the Gn domain III, implying that Gn is a desired target for developing vaccines and neutralizing antibodies against RVFV.

Rift Valley fever virus (RVFV), which causes Rift Valley fever (RVF), is a mosquito-borne zoonotic pathogen that causes serious morbidity and mortality in livestock and humans. RVF is a World Health Organization (WHO) priority disease and, together with rabies, is a major health burden in Africa. Here, we present the development and characterization of an inactivated recombinant RVFV and rabies virus (RABV) vaccine candidate (rSRV9-eGn). Immunization with rSRV9-eGn stimulated the production of RVFV-specific IgG antibodies and induced humoral and cellular immunity in mice but did not induce the production of neutralizing antibodies. IgG1 and IgG2a were the main isotypes observed by IgG subtype detection, and IgG3 antibodies were not detected. The ratios of IgG1/IgG2a > 1 indicated a Type 2 humoral immune response. An effective vaccine is intended to establish a long-lived population of memory T cells, and mice generated memory cells among the proliferating T cell population after immunization with rSRV9-eGn, with effector memory T cells (T EM ) as the major population. Due to the lack of prophylactic treatment experiments, it is impossible to predict whether this vaccine can protect animals from RVFV infection with only high titres of anti-RVFV IgG antibodies and no neutralizing antibodies induced, and thus, protection confirmation needs further verification. However, this RVFV vaccine designed with RABV as the vector provides ideas for the development of vaccines that prevent RVFV and RABV infections., Competing Interests: The authors declare that they have no conflicts of interest.

The increasing risk of Rift Valley fever virus (RVFV) infection as a global veterinary and public health threat demands the development of safe and accurate diagnostic tests. The aim of this study was to assess the suitability of a baculovirus expression system to produce recombinant RVFV nucleoprotein (N) for use as serodiagnostic antigen in an indirect enzyme-linked immunosorbent assay (ELISA). The ability of the recombinant N antigen to detect RVFV antibody responses was evaluated in ELISA format using antisera from sheep and cattle experimentally infected with two genetically distinct wild-type RVFV strains and sera from indigenous sheep and goat populations exposed to natural RVFV field infection in The Gambia. The recombinant N exhibited specific reactivity with the N-specific monoclonal antibody and various hyperimmune serum samples from ruminants. The indirect ELISA detected N-specific antibody responses in animals with 100% sensitivity compared to the plaque reduction neutralization test (6 to 21 days postinfection) and with 97% and 100% specificity in sheep and cattle, respectively. There was a high level of correlation between the indirect N ELISA and the virus neutralization test for sheep sera ( R 2 = 0.75; 95% confidence interval [CI] = 0.73 to 0.92) and cattle sera ( R 2 = 0.80; 95% CI = 0.67 to 0.97); in addition, the N-specific ELISA detected RVFV seroprevalence levels of 26.1% and 54.3% in indigenous sheep and goats, respectively, in The Gambia. The high specificity and correlation with the virus neutralization test support the idea of the feasibility of using the recombinant baculovirus-expressed RVFV N-based indirect ELISA to assess RVFV seroprevalence in livestock in areas of endemicity and nonendemicity., (Copyright © 2019 American Society for Microbiology.)

Following the Ebola outbreak in Western Africa in 2013-16, a global effort has taken place for preparedness for future outbreaks. As part of this response, the development of vaccines, treatments and diagnostic tools has been accelerated, especially towards pathogens listed as likely to cause an epidemic and for which there are no current treatments. Several of the priority pathogens identified by the World Health Organisation are haemorrhagic fever viruses. This review provides information on the role of reference materials as an enabling tool for the development and evaluation of assays, and ultimately vaccines and treatments. The types of standards available are described, along with how they can be applied for assay harmonisation through calibration as a relative potency to a common arbitrary unitage system (WHO International Unit). This assures that assay metrology is accurate and robust. We describe reference materials that have been or are being developed for haemorrhagic fever viruses and consider the issues surrounding their production, particularly that of biosafety where the viruses require specialised containment facilities. Finally, we advocate the use of reference materials at early stages, including research and development, as this helps produce reliable assays and can smooth the path to regulatory approval.

Tanzania is one of the sub-Saharan countries that have experienced a number of Rift Valley fever (RVF) outbreaks at intervals of 10-20 years since the first isolation of the virus during the early 1930s. Recent studies have reported serological evidence of inter epizootic/epidemic period circulation of RVF virus (RVFV) in livestock and humans. The aim of this study was to conduct a cross-sectional survey in Tanzania during 2015/16 to further explore the possibility that RVFV was circulating among cattle during the Inter epizootic/epidemic period. A total of 443 cattle samples were collected in Manyara, Dodoma, Singida, and Mbeya regions of Tanzania. The samples were tested for RVFV antibodies using a commercial ELISA kit and a plaque reduction neutralization test. Serum samples were also tested for RVFV viral RNA by an reverse transcription polymerase chain reaction (RT-PCR) assay. An overall RVFV seroprevalence rate of 7.7% (34/443) was detected by ELISA among cattle in all four regions. The Mbeya region cattle had the highest seroprevalence of 26.4% (23/87), followed by Dodoma 5.9% (10/171) and lastly Singida 0.9% (1/101). Of 33 ELISA antibody-positive samples, only 0.2% (1/443) had IgM antibody. Of 36 ELISA antibody-positive and doubtful samples, 32 were positive for neutralizing antibody with titers between 10 and > 10,240. None of the samples were positive for RVFV viral RNA by RT-PCR. The detection of RVFV antibodies in cattle suggested that these animals were involved in an enzootic cycle during the interepidemic period and that the high antibody titers may confer protection of cattle against RVFV.

Rift Valley fever virus (RVFV) is an important mosquito-borne pathogen with devastating impacts on agriculture and public health. With outbreaks being reported beyond the continent of Africa to the Middle East, there is great concern that RVFV will continue to spread to non-endemic areas such as the Americas and Europe. There is a need for safe and high throughput serological assays for rapid detection of RVFV during outbreaks and for surveillance. We evaluated a multiplexing fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies in ruminant sera against the RVFV nucleocapsid Np, glycoprotein Gn, and non-structural protein NSs. Sheep and cattle sera from a region in Kenya with previous outbreaks were tested by FMIA and two commercially available competitive ELISAs (BDSL and IDvet). Our results revealed strong detection of RVFV antibodies against the Np, Gn and NSs antigen targets. Additionally, testing of samples with FMIA Np and Gn had 100% agreement with the IDvet ELISA. The targets developed in the FMIA assay provided a basis for a larger ruminant disease panel that can simultaneously screen several abortive and zoonotic pathogens., (Copyright © 2019. Published by Elsevier B.V.)

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that causes substantial morbidity and mortality in livestock and humans. To date, there are no licensed human vaccines or therapeutics available. Here, we report the isolation of monoclonal antibodies from a convalescent patient, targeting the RVFV envelope proteins Gn and Gc. The Gn-specific monoclonal antibodies exhibited much higher neutralizing activities in vitro and protection efficacies in mice against RVFV infection, compared to the Gc-specific monoclonal antibodies. The Gn monoclonal antibodies were found to interfere with soluble Gn binding to cells and prevent infection by blocking the attachment of virions to host cells. Structural analysis of Gn complexed with four Gn-specific monoclonal antibodies resulted in the definition of three antigenic patches (A, B and C) on Gn domain I. Both patches A and B are major neutralizing epitopes. Our results highlight the potential of antibody-based therapeutics and provide a structure-based rationale for designing vaccines against RVFV.

Rift Valley fever virus (RVFV) causes severe disease in livestock concurrent with zoonotic transmission to humans. A subset of people infected with RVFV develop encephalitis, and significant gaps remain in our knowledge of how RVFV causes pathology in the brain. We previously found that, in Lewis rats, subcutaneous inoculation with RVFV resulted in subclinical disease while inhalation of RVFV in a small particle aerosol caused fatal encephalitis. Here, we compared the disease course of RVFV in Lewis rats after each different route of inoculation in order to understand more about pathogenic mechanisms of fatal RVFV encephalitis. In aerosol-infected rats with lethal encephalitis, neutrophils and macrophages were the major cell types infiltrating the CNS, and this was concomitant with microglia activation and extensive cytokine inflammation. Despite this, prevention of neutrophil infiltration into the brain did not ameliorate disease. Unexpectedly, in subcutaneously-inoculated rats with subclinical disease, detectable viral RNA was found in the brain along with T-cell infiltration. This study sheds new light on the pathogenic mechanisms of RVFV encephalitis., Competing Interests: The authors have declared that no competing interests exist.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus and hemorrhagic fever agent, which causes abortion storms in farmed small ruminants and potentially causes miscarriages in humans. Although live-attenuated vaccines are available for animals, they can only be used in endemic areas and there are currently no commercially available vaccines for humans. Here the authors describe the production of chimaeric RVFV virus-like particles transiently expressed in Nicotiana benthamiana by Agrobacterium tumefaciens-mediated gene transfer. The glycoprotein (Gn) gene is modified by removing its ectodomain (Gne) and fusing it to the transmembrane domain and cytosolic tail-encoding region of avian influenza H5N1 hemagglutinin. This is expressed transiently in N. benthamiana with purified protein yields calculated to be ≈57 mg kg -1 fresh weight. Transmission electron microscopy shows putative chimaeric RVFV Gne-HA particles of 49-60 nm which are immunogenic, eliciting Gn-specific antibody responses in vaccinated mice without the use of adjuvant. To our knowledge, this is the first demonstration of the synthesis of Gne-HA chimaeric RVFV VLPs and the first demonstration of a detectable yield of RVFV Gn in plants., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Vaccination of domestic ruminants is considered to be an effective strategy for protecting these animals against Rift Valley fever (RVF), but available vaccines have limitations. Therefore, the aim of this study was to determine the safety and immunogenicity of RVF virus (RVFV) mutagenesis passage 12 (MP-12) and arMP-12ΔNSm21/384 vaccine candidates in goats (Capra aegagrus hircus) in Tanzania. Goats were vaccinated intramuscularly with RVFV MP-12 or arMP-12ΔNSm21/384, and then on Day 87 post-vaccination (PV) all animals were revaccinated using the RVFV MP-12 vaccine candidate. Serum samples were collected from the animals before and after vaccination at various intervals to test for RVFV using a Vero cell culture assay and reverse transcription polymerase chain reaction and for RVFV-neutralising antibody using a plaque reduction neutralisation assay. Serum samples collected before vaccination on Days -14 and 0, and on Days 3, 4 and 5 PV were negative for RVFV and neutralising antibody. All animals remained healthy, and viremia was not detected in any of the animals. Rift Valley fever virus antibody was first detected on Day 5 PV at a 1:10 dilution in five of five animals vaccinated with the MP-12 vaccine and in five of eight animals vaccinated with arMP-12ΔNSm21/384. Titres then increased and were sustained at 1:40 to 1:640 through to Day 87 PV. All animals that were revaccinated on Day 87 PV with MP-12 developed antibody titres ranging from 1:160 to as high as 1:10 240 on Days 14 and 21 PV. Although the antibody titres for goats vaccinated with RVF MP-12 were slightly higher than titres elicited by the arMP-12ΔNSm21/384 vaccine, these findings demonstrated that both vaccines are promising candidates for the prevention of RVF among Tansanian goats.

The risk of exposure of slaughterhouse workers to Rift Valley fever (RVF) virus-infected animals in Nigeria was assessed by determining the prevalence of anti-RVF IgM in cattle, goats and sheep slaughtered in a major abattoir in Ibadan, Nigeria. Blood samples were collected from 290 animals in Bodija Municipal abattoir, Ibadan, Nigeria in January and February 2017 and analyzed for the presence of RVF virus using IgM Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for detection of the virus RNA. Descriptive statistics was used to analyze data. Overall, an IgM prevalence of 0.7% (2/290) was found among the blood samples of the animals, suggesting recent exposure to the virus. Antibody was detected in the sera from a cow and one goat. RVF virus RNA was not detected in the 2 IgM positive blood samples. There was no statistically significant relationship between RVF IgM infection and some variables of the animals, including age, sex and breed ( p ≥ 0.05). Results of this study indicate active RVF virus transmission in domestic livestock in Nigeria. The study emphasizes the need to embark on monitoring of human and animal populations to prevent outbreak of the virus in the country.

Rift Valley fever virus (RVFV), a mosquito-borne, zoonotic pathogen, is endemic to sub-Saharan Africa and has spread beyond the continent to the Arabian Peninsula. The high likelihood of RVFV's spread to other non-endemic countries spurs the need for development and implementation of rapid diagnostic tests and surveillance programs. In this preliminary evaluation, we assessed the diagnostic accuracy and precision of a recombinant RVFV nucleoprotein based competitive ELISA (cELISA) assay to detect RVFV antibodies compared to a plaque reduction neutralization test (PRNT 80 ), using sera of cattle and sheep that were experimentally infected with either the MP-12 RVFV vaccine or a wild-type RVFV strain, as well as using known RVFV negative sera. With the manufacturer recommended 60% inhibition for the cELISA and a 1:40 titer for the PRNT 80 , the sensitivity and specificity of the cELISA assay was determined to be 95.1% (95% CI = 83.5-99.4%) and 91.8% (95% CI = 85.0-96.2%), respectively. Antibodies to RVFV were first detected at 5 days post inoculation (dpi) in both species' sera. The prototype cELISA is an easy to implement, sensitive, specific, and safe test for the detection of antibodies to RVFV that can be used in surveillance programs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Rift Valley fever phlebovirus (RVFV), the causative agent of an emerging zoonotic disease in Africa and Arabia, can infect a variety of species, predominantly ruminants, camelids, and humans. While clinical symptoms are mostly absent in adult ruminants and camelids, RVFV infection may lead to a serious, sometimes fatal disease in humans. Virus transmissions between individuals and between species mainly occur through mosquito bites, but direct or even indirect contact with infectious materials may also result in infection. Although the main reservoir of the virus is not yet identified, small mammals such as rodents and bats may act as amplifying hosts. We therefore inoculated Rousettus aegyptiacus fruit bats that are abundant in northern Africa with the vaccine strain MP-12, in order to elucidate the general competence of this species for virus propagation and transmission. We were able to detect the RVFV genome in the spleen of each of these animals, and re-isolated the virus from the spleen and liver of some animals. Moreover, we were able to identify the Gc RVFV surface antigen in mild subacute multifocal necrotizing hepatic lesions of one bat which was sacrificed 7 days post exposure. These findings demonstrate that Ro u settus aegyptiacus fruit bats can propagate RVFV., Competing Interests: The authors declare no conflicts of interest.

Rift Valley fever virus (RVFV) is an arbovirus that causes disease in livestock and humans in Africa and the Middle East. While human disease is typically mild and self-limiting, some individuals develop severe manifestations, such as hepatitis, hemorrhagic fever, or encephalitis. Encephalitis occurs 2 to 3 weeks after acute illness; therefore, we hypothesized that it was a result of an inadequate adaptive immunity. To test this hypothesis in vivo , we used an attenuated virus (DelNSsRVFV) that does not typically cause disease in mice. We first characterized the normal immune response to infection with DelNSsRVFV in immunocompetent mice and noted expansion of natural killer cells and monocytes, as well as activation of both CD8 and CD4 T cells. Depleting C57BL/6 mice of CD4 T cells prior to DelNSsRVFV infection resulted in encephalitis in 30% of the mice; in encephalitic mice, we noted infiltration of T cells and inflammatory monocytes into the brain. CD4 and CD8 codepletion in C57BL/6 mice, as well as CD4 depletion in CCR2 knockout mice, increased the frequency of encephalitis, demonstrating that these cell types normally contributed to the prevention of disease. Encephalitic mice had similar viral RNA loads in the brain regardless of which cell types were depleted, suggesting that CD4 T cells, CD8 T cells, and inflammatory monocytes did little to control viral replication in the brain. CD4-depleted mice exhibited diminished humoral and T cell memory responses, suggesting that these immune mechanisms contributed to peripheral control of virus, thus preventing infection of the brain. IMPORTANCE RVFV is found in Africa and the Middle East and is transmitted by mosquitos or through contact with infected animals. Infected individuals can develop mild disease or more severe forms, such as hepatitis or encephalitis. In order to understand why some individuals develop encephalitis, we first need to know which immune functions protect those who do not develop this form of disease. In this study, we used a mouse model of RVFV infection to demonstrate that CD4 T cells, CD8 T cells, and monocytes all contribute to prevention of encephalitis. Their likely mechanism of action is preventing RVFV from ever reaching the brain., (Copyright © 2018 American Society for Microbiology.)

Background: Rift Valley Fever (RVF) is a mosquito-borne viral zoonosis. To detect RVF virus (RVFV) infection, indirect immunoglobulin G (IgG) and immunoglobulin M (IgM) enzyme linked immunosorbent assays (ELISAs) which utilize recombinant RVFV nucleocapsid (RVFV-N) protein as assay antigen, have reportedly been used, however, there is still a need to develop more sensitive and specific methods of detection., Methods: RVFV-N protein was expressed in Escherichia coli (E. coli) and purified by histidine-tag based affinity chromatography. This recombinant RVFV-N (rRVFV-N) protein was then used as antigen to develop an IgG sandwich ELISA and IgM capture ELISAs for human sera. Ninety six serum samples collected from healthy volunteers during the RVF surveillance programme in Kenya in 2013, and 93 serum samples collected from RVF-suspected patients during the 2006-2007 RVF outbreak in Kenya were used respectively, to evaluate the newly established rRVFV-N protein-based IgG sandwich ELISA and IgM capture ELISA systems in comparison with the inactivated virus-based ELISA systems., Results: rRVFV-N protein-based-IgG sandwich ELISA and IgM capture ELISA for human sera were established. Both the new ELISA systems were in 100% concordance with the inactivated virus-based ELISA systems, with a sensitivity and specificity of 100%., Conclusions: Recombinant RVFV-N is a safe and affordable antigen for RVF diagnosis. Our rRVFV-N-based ELISA systems are safe and reliable tools for diagnosis of RVFV infection in humans and especially useful in large-scale epidemiological investigation and for application in developing countries.

Rift Valley fever phlebovirus (RVFV) is a pathogen of Rift Valley fever, which is a mosquito-borne zoonotic disease for domestic livestock and humans in African countries. Currently, no approved vaccine is available for use in non-endemic areas. The MP-12 strain is so far the best live attenuated RVFV vaccine candidate because of its good protective efficacy in animal models. However, there are safety concerns for use of MP-12 in humans. We previously developed a single-cycle replicable MP-12 (scMP-12) which lacks NSs gene and undergoes only a single round of viral replication because of its impaired ability to induce membrane-membrane fusion. In the present study, we generated an scMP-12 mutant (scMP-12-mutNSs) carrying a mutant NSs, which degrades double-stranded RNA-dependent protein kinase R but does not inhibit host transcription. Immunization of mice with a single dose (10 5 PFU) of scMP-12-mutNSs elicited RVFV neutralizing antibodies and high titers of anti-N IgG production and fully protected the mice from lethal wild-type RVFV challenge. Immunogenicity and protective efficacy of scMP-12-mutNSs were better than scMP-12, demonstrating that scMP-12-mutNSs is a more efficacious vaccine candidate than scMP-12. Furthermore, our data suggested that RVFV vaccine efficacy can be improved by using this specific NSs mutant.

Background: Tanzania is among the Rift Valley fever (RVF) epizootic/endemic countries in sub Saharan Africa, where RVF disease outbreaks occur within a range of 3 to 17-year intervals. Detection of Rift Valley fever virus (RVFV) antibodies in animals in regions with no previous history of outbreaks raises the question of whether the disease is overlooked due to lack-of effective surveillance systems, or if there are strains of RVFV with low pathogenicity. Furthermore, which vertebrate hosts are involved in the inter-epidemic and inter-epizootic maintenance of RVFV? In our study region, the Kyela and Morogoro districts in Tanzania, no previous RVF outbreaks have been reported., Methodology: The study was conducted from June 2014 to October 2015 in the Kyela and Morogoro districts, Tanzania. Samples (n = 356) were retrieved from both the local breed of zebu cattle (Bos indicus) and Bos indicus/Bos Taurus cross breed. RVFV antibodies were analyzed by two enzyme-linked immunosorbent assay (ELISA) approaches. Initially, samples were analyzed by a RVFV multi-species competition ELISA (cELISA), which detected both RVFV IgG and IgM antibodies. All serum samples that were positive with the cELISA method were specifically analysed for the presence of RVFV IgM antibodies to trace recent infection. A plaque reduction neutralization assay (PRNT80) was performed to determine presence of RVFV neutralizing antibodies in all cELISA positive samples., Findings: Overall RVFV seroprevalence rate in cattle by cELISA in both districts was 29.2% (104 of 356) with seroprevalence rates of 33% (47/147) in the Kyela district and 27% (57/209) in the Morogoro district. In total, 8.4% (30/356) of all cattle sampled had RVFV IgM antibodies, indicating current disease transmission. When segregated by districts, the IgM antibody seroprevalence was 2.0% (3/147) and 12.9% (27/209) in Kyela and Morogoro districts respectively. When the 104 cELISA positive samples were analyzed by PRNT80 to confirm that RVFV-specific antibodies were present, the majority (89%, 93/104) had RVFV neutralising antibodies., Conclusion: The results provided evidence of widespread prevalence of RVFV antibody among cattle during an inter-epizootic/inter-epidemic period in Tanzania in regions with no previous history of outbreaks. There is a need for further investigations of RVFV maintenance and transmission in vertebrates and vectors during the long inter-epizootic/inter-epidemic periods., Competing Interests: The authors have declared that no competing interests exist.

Rift Valley fever virus is an arbovirus found in Africa and the Middle East. Most infected individuals experience a mild self-limiting illness; however, some develop severe disease including hepatitis, hemorrhagic fever, or encephalitis. The biological reasons for these marked differences in disease manifestation are unknown. In this study, we evaluate 32 biomarkers in serum of 26 patients from an outbreak that occurred in Saudi Arabia in 2000-2001. Eleven biomarkers correlated with viral RNA. Thirteen biomarkers were associated with a fatal outcome. No associations of biomarkers and hemorrhage or central nervous system disease were identified in this cohort.

The present study was planned to examine the effect of butylated hydroxy toluene (BHT) on the immune response of Rift Valley fever vaccine (RVFV) in Swiss mice. Animals were divided into four equal groups. The first group was kept as negative control. The 2nd group was orally administrated with the acceptable daily intake (ADI) of BHT 0.3 mg/kg b.wt. daily for 21 days and the 3rd group were vaccinated only by inactivated RVFV at a dose of 0.2 ml I/P two times. The 4th group was orally administrated BHT as in the 2nd group and vaccinated by inactivated RVFV as in the 4th group. Blood samples were collected from all groups two weeks from booster vaccination. The cellular immunity was determined by leucocytic indices and the neutrophil-lymphocyte ratio (NLR) whereas, humoral immunity was evaluated with IgG antibodies titer using enzyme-linked immune-sorbent assay (ELISA) test, serum neutralization test (SNT) and challenge test. BHT induced leucopenia, neutrophilia and marked lymphocytopenia in both non-vaccinated and vaccinated mice. Moreover, BHT significantly decreased the efficiency of vaccination by inducing 70% cytopathic effect (CPE) in the infected cell cultures and increasing the ED 50 value of RVFV vaccine. The present study indicates that BHT possesses a potential for decreasing both cellular and humoral mediated mechanisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Rift Valley fever virus (RVFV) is a zoonotic arbovirus that causes severe disease in livestock and humans. The virus has caused recurrent outbreaks in Africa and the Arabian Peninsula since its discovery in 1931. This review sought to evaluate RVFV seroprevalence across the African continent in livestock, wildlife and humans in order to understand the spatio-temporal distribution of RVFV seroprevalence and to identify knowledge gaps and areas requiring further research. Risk factors associated with seropositivity were identified and study designs evaluated to understand the validity of their results., Methodology: The Preferred Reporting of Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were used to produce a protocol to systematically search for RVFV seroprevalence studies in PubMed and Web of Science databases. The Strengthening the Reporting of Observational studies in Epidemiology (STROBE) statement guided the evaluation of study design and analyses., Principal Findings: A total of 174 RVFV seroprevalence studies in 126 articles fulfilled the inclusion criteria. RVFV seroprevalence was recorded in 31 African countries from 1968 to 2016 and varied by time, species and country. RVFV seroprevalence articles including either livestock and humans or livestock and wildlife seroprevalence records were limited in number (8/126). No articles considered wildlife, livestock and human seroprevalence concurrently, nor wildlife and humans alone. Many studies did not account for study design bias or the sensitivity and specificity of diagnostic tests., Conclusions: Future research should focus on conducting seroprevalence studies at the wildlife, livestock and human interface to better understand the nature of cross-species transmission of RVFV. Reporting should be more transparent and biases accounted for in future seroprevalence research to understand the true burden of disease on the African continent., Competing Interests: The authors have declared that no competing interests exist.

Rift Valley fever (RVF) in humans is usually mild, but, in a subset of cases, can progress to severe hepatic and neurological disease. Rodent models of RVF generally develop acute severe clinical disease. Here, we inoculated humanized NSG-SGM3 mice with Rift Valley fever virus (RVFV) to investigate whether the presence of human immune cells in mice would alter the progression of RVFV infection to more closely model human disease. Despite increased human cytokine expression, including responses mirroring those seen in human disease, and decreased hepatic viral RNA levels at terminal euthanasia, both high- and low-dose RVFV inoculation resulted in lethal disease in all mice with comparable time-to-death as unengrafted mice., Competing Interests: The authors have declared that no competing interests exist.

Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that has caused large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Currently, no licensed vaccine or therapeutics exists to treat this potentially deadly disease. The explosive nature of RVFV outbreaks and the severe consequences of its accidental or intentional introduction into RVFV-free areas provide the impetus for the development of novel vaccine candidates for use in both livestock and humans. Rationally designed vaccine candidates using reverse genetics have been used to develop deletion mutants of two known RVFV virulence factors, the NSs and NSm genes. These recombinant viruses were demonstrated to be protective and immunogenic in rats, mice, and sheep, without producing clinical illness in these animals. Here, we expand upon those findings and evaluate the single deletion mutant (ΔNSs rRVFV) and double deletion mutant (ΔNSs-ΔNSm rRVFV) vaccine candidates in the common marmoset (Callithrix jacchus), a non-human primate (NHP) model resembling severe human RVF disease. We demonstrate that both the ΔNSs and ΔNSs-ΔNSm rRVFV vaccine candidates were found to be safe and immunogenic in the current study. The vaccinated animals received a single dose of vaccine that led to the development of a robust antibody response. No vaccine-induced adverse reactions, signs of clinical illness or infectious virus were detected in the vaccinated marmosets. All vaccinated animals that were subsequently challenged with RVFV were protected against viremia and liver disease. In summary, our results provide the basis for further development of the ΔNSs and ΔNSs-ΔNSm rRVFV as safe and effective human RVFV vaccines for this significant public health threat., Competing Interests: The authors have declared that no competing interests exist.

Rift Valley fever virus is an arbovirus that affects both livestock and humans throughout Africa and in the Middle East. Despite its endemicity throughout Africa, it is a rare event to identify an infected individual during the acute phase of the disease and an even rarer event to collect serial blood samples from the affected patient. Severely affected patients can present with hemorrhagic manifestations of disease. In this study we identified three Ugandan men with RVFV disease that was accompanied by hemorrhagic manifestations. Serial blood samples from these men were analyzed for a series of biomarkers specific for various aspects of human pathophysiology including inflammation, endothelial function and coagulopathy. There were significant differences between biomarker levels in controls and cases both early during the illness and after clearance of viremia. Positive correlation of viral load with markers of inflammation (IP-10, CRP, Eotaxin, MCP-2 and Granzyme B), markers of fibrinolysis (tPA and D-dimer), and markers of endothelial function (sICAM-1) were all noted. However, and perhaps most interesting given the fact that these individuals exhibited hemorrhagic manifestations of disease, was the finding of a negative correlation between viral load and P-selectin, ADAMTS13, and fibrinogen all of which are associated with coagulation pathways occurring on the endothelial surface.

Rift Valley fever (RVF) is a zoonotic mosquito-borne bunyaviral disease associated with high abortion rates, neonatal deaths, and fetal malformations in ruminants, and mild to severe disease in humans. Outbreaks of RVF cause huge economic losses and public health impacts in endemic countries in Africa and the Arabian Peninsula. A proper vaccination strategy is important for preventing or minimizing outbreaks. Vaccination against RVF is not practiced in many countries, however, due to absence or irregular occurrences of outbreaks, despite serological evidence of RVF viral activity. Nonetheless, effective vaccination strategies, and functional national and international multi-disciplinary networks, remain crucial for ensuring availability of vaccines and supporting execution of vaccination in high risk areas for efficient response to RVF alerts and outbreaks., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The mosquito-borne Rift Valley fever virus (RVFV) causes severe diseases in domesticated animals including cattle, sheep, camels and goats. Capripoxviruses (CPV) are suitable vectors for multivalent vaccine development. A recombinant rKS1-based CPV expressing the gene encoding the viral glycoprotein Gn of RVFV has been shown to induce protection in mice and sheep. The aim of this study was to evaluate the immunogenicity induced by this candidate vaccine in goats, and the level of cytokines produced by RVFV-specific Th1 and Th2 lymphocytes. The results of this study suggest that Th2 mediates immunity mainly through the significant production of IL4, which, coupled with a decrease in IFN-γ, may be involved in the replication of the capripoxvirus expressing the G N of RVFV. CD4+ cells may play the role of helper cells in B cell responses and neutralizing antibody production in the anti-CPV humoral response, leading to strong immunity against RVFV., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Rift Valley Fever (RVF) is an emerging arboviral disease of livestock and humans. Although the disease is caused by a mosquito-borne virus, humans are infected through contact with, or inhalation of, virus-laden particles from contaminated animal carcasses. Some individuals infected with RVF virus (RVFV) develop meningoencephalitis, resulting in morbidity and mortality. Little is known about the pathogenic mechanisms that lead to neurologic sequelae, and thus, animal models that represent human disease are needed. African green monkeys (AGM) exposed to aerosols containing RVFV develop a reproducibly lethal neurological disease that resembles human illness. To understand the disease process and identify biomarkers of lethality, two groups of 5 AGM were infected by inhalation with either a lethal or a sublethal dose of RVFV. Divergence between lethal and sublethal infections occurred as early as 2 days postinfection (dpi), at which point CD8 + T cells from lethally infected AGM expressed activated caspase-3 and simultaneously failed to increase levels of major histocompatibility complex (MHC) class II molecules, in contrast to surviving animals. At 4 dpi, lethally infected animals failed to demonstrate proliferation of total CD4 + and CD8 + T cells, in contrast to survivors. These marked changes in peripheral blood cells occur much earlier than more-established indicators of severe RVF disease, such as granulocytosis and fever. In addition, an early proinflammatory (gamma interferon [IFN-γ], interleukin 6 [IL-6], IL-8, monocyte chemoattractant protein 1 [MCP-1]) and antiviral (IFN-α) response was seen in survivors, while very late cytokine expression was found in animals with lethal infections. By characterizing immunological markers of lethal disease, this study furthers our understanding of RVF pathogenesis and will allow the testing of therapeutics and vaccines in the AGM model. IMPORTANCE Rift Valley Fever (RVF) is an important emerging viral disease for which we lack both an effective human vaccine and treatment. Encephalitis and neurological disease resulting from RVF lead to death or significant long-term disability for infected people. African green monkeys (AGM) develop lethal neurological disease when infected with RVF virus by inhalation. Here we report the similarities in disease course between infected AGM and humans. For the first time, we examine the peripheral immune response during the course of infection in AGM and show that there are very early differences in the immune response between animals that survive infection and those that succumb. We conclude that AGM are a novel and suitable monkey model for studying the neuropathogenesis of RVF and for testing vaccines and therapeutics against this emerging viral pathogen., (Copyright © 2018 American Society for Microbiology.)

Livestock handlers are occupationally exposed to Rift Valley fever virus (RVFV) because of frequent and longer contact with mosquito vectors, animal blood and other tissues. We determined the seroprevalence of RVF virus infection among livestock handlers in Ibadan, Nigeria. Blood samples were collected from 265 workers in two major livestock markets and a livestock farm. Questionnaires were administered to obtain information on risk factors that may be associated with RVF virus transmission. The blood samples were tested using enzyme-linked immunosorbent assays (ELISA) to detect multispecies anti-RVF virus IgG/IgM and human anti-RVF virus IgM. In total, 14 (5.3%) of the 265 participants tested positive for anti-RVFV IgG. Seropositive individuals were more among livestock keepers (5.6%) than butchers (3.6%). Workers that frequently visited the livestock rearing areas of the northern part of Nigeria had a statistically significant (P = 0.004) higher rate of infection compared to those that did not. Very few (0.8%) of these workers knew about RVF virus while none had been vaccinated against the virus infection. This study indicates that RVF virus continues to circulate sub-clinically in Nigeria, thus the need to educate and vaccinate individuals who are occupationally at risk of infection.