Vaccination with DNA Plasmids Expressing Gn Coupled to C3d or Alphavirus Replicons Expressing Gn Protects Mice against Rift Valley Fever Virus

Background

Rift Valley fever (RVF) is an arthropod-borne viral zoonosis. Rift Valley fever virus (RVFV) is an important biological threat with the potential to spread to new susceptible areas. In addition, it is a potential biowarfare agent.

Methodology/Principal Findings

We developed two potential vaccines, DNA plasmids and alphavirus replicons, expressing the Gn glycoprotein of RVFV alone or fused to three copies of complement protein, C3d. Each vaccine was administered to mice in an all DNA, all replicon, or a DNA prime/replicon boost strategy and both the humoral and cellular responses were assessed. DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited high titer neutralizing antibodies that were similar to titers elicited by the live-attenuated MP12 virus. Mice vaccinated with an inactivated form of MP12 did elicit high titer antibodies, but these antibodies were unable to neutralize RVFV infection. However, only vaccine strategies incorporating alphavirus replicons elicited cellular responses to Gn. Both vaccines strategies completely prevented weight loss and morbidity and protected against lethal RVFV challenge. Passive transfer of antisera from vaccinated mice into naïve mice showed that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited antibodies that protected mice as well as sera from mice immunized with MP12.

Conclusion/Significance

These results show that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn administered alone or in a DNA prime/replicon boost strategy are effective RVFV vaccines. These vaccine strategies provide safer alternatives to using live-attenuated RVFV vaccines for human use.     

Aedes Mosquito Saliva Modulates Rift Valley Fever Virus Pathogenicity

Background

Rift Valley fever (RVF) is a severe mosquito-borne disease affecting humans and domestic ruminants. Mosquito saliva contains compounds that counteract the hemostatic, inflammatory, and immune responses of the host. Modulation of these defensive responses may facilitate virus infection. Indeed, Aedes mosquito saliva played a crucial role in the vector''s capacity to effectively transfer arboviruses such as the Cache Valley and West Nile viruses. The role of mosquito saliva in the transmission of Rift Valley fever virus (RVFV) has not been investigated.

Objective

Using a murine model, we explored the potential for mosquitoes to impact the course of RVF disease by determining whether differences in pathogenesis occurred in the presence or absence of mosquito saliva and salivary gland extract.

Methods

C57BL/6NRJ male mice were infected with the ZH548 strain of RVFV via intraperitoneal or intradermal route, or via bites from RVFV-exposed mosquitoes. The virus titers in mosquitoes and mouse organs were determined by plaque assays.

Findings

After intraperitoneal injection, RVFV infection primarily resulted in liver damage. In contrast, RVFV infection via intradermal injection caused both liver and neurological symptoms and this route best mimicked the natural infection by mosquitoes. Co-injections of RVFV with salivary gland extract or saliva via intradermal route increased the mortality rates of mice, as well as the virus titers measured in several organs and in the blood. Furthermore, the blood cell counts of infected mice were altered compared to those of uninfected mice.

Interpretation

Different routes of infection determine the pattern in which the virus spreads and the organs it targets. Aedes saliva significantly increases the pathogenicity of RVFV.     

Broad Spectrum Antiviral Activity of Favipiravir (T-705): Protection from Highly Lethal Inhalational Rift Valley Fever

Background

Development of antiviral drugs that have broad-spectrum activity against a number of viral infections would be of significant benefit. Due to the evolution of resistance to currently licensed antiviral drugs, development of novel anti-influenza drugs is in progress, including Favipiravir (T-705), which is currently in human clinical trials. T-705 displays broad-spectrum in vitro activity against a number of viruses, including Rift Valley Fever virus (RVFV). RVF is an important neglected tropical disease that causes human, agricultural, and economic losses in endemic regions. RVF has the capacity to emerge in new locations and also presents a potential bioterrorism threat. In the current study, the in vivo efficacy of T-705 was evaluated in Wistar-Furth rats infected with the virulent ZH501 strain of RVFV by the aerosol route.

Methodology/Principal Findings

Wistar-Furth rats are highly susceptible to a rapidly lethal disease after parenteral or inhalational exposure to the pathogenic ZH501 strain of RVFV. In the current study, two experiments were performed: a dose-determination study and a delayed-treatment study. In both experiments, all untreated control rats succumbed to disease. Out of 72 total rats infected with RVFV and treated with T-705, only 6 succumbed to disease. The remaining 66 rats (92%) survived lethal infection with no significant weight loss or fever. The 6 treated rats that succumbed survived significantly longer before succumbing to encephalitic disease.

Conclusions/Significance

Currently, there are no licensed antiviral drugs for treating RVF. Here, T-705 showed remarkable efficacy in a highly lethal rat model of Rift Valley Fever, even when given up to 48 hours post-infection. This is the first study to show protection of rats infected with the pathogenic ZH501 strain of RVFV. Our data suggest that T-705 has potential to be a broad-spectrum antiviral drug.     

A Single Immunization with MVA Expressing GnGc Glycoproteins Promotes Epitope-specific CD8+-T Cell Activation and Protects Immune-competent Mice against a Lethal RVFV Infection

Background

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen causing an important disease in ruminants often transmitted to humans after epizootic outbreaks in African and Arabian countries. To help combat the spread of the disease, prophylactic measures need to be developed and/or improved.

Methodology/Principal Findings

In this work, we evaluated the immunogenicity and protective efficacy of recombinant plasmid DNA and modified vaccinia virus Ankara (rMVA) vectored vaccines against Rift Valley fever in mice. These recombinant vaccines encoded either of two components of the Rift Valley fever virus: the viral glycoproteins (Gn/Gc) or the nucleoprotein (N). Following lethal challenge with live RVFV, mice immunized with a single dose of the rMVA-Gn/Gc vaccine showed no viraemia or clinical manifestation of disease, but mounted RVFV neutralizing antibodies and glycoprotein specific CD8+ T-cell responses. Neither DNA-Gn/Gc alone nor a heterologous prime-boost immunization schedule (DNA-Gn/Gc followed by rMVAGn/Gc) was better than the single rMVA-Gn/Gc immunization schedule with regards to protective efficacy. However, the rMVA-Gn/Gc vaccine failed to protect IFNAR^−/− mice upon lethal RVFV challenge suggesting a role for innate responses in protection against RVFV. Despite induction of high titer antibodies against the RVFV nucleoprotein, the rMVA-N vaccine, whether in homologous or heterologous prime-boost schedules with the corresponding recombinant DNA vaccine, only conferred partial protection to RVFV challenge.

Conclusions/Significance

Given the excellent safety profile of rMVA based vaccines in humans and animals, our data supports further development of rMVA-Gn/Gc as a vaccine strategy that can be used for the prevention of Rift Valley fever in both humans and livestock.     

Rift Valley fever virus 78kDa envelope protein attenuates virus replication in macrophage-derived cell lines and viral virulence in mice

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus with a wide host range including ruminants and humans. RVFV outbreaks have had devastating effects on public health and the livestock industry in African countries. However, there is no approved RVFV vaccine for human use in non-endemic countries and no FDA-approved antiviral drug for RVFV treatment. The RVFV 78kDa protein (P78), which is a membrane glycoprotein, plays a role in virus dissemination in the mosquito host, but its biological role in mammalian hosts remains unknown. We generated an attenuated RVFV MP-12 strain-derived P78-High virus and a virulent ZH501 strain-derived ZH501-P78-High virus, both of which expressed a higher level of P78 and carried higher levels of P78 in the virion compared to their parental viruses. We also generated another MP-12-derived mutant virus (P78-KO virus) that does not express P78. MP-12 and P78-KO virus replicated to similar levels in fibroblast cell lines and Huh7 cells, while P78-High virus replicated better than MP-12 in Vero E6 cells, fibroblast cell lines, and Huh7 cells. Notably, P78-High virus and P78-KO virus replicated less efficiently and more efficiently, respectively, than MP-12 in macrophage cell lines. ZH501-P78-High virus also replicated poorly in macrophage cell lines. Our data further suggest that inefficient binding of P78-High virus to the cells led to inefficient virus internalization, low virus infectivity and reduced virus replication in a macrophage cell line. P78-High virus and P78-KO virus showed lower and higher virulence than MP-12, respectively, in young mice. ZH501-P78-High virus also exhibited lower virulence than ZH501 in mice. These data suggest that high levels of P78 expression attenuate RVFV virulence by preventing efficient virus replication in macrophages. Genetic alteration leading to increased P78 expression may serve as a novel strategy for the attenuation of RVFV virulence and generation of safe RVFV vaccines.     

Chemotactic and inflammatory responses in the liver and brain are associated with pathogenesis of Rift Valley fever virus infection in the mouse

Rift Valley fever virus (RVFV) is a major human and animal pathogen associated with severe disease including hemorrhagic fever or encephalitis. RVFV is endemic to parts of Africa and the Arabian Peninsula, but there is significant concern regarding its introduction into non-endemic regions and the potentially devastating effect to livestock populations with concurrent infections of humans. To date, there is little detailed data directly comparing the host response to infection with wild-type or vaccine strains of RVFV and correlation with viral pathogenesis. Here we characterized clinical and systemic immune responses to infection with wild-type strain ZH501 or IND vaccine strain MP-12 in the C57BL/6 mouse. Animals infected with live-attenuated MP-12 survived productive viral infection with little evidence of clinical disease and minimal cytokine response in evaluated tissues. In contrast, ZH501 infection was lethal, caused depletion of lymphocytes and platelets and elicited a strong, systemic cytokine response which correlated with high virus titers and significant tissue pathology. Lymphopenia and platelet depletion were indicators of disease onset with indications of lymphocyte recovery correlating with increases in G-CSF production. RVFV is hepatotropic and in these studies significant clinical and histological data supported these findings; however, significant evidence of a pro-inflammatory response in the liver was not apparent. Rather, viral infection resulted in a chemokine response indicating infiltration of immunoreactive cells, such as neutrophils, which was supported by histological data. In brains of ZH501 infected mice, a significant chemokine and pro-inflammatory cytokine response was evident, but with little pathology indicating meningoencephalitis. These data suggest that RVFV pathogenesis in mice is associated with a loss of liver function due to liver necrosis and hepatitis yet the long-term course of disease for those that might survive the initial hepatitis is neurologic in nature which is supported by observations of human disease and the BALB/c mouse model.     

Rift Valley Fever Virus Incorporates the 78 kDa Glycoprotein into Virions Matured in Mosquito C6/36 Cells

Rift Valley fever virus (RVFV), genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment coding for four proteins: nonstructural NSm protein, two glycoproteins Gn and Gc and large 78 kDa glycoprotein (LGp) of unknown function. Goat anti-RVFV polyclonal antibody and mouse monoclonal antibody, generated against a polypeptide unique to the LGp within the RVFV proteome, detected this protein in gradient purified RVFV ZH501 virions harvested from mosquito C6/36 cells but not in virions harvested from the mammalian Vero E6 cells. The incorporation of LGp into the mosquito cell line - matured virions was confirmed by immune-electron microscopy. The LGp was incorporated into the virions immediately during the first passage in C6/36 cells of Vero E6 derived virus. Our data indicate that LGp is a structural protein in C6/36 mosquito cell generated virions. The protein may aid the transmission from the mosquitoes to the ruminant host, with a possible role in replication of RVFV in the mosquito host. To our knowledge, this is a first report of different protein composition between virions formed in insect C6/36 versus mammalian Vero E6 cells.     

Genetic Subpopulations of Rift Valley Fever Virus Strains ZH548 and MP-12 and Recombinant MP-12 Strains

Rift Valley fever virus strain MP-12 was generated by serial plaque passages of parental strain ZH548 12 times in MRC-5 cells in the presence of a chemical mutagen, 5-fluorouracil. As a result, MP-12 encoded 4, 9, and 10 mutations in the S, M, and L segments, respectively. Among them, mutations in the M and L segments were responsible for attenuation, while the MP-12 S segment still encoded a virulent phenotype. We performed high-throughput sequencing of MP-12 vaccine, ZH548, and recombinant MP-12 (rMP-12) viruses. We found that rMP-12 contains very low numbers of viral subpopulations, while MP-12 and ZH548 contain 2 to 4 times more viral genetic subpopulations than rMP-12. MP-12 genetic subpopulations did not encode the ZH548 sequence at the 23 MP-12 consensus mutations. On the other hand, 4 and 2 mutations in M and L segments of MP-12 were found in ZH548 subpopulations. Thus, those 6 mutations were no longer MP-12-specific mutations. ZH548 encoded several unique mutations compared to other Egyptian strains, i.e., strains ZH501, ZH1776, and ZS6365. ZH548 subpopulations shared nucleotides at the mutation site common with those in the Egyptian strains, while MP-12 subpopulations did not share those nucleotides. Thus, MP-12 retains unique genetic subpopulations and has no evidence of reversion to the ZH548 sequence in the subpopulations. This study provides the first information regarding the genetic subpopulations of RVFV and shows the genetic stability of the MP-12 vaccine manufactured in MRC-5 cells.     

Tissue tropism and target cells of NSs-deleted rift valley fever virus in live immunodeficient mice

Background

Rift Valley fever virus (RVFV) causes disease in livestock and humans. It can be transmitted by mosquitoes, inhalation or physical contact with the body fluids of infected animals. Severe clinical cases are characterized by acute hepatitis with hemorrhage, meningoencephalitis and/or retinitis. The dynamics of RVFV infection and the cell types infected in vivo are poorly understood.

Methodology/Principal Findings

RVFV strains expressing humanized Renilla luciferase (hRLuc) or green fluorescent protein (GFP) were generated and inoculated to susceptible Ifnar1-deficient mice. We investigated the tissue tropism in these mice and the nature of the target cells in vivo using whole-organ imaging and flow cytometry. After intraperitoneal inoculation, hRLuc signal was observed primarily in the thymus, spleen and liver. Macrophages infiltrating various tissues, in particular the adipose tissue surrounding the pancreas also expressed the virus. The liver rapidly turned into the major luminescent organ and the mice succumbed to severe hepatitis. The brain remained weakly luminescent throughout infection. FACS analysis in RVFV-GFP-infected mice showed that the macrophages, dendritic cells and granulocytes were main target cells for RVFV. The crucial role of cells of the monocyte/macrophage/dendritic lineage during RVFV infection was confirmed by the slower viral dissemination, decrease in RVFV titers in blood, and prolonged survival of macrophage- and dendritic cell-depleted mice following treatment with clodronate liposomes. Upon dermal and nasal inoculations, the viral dissemination was primarily observed in the lymph node draining the injected ear and in the lungs respectively, with a significant increase in survival time.

Conclusions/Significance

These findings reveal the high levels of phagocytic cells harboring RVFV during viral infection in Ifnar1-deficient mice. They demonstrate that bioluminescent and fluorescent viruses can shed new light into the pathogenesis of RVFV infection.     

The Use of NanoTrap Particles as a Sample Enrichment Method to Enhance the Detection of Rift Valley Fever Virus

Background

Rift Valley Fever Virus (RVFV) is a zoonotic virus that is not only an emerging pathogen but is also considered a biodefense pathogen due to the threat it may cause to public health and national security. The current state of diagnosis has led to misdiagnosis early on in infection. Here we describe the use of a novel sample preparation technology, NanoTrap particles, to enhance the detection of RVFV. Previous studies demonstrated that NanoTrap particles lead to both 100 percent capture of protein analytes as well as an improvement of more than 100-fold in sensitivity compared to existing methods. Here we extend these findings by demonstrating the capture and enrichment of viruses.

Results

Screening of NanoTrap particles indicated that one particle, NT53, was the most efficient at RVFV capture as demonstrated by both qRT-PCR and plaque assays. Importantly, NT53 capture of RVFV resulted in greater than 100-fold enrichment from low viral titers when other diagnostics assays may produce false negatives. NT53 was also capable of capturing and enhancing RVFV detection from serum samples. RVFV that was inactivated through either detergent or heat treatment was still found bound to NT53, indicating the ability to use NanoTrap particles for viral capture prior to transport to a BSL-2 environment. Furthermore, both NP-40-lysed virus and purified RVFV RNA were bound by NT53. Importantly, NT53 protected viral RNA from RNase A degradation, which was not observed with other commercially available beads. Incubation of RVFV samples with NT53 also resulted in increased viral stability as demonstrated through preservation of infectivity at elevated temperatures. Finally, NanoTrap particles were capable of capturing VEEV and HIV, demonstrating the broad applicability of NanoTrap particles for viral diagnostics.

Conclusion

This study demonstrates NanoTrap particles are capable of capturing, enriching, and protecting RVFV virions. Furthermore, the use of NanoTrap particles can be extended to a variety of viruses, including VEEV and HIV.     

Rift Valley Fever Virus Infection in Golden Syrian Hamsters

Rift Valley fever virus (RVFV) is a formidable pathogen that causes severe disease and abortion in a variety of livestock species and a range of disease in humans that includes hemorrhagic fever, fulminant hepatitis, encephalitis and blindness. The natural transmission cycle involves mosquito vectors, but exposure can also occur through contact with infected fluids and tissues. The lack of approved antiviral therapies and vaccines for human use underlies the importance of small animal models for proof-of-concept efficacy studies. Several mouse and rat models of RVFV infection have been well characterized and provide useful systems for the study of certain aspects of pathogenesis, as well as antiviral drug and vaccine development. However, certain host-directed therapeutics may not act on mouse or rat pathways. Here, we describe the natural history of disease in golden Syrian hamsters challenged subcutaneously with the pathogenic ZH501 strain of RVFV. Peracute disease resulted in rapid lethality within 2 to 3 days of RVFV challenge. High titer viremia and substantial viral loads were observed in most tissues examined; however, histopathology and immunostaining for RVFV antigen were largely restricted to the liver. Acute hepatocellular necrosis associated with a strong presence of viral antigen in the hepatocytes indicates that fulminant hepatitis is the likely cause of mortality. Further studies to assess the susceptibility and disease progression following respiratory route exposure are warranted. The use of the hamsters to model RVFV infection is suitable for early stage antiviral drug and vaccine development studies.     

Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells

Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-β subunit can be observed in MP-12-infected cells, which we have labeled IKK-β2. The IKK-β2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-β2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-β2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets.     

Deletion of the NSm Virulence Gene of Rift Valley Fever Virus Inhibits Virus Replication in and Dissemination from the Midgut of Aedes aegypti Mosquitoes

Background

Previously, we investigated the role of the Rift Valley fever virus (RVFV) virulence genes NSs and NSm in mosquitoes and demonstrated that deletion of NSm significantly reduced the infection, dissemination, and transmission rates of RVFV in Aedes aegypti mosquitoes. The specific aim of this study was to further characterize midgut infection and escape barriers of RVFV in Ae. aegypti infected with reverse genetics-generated wild type RVFV (rRVF-wt) or RVFV lacking the NSm virulence gene (rRVF-ΔNSm) by examining sagittal sections of infected mosquitoes for viral antigen at various time points post-infection.

Methodology and Principal Findings

Ae. aegypti mosquitoes were fed an infectious blood meal containing either rRVF-wt or rRVF-ΔNSm. On days 0, 1, 2, 3, 4, 6, 8, 10, 12, and 14 post-infection, mosquitoes from each experimental group were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and examined for RVFV antigen by immunofluorescence assay. Remaining mosquitoes at day 14 were assayed for infection, dissemination, and transmission. Disseminated infections were observed in mosquitoes as early as three days post infection for both virus strains. However, infection rates for rRVF-ΔNSm were statistically significantly less than for rRVF-wt. Posterior midgut infections in mosquitoes infected with rRVF-wt were extensive, whereas midgut infections of mosquitoes infected with rRVF-ΔNSm were confined to one or a few small foci.

Conclusions/Significance

Deletion of NSm resulted in the reduced ability of RVFV to enter, replicate, and disseminate from the midgut epithelial cells. NSm appears to have a functional role in the vector competence of mosquitoes for RVFV at the level of the midgut barrier.     

Rift Valley Fever Virus Seroprevalence in Human Rural Populations of Gabon

Background

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis caused by a phlebovirus and transmitted by Aedes mosquitoes. Humans can also be infected through direct contact with blood (aerosols) or tissues (placenta, stillborn) of infected animals. Although severe clinical cases can be observed, infection with RVF virus (RVFV) in humans is, in most cases, asymptomatic or causes a febrile illness without serious symptoms. In small ruminants RVFV mainly causes abortion and neonatal death. The distribution of RVFV has been well documented in many African countries, particularly in the north (Egypt, Sudan), east (Kenya, Tanzania, Somalia), west (Senegal, Mauritania) and south (South Africa), but also in the Indian Ocean (Madagascar, Mayotte) and the Arabian Peninsula. In contrast, the prevalence of RVFV has rarely been investigated in central African countries.

Methodology/Principal Findings

We therefore conducted a large serological survey of rural populations in Gabon, involving 4,323 individuals from 212 randomly selected villages (10.3% of all Gabonese villages). RVFV-specific IgG was found in a total of 145 individuals (3.3%) suggesting the wide circulation of Rift Valley fever virus in Gabon. The seroprevalence was significantly higher in the lakes region than in forest and savannas zones, with respective rates of 8.3%, 2.9% and 2.2%. In the lakes region, RVFV-specific IgG was significantly more prevalent in males than in females (respectively 12.8% and 3.8%) and the seroprevalence increased gradually with age in males but not in females.

Conclusions/Significance

Although RVFV was suggested to circulate at a relatively high level in Gabon, no outbreaks or even isolated cases have been documented in the country. The higher prevalence in the lakes region is likely to be driven by specific ecologic conditions favorable to certain mosquito vector species. Males may be more at risk of infection than females because they spend more time farming and hunting outside the villages, where they may be more exposed to mosquito bites and infected animals. Further investigations are needed to determine the putative sylvan cycle of RVFV, including the mosquito species and the reservoir role of wild animals in the viral maintenance cycle.     

Rift Valley Fever Virus Encephalitis Is Associated with an Ineffective Systemic Immune Response and Activated T Cell Infiltration into the CNS in an Immunocompetent Mouse Model

Background

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in livestock and humans throughout Africa and the Arabian Peninsula. In people, RVFV generally causes a self-limiting febrile illness but in a subset of individuals, it progresses to more serious disease. One manifestation is a delayed-onset encephalitis that can be fatal or leave the afflicted with long-term neurologic sequelae. In order to design targeted interventions, the basic pathogenesis of RVFV encephalitis must be better understood.

Methodology/Principal Findings

To characterize the host immune responses and viral kinetics associated with fatal and nonfatal infections, mice were infected with an attenuated RVFV lacking NSs (ΔNSs) that causes lethal disease only when administered intranasally (IN). Following IN infection, C57BL/6 mice developed severe neurologic disease and succumbed 7–9 days post-infection. In contrast, inoculation of ΔNSs virus subcutaneously in the footpad (FP) resulted in a subclinical infection characterized by a robust immune response with rapid antibody production and strong T cell responses. IN-inoculated mice had delayed antibody responses and failed to clear virus from the periphery. Severe neurological signs and obtundation characterized end stage-disease in IN-inoculated mice, and within the CNS, the development of peak virus RNA loads coincided with strong proinflammatory responses and infiltration of activated T cells. Interestingly, depletion of T cells did not significantly alter survival, suggesting that neurologic disease is not a by-product of an aberrant immune response.

Conclusions/Significance

Comparison of fatal (IN-inoculated) and nonfatal (FP-inoculated) ΔNSs RVFV infections in the mouse model highlighted the role of the host immune response in controlling viral replication and therefore determining clinical outcome. There was no evidence to suggest that neurologic disease is immune-mediated in RVFV infection. These results provide important insights for the future design of vaccines and therapeutic options.     

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.     

Rift Valley Fever Virus Circulating among Ruminants,Mosquitoes and Humans in the Central African Republic

BackgroundRift Valley fever virus (RVFV) causes a viral zoonosis, with discontinuous epizootics and sporadic epidemics, essentially in East Africa. Infection with this virus causes severe illness and abortion in sheep, goats, and cattle as well as other domestic animals. Humans can also be exposed through close contact with infectious tissues or by bites from infected mosquitoes, primarily of the Aedes and Culex genuses. Although the cycle of RVFV infection in savannah regions is well documented, its distribution in forest areas in central Africa has been poorly investigated.

Methodology/Principal Findings

To evaluate current circulation of RVFV among livestock and humans living in the Central African Republic (CAR), blood samples were collected from sheep, cattle, and goats and from people at risk, such as stock breeders and workers in slaughterhouses and livestock markets. The samples were tested for anti-RVFV immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies. We also sequenced the complete genomes of two local strains, one isolated in 1969 from mosquitoes and one isolated in 1985 from humans living in forested areas. The 1271 animals sampled comprised 727 cattle, 325 sheep, and 219 goats at three sites. The overall seroprevalence of anti-RVFV IgM antibodies was 1.9% and that of IgG antibodies was 8.6%. IgM antibodies were found only during the rainy season, but the frequency of IgG antibodies did not differ significantly by season. No evidence of recent RVFV infection was found in 335 people considered at risk; however, 16.7% had evidence of past infection. Comparison of the nucleotide sequences of the strains isolated in the CAR with those isolated in other African countries showed that they belonged to the East/Central African cluster.

Conclusion and significance

This study confirms current circulation of RVFV in CAR. Further studies are needed to determine the potential vectors involved and the virus reservoirs.     

Acid-Activated Structural Reorganization of the Rift Valley Fever Virus Gc Fusion Protein

The entry of the enveloped Rift Valley fever virus (RVFV) into its host cell is mediated by the viral glycoproteins Gn and Gc. We investigated the RVFV entry process and, in particular, its pH-dependent activation mechanism using our recently developed nonspreading-RVFV-particle system. Entry of the virus into the host cell was efficiently inhibited by lysosomotropic agents that prevent endosomal acidification and by compounds that interfere with dynamin- and clathrin-dependent endocytosis. Exposure of plasma membrane-bound virions to an acidic pH (<pH 6) equivalent to the pH of late endolysosomal compartments allowed the virus to bypass the endosomal route of infection. Acid exposure of virions in the absence of target membranes triggered the class II-like Gc fusion protein to form extremely stable oligomers that were resistant to SDS and temperature dissociation and concomitantly compromised virus infectivity. By targeted mutagenesis of conserved histidines in Gn and Gc, we demonstrated that mutation of a single histidine (H857) in Gc completely abrogated virus entry, as well as acid-induced Gc oligomerization. In conclusion, our data suggest that after endocytic uptake, RVFV traffics to the acidic late endolysosomal compartments, where histidine protonation drives the reorganization of the Gc fusion protein that leads to membrane fusion.     

Host alternation is necessary to maintain the genome stability of rift valley fever virus

Background

Most arthropod-borne viruses (arboviruses) are RNA viruses, which are maintained in nature by replication cycles that alternate between arthropod and vertebrate hosts. Arboviruses appear to experience lower rates of evolution than RNA viruses that replicate in a single host. This genetic stability is assumed to result from a fitness trade-off imposed by host alternation, which constrains arbovirus genome evolution. To test this hypothesis, we used Rift Valley fever virus (RVFV), an arbovirus that can be transmitted either directly (between vertebrates during the manipulation of infected tissues, and between mosquitoes by vertical transmission) or indirectly (from one vertebrate to another by mosquito-borne transmission).

Methodology/Principal Findings

RVFV was serially passaged in BHK21 (hamster) or Aag2 (Aedes aegypti) cells, or in alternation between the two cell types. After 30 passages, these single host-passaged viruses lost their virulence and induced protective effects against a challenge with a virulent virus. Large deletions in the NSs gene that encodes the virulence factor were detectable from the 15^th serial passage onwards in BHK21 cells and from the 10^th passage in Aag2 cells. The phosphoprotein NSs is not essential to viral replication allowing clones carrying deletions in NSs to predominate as they replicate slightly more rapidly. No genetic changes were found in viruses that were passaged alternately between arthropod and vertebrate cells. Furthermore, alternating passaged viruses presenting complete NSs gene remained virulent after 30 passages.

Conclusions/Significance

Our results strongly support the view that alternating replication is necessary to maintain the virulence factor carried by the NSs phosphoprotein.