Rift Valley Fever Virus Strain MP-12 Enters Mammalian Host Cells via Caveola-Mediated Endocytosis

Rift Valley fever virus (RVFV) is a zoonotic pathogen capable of causing serious morbidity and mortality in both humans and livestock. The lack of efficient countermeasure strategies, the potential for dispersion into new regions, and the pathogenesis in humans and livestock make RVFV a serious public health concern. The receptors, cellular factors, and entry pathways used by RVFV and other members of the family Bunyaviridae remain largely uncharacterized. Here we provide evidence that RVFV strain MP-12 uses dynamin-dependent caveola-mediated endocytosis for cell entry. Caveolae are lipid raft domains composed of caveolin (the main structural component), cholesterol, and sphingolipids. Caveola-mediated endocytosis is responsible for the uptake of a wide variety of host ligands, as well as bacteria, bacterial toxins, and a number of viruses. To determine the cellular entry mechanism of RVFV, we used small-molecule inhibitors, RNA interference (RNAi), and dominant negative (DN) protein expression to inhibit the major mammalian cell endocytic pathways. Inhibitors and RNAi specific for macropinocytosis and clathrin-mediated endocytosis had no effect on RVFV infection. In contrast, inhibitors of caveola-mediated endocytosis, and RNAi targeted to caveolin-1 and dynamin, drastically reduced RVFV infection in multiple cell lines. Expression of DN caveolin-1 also reduced RVFV infection significantly, while expression of DN EPS15, a protein required for the assembly of clathrin-coated pits, and DN PAK-1, an obligate mediator of macropinocytosis, had no significant impact on RVFV infection. These results together suggest that the primary mechanism of RVFV MP-12 uptake is dynamin-dependent, caveolin-1-mediated endocytosis.     

Nuclear Relocalization of Polyadenylate Binding Protein during Rift Valley Fever Virus Infection Involves Expression of the NSs Gene

Rift Valley fever virus (RVFV), an ambisense member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever, an important zoonotic infection in Africa and the Middle East. Phlebovirus proteins are translated from virally transcribed mRNAs that, like host mRNA, are capped but, unlike host mRNAs, are not polyadenylated. Here, we investigated the role of PABP1 during RVFV infection of HeLa cells. Immunofluorescence studies of infected cells demonstrated a gross relocalization of PABP1 to the nucleus late in infection. Immunofluorescence microscopy studies of nuclear proteins revealed costaining between PABP1 and markers of nuclear speckles. PABP1 relocalization was sharply decreased in cells infected with a strain of RVFV lacking the gene encoding the RVFV nonstructural protein S (NSs). To determine whether PABP1 was required for RVFV infection, we measured the production of nucleocapsid protein (N) in cells transfected with small interfering RNAs (siRNAs) targeting PABP1. We found that the overall percentage of RVFV N-positive cells was not changed by siRNA treatment, indicating that PABP1 was not required for RVFV infection. However, when we analyzed populations of cells producing high versus low levels of PABP1, we found that the percentage of RVFV N-positive cells was decreased in cell populations producing physiologic levels of PABP1 and increased in cells with reduced levels of PABP1. Together, these results suggest that production of the NSs protein during RVFV infection leads to sequestration of PABP1 in the nuclear speckles, creating a state within the cell that favors viral protein production.     

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

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

Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes – NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.     

Evidence for Circulation of the Rift Valley Fever Virus among Livestock in the Union of Comoros

Rift Valley fever virus (RVFV) is an arthropod-borne phlebovirus reported to be circulating in most parts of Africa. Since 2009, RVFV has been suspected of continuously circulating in the Union of Comoros. To estimate the incidence of RVFV antibody acquisition in the Comorian ruminant population, 191 young goats and cattle were selected in six distinct zones and sampled periodically from April 2010 to August 2011. We found an estimated incidence of RVFV antibody acquisition of 17.5% (95% confidence interval (CI): [8.9–26.1]) with a significant difference between islands (8.2% in Grande Comore, 72.3% in Moheli and 5.8% in Anjouan). Simultaneously, a longitudinal entomological survey was conducted and ruminant trade-related information was collected. No RVFV RNA was detected out of the 1,568 blood-sucking caught insects, including three potential vectors of RVFV mosquito species. Our trade survey suggests that there is a continuous flow of live animals from eastern Africa to the Union of Comoros and movements of ruminants between the three Comoro islands. Finally, a cross-sectional study was performed in August 2011 at the end of the follow-up. We found an estimated RVFV antibody prevalence of 19.3% (95% CI: [15.6%–23.0%]). Our findings suggest a complex RVFV epidemiological cycle in the Union of Comoros with probable inter-islands differences in RVFV circulation patterns. Moheli, and potentially Anjouan, appear to be acting as endemic reservoir of infection whereas RVFV persistence in Grande Comore could be correlated with trade in live animals with the eastern coast of Africa. More data are needed to estimate the real impact of the disease on human health and on the national economy.     

Aerosol Exposure to Rift Valley Fever Virus Causes Earlier and More Severe Neuropathology in the Murine Model,which Has Important Implications for Therapeutic Development

Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that can cause severe disease including acute-onset hepatitis, delayed-onset encephalitis, retinitis and blindness, or a hemorrhagic syndrome. Currently, no licensed vaccine or therapeutics exist to treat this potentially deadly disease. Detailed studies describing the pathogenesis of RVFV following aerosol exposure have not been completed and candidate therapeutics have not been evaluated following an aerosol exposure. These studies are important because while mosquito transmission is the primary means for human infection, it can also be transmitted by aerosol or through mucosal contact. Therefore, we directly compared the pathogenesis of RVFV following aerosol exposure to a subcutaneous (SC) exposure in the murine model by analyzing survival, clinical observations, blood chemistry, hematology, immunohistochemistry, and virus titration of tissues. Additionally, we evaluated the effectiveness of the nucleoside analog ribavirin administered prophylactically to treat mice exposed by aerosol and SC. The route of exposure did not significantly affect the survival, chemistry or hematology results of the mice. Acute hepatitis occurred despite the route of exposure. However, the development of neuropathology occurred much earlier and was more severe in mice exposed by aerosol compared to SC exposed mice. Mice treated with ribavirin and exposed SC were partially protected, whereas treated mice exposed by aerosol were not protected. Early and aggressive viral invasion of brain tissues following aerosol exposure likely played an important role in ribavirin''s failure to prevent mortality among these animals. Our results highlight the need for more candidate antivirals to treat RVFV infection, especially in the case of a potential aerosol exposure. Additionally, our study provides an account of the key pathogenetic differences in RVF disease following two potential exposure routes and provides important insights into the development and evaluation of potential vaccines and therapeutics to treat RVFV infection.     

AMP-Activated Kinase Restricts Rift Valley Fever Virus Infection by Inhibiting Fatty Acid Synthesis

The cell intrinsic innate immune responses provide a first line of defense against viral infection, and often function by targeting cellular pathways usurped by the virus during infection. In particular, many viruses manipulate cellular lipids to form complex structures required for viral replication, many of which are dependent on de novo fatty acid synthesis. We found that the energy regulator AMPK, which potently inhibits fatty acid synthesis, restricts infection of the Bunyavirus, Rift Valley Fever Virus (RVFV), an important re-emerging arthropod-borne human pathogen for which there are no effective vaccines or therapeutics. We show restriction of RVFV both by AMPK and its upstream activator LKB1, indicating an antiviral role for this signaling pathway. Furthermore, we found that AMPK is activated during RVFV infection, leading to the phosphorylation and inhibition of acetyl-CoA carboxylase, the first rate-limiting enzyme in fatty acid synthesis. Activating AMPK pharmacologically both restricted infection and reduced lipid levels. This restriction could be bypassed by treatment with the fatty acid palmitate, demonstrating that AMPK restricts RVFV infection through its inhibition of fatty acid biosynthesis. Lastly, we found that this pathway plays a broad role in antiviral defense since additional viruses from disparate families were also restricted by AMPK and LKB1. Therefore, AMPK is an important component of the cell intrinsic immune response that restricts infection through a novel mechanism involving the inhibition of fatty acid metabolism.     

Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon

As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed a tissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening. Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities and identified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells. Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for early steps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for the development of anti-RVFV therapeutics.     

Vector competence of Culex antennatus and Anopheles coustani mosquitoes for Rift Valley fever virus in Madagascar

Culex antennatus (Diptera: Culicidae), Anopheles coustani (Diptera: Culicidae) and Anopheles squamosus/cydippis were found to be infected with Rift Valley fever virus (RVFV) during an epidemic that occurred in 2008 and 2009 in Madagascar. To understand the roles played by Cx. antennatus and An. coustani in virus maintenance and transmission, RVFV vector competence was assessed in each species. Mosquito body parts and saliva of mosquitoes that fed on RVFV‐infected blood were tested for RVFV using real‐time quantitative polymerase chain reaction (RT‐qPCR) assays. Overall, viral RNA was detected in body parts and saliva at 5 days post‐infection (d.p.i.) in both species. At 5 d.p.i., infection rates were 12.5% (3/24) and 15.8% (6/38), disseminated infection rates were 100% (3/3) and 100% (6/6), transmission rates were 33.3% (1/3) and 83.3% (5/6), and transmission efficiencies were 4.2% (1/24) and 13.2% (5/38) in Cx. antennatus and An. coustani, respectively. Although RVFV detected in saliva did not propagate on to Vero cells, these results support potential roles for these two mosquito species in the transmission of RVFV.     

The Nucleocapsid Protein of Rift Valley Fever Virus Is a Potent Human CD8+ T Cell Antigen and Elicits Memory Responses

There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8⁺ T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8⁺ T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N_121–129) and IL9 (ILDAHSLYL, N_165–173), were defined. VT9- and IL9-specific CD8⁺ T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8⁺ T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8⁺ T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8⁺ T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens.     

Development of a Novel,Single-Cycle Replicable Rift Valley Fever Vaccine

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is an arbovirus that causes severe disease in humans and livestock in sub-Saharan African countries. Although the MP-12 strain of RVFV is a live attenuated vaccine candidate, neuroinvasiveness and neurovirulence of MP-12 in mice may be a concern when vaccinating certain individuals, especially those that are immunocompromised. We have developed a novel, single-cycle replicable MP-12 (scMP-12), which carries an L RNA, M RNA mutant encoding a mutant envelope protein lacking an endoplasmic reticulum retrieval signal and defective for membrane fusion function, and S RNA encoding N protein and green fluorescent protein. The scMP-12 underwent efficient amplification, then formed plaques and retained the introduced mutation after serial passages in a cell line stably expressing viral envelope proteins. However, inoculation of the scMP-12 into naïve cells resulted in a single round of viral replication, and production of low levels of noninfectious virus-like particles. Intracranial inoculation of scMP-12 into suckling mice did not cause clinical signs or death, a finding which demonstrated that the scMP-12 lacked neurovirulence. Mice immunized with a single dose of scMP-12 produced neutralizing antibodies, whose titers were higher than in mice immunized with replicon particles carrying L RNA and S RNA encoding N protein and green fluorescent protein. Moreover, 90% of the scMP-12-immunized mice were protected from wild-type RVFV challenge by efficiently suppressing viremia and replication of the challenge virus in the liver and the spleen. These data demonstrated that scMP-12 is a safe and immunogenic RVFV vaccine candidate.     

Rift valley fever: recent insights into pathogenesis and prevention

Rift Valley fever virus (RVFV) is a zoonotic pathogen that primarily affects ruminants but can also be lethal in humans. A negative-stranded RNA virus of the family Bunyaviridae, this pathogen is transmitted mainly via mosquito vectors. RVFV has shown the ability to inflict significant damage to livestock and is also a threat to public health. While outbreaks have traditionally occurred in sub-Saharan Africa, recent outbreaks in the Middle East have raised awareness of the potential of this virus to spread to Europe, Asia, and the Americas. Although the virus was initially characterized almost 80 years ago, the only vaccine approved for widespread veterinary use is an attenuated strain that has been associated with significant pathogenic side effects. However, increased understanding of the molecular biology of the virus over the last few years has led to recent advances in vaccine design and has enabled the development of more-potent prophylactic measures to combat infection. In this review, we discuss several aspects of RVFV, with particular emphasis on the molecular components of the virus and their respective roles in pathogenesis and an overview of current vaccine candidates. Progress in understanding the epidemiology of Rift Valley fever has also enabled prediction of potential outbreaks well in advance, thus providing another tool to combat the physical and economic impact of this disease.     

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

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

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)^FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCF^FBXW11 E3 ligase. We further show that disrupting the assembly of the SCF^FBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCF^FBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCF^FBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.