IFITM-2 and IFITM-3 but Not IFITM-1 Restrict Rift Valley Fever Virus

We show that interferon-induced transmembrane protein 1 (IFITM-1), IFITM-2, and IFITM-3 exhibit a broad spectrum of antiviral activity against several members of the Bunyaviridae family, including Rift Valley fever virus (RVFV), La Crosse virus, Andes virus, and Hantaan virus, all of which can cause severe disease in humans and animals. We found that RVFV was restricted by IFITM-2 and -3 but not by IFITM-1, whereas the remaining viruses were equally restricted by all IFITMs. Indeed, at low doses of alpha interferon (IFN-α), IFITM-2 and -3 mediated more than half of the antiviral activity of IFN-α against RVFV. IFITM-2 and -3 restricted RVFV infection mostly by preventing virus membrane fusion with endosomes, while they had no effect on virion attachment to cells, endocytosis, or viral replication kinetics. We found that large fractions of IFITM-2 and IFITM-3 occupy vesicular compartments that are distinct from the vesicles coated by IFITM-1. In addition, although overexpression of all IFITMs expanded vesicular and acidified compartments within cells, there were marked phenotypic differences among the vesicular compartments occupied by IFITMs. Collectively, our data provide new insights into the possible mechanisms by which the IFITM family members restrict distinct viruses.     

A cellular function is required for pseudorabies virus envelope glycoprotein processing and virus egress.

The mouse L-cell mutant gro29 is defective for egress of herpes simplex virus type 1 (HSV-1) virions and is significantly reduced in HSV-1 glycoprotein export (B. W. Banfield and F. Tufaro, J. Virol. 64:5716-5729, 1990). In this report, we demonstrate that pseudorabies virus (PRV), a distantly related alphaherpesvirus, shows a distinctive set of defects after infection of gro29 cells. Specifically, we identify defects in the rate and extent of viral glycoprotein export, infectious particle formation, plaque formation, and virus egress. The initial rate of viral glycoprotein synthesis was unaffected in gro29 cells, but the extent of export from the endoplasmic reticulum to the Golgi apparatus was impaired and export through the Golgi apparatus became essentially blocked late in infection. Moreover, by using a secreted variant of a viral membrane protein, we found that export from the Golgi apparatus out of the cell was also defective in gro29 cells. PRV does not form plaques on gro29 monolayers. A low level of infectious virus is formed and released early after infection, but further virus egress is blocked. Taken together, these observations suggest that the gro29 phenotype involves either multiple proteins or a single protein used at multiple steps in viral glycoprotein export and virus egress from cells. Moreover, this host cell protein is required by both HSV and PRV for efficient propagation in infected cells.     

Identification of Three Antiviral Inhibitors against Japanese Encephalitis Virus from Library of Pharmacologically Active Compounds 1280

Japanese encephalitis virus (JEV) can cause severe central nervous disease with a high mortality rate. There is no antiviral drug available for JEV-specific treatment. In this study, a cytopathic-effect-based, high-throughput screening assay was developed and applied to screen JEV inhibitors from Library of Pharmacologically Active Compounds 1280. The antiviral effects of three hit compounds including FGIN-1-27, cilnidipine, and niclosamide were evaluated in cells by western blotting, indirect immunofluorescence assay, and plaque reduction assay. A time-of-addition assay proved that all three compounds inhibited JEV at the stage of replication. The EC50s of FGIN-1-27, cilnidipine, and niclosamide were 3.21, 6.52, and 5.80 µM, respectively, while the selectivity indexes were 38.79, 30.67, and 7.49. FGIN-1-27 and cilnidipine have high efficiency and selectivity against JEV. This study provided two JEV antiviral inhibitors as candidates for treatment of JEV infection.     

The frequency of Familial Mediterranean fever gene mutations and genotypes at Kirikkale and comparison with the mean of regional MEFV mutation frequency of Turkey

In this study we have retrospectively analysed the mutation spectrum of the 351 Familial Mediterranean fever patients referred to K?r?kkale University Faculty of Medicine, Department of Medical Genetics Laboratory over a period of 5 years and compared them with Turkey’s mean. We have found 11 different mutations, including rare mutations such as F479L, K695R, M680I(G/A) and 45 different genotypes showing the heterogeneity of MEFV mutations in Central Anatolia. The most three prevalent mutations were M694V (14.8 %), E148Q (7.1 %) and M680I(G/C) (4.1 %) in accordance with the literature. We have also investigated R202Q in our routine molecular diagnosis. Mutation causing R202Q (c.605G > A) change was described as a frequent polymorphism and G allele was found in linkage disequilibrium (LD) with M694V. There are limited number of studies investigating R202Q, some of them implicate that its homozygote state is disease causing. We showed the high frequency of R202Q (23.7 %) with and without M694V in all the groups analysed and its high LD rate with M694V in the diagnosed group. Our study is reflecting the mutational heterogeneity of MEFV and summarize mutational spectrum of Turkey’s geographical regions and overall Turkey.     

Quinolylhydrazones as novel inhibitors of Plasmodium falciparum serine protease PfSUB1

Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) is a serine protease that plays key roles in the egress of the parasite from red blood cells and in preparing the released merozoites for the subsequent invasion of new erythrocytes. The development of potent and selective PfSUB1 inhibitors could pave the way to the discovery of potential antimalarial drugs endowed with an innovative mode of action and consequently able to overcome the current problems of resistance to established chemotherapies. Through the screening of a proprietary library of compounds against PfSUB1, we identified hydrazone 2 as a hit compound. Here we report a preliminary investigation of the structure-activity relationships for a class of PfSUB1 inhibitors related to our identified hit.     

Identification of Rift Valley Fever Virus Nucleocapsid Protein-RNA Binding Inhibitors Using a High-Throughput Screening Assay

Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection, and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential antiviral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug-screening assay and tested 26 424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of Food and Drug Administration-approved drugs, druglike molecules, and natural product extracts, we identified several lead compounds that are promising candidates for medicinal chemistry.     

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.     

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.     

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.     

Discovery of novel benzoquinazolinones and thiazoloimidazoles, inhibitors of influenza H5N1 and H1N1 viruses, from a cell-based high-throughput screen

A highly reproducible and robust cell-based high-throughput screening (HTS) assay was adapted for screening of small molecules for antiviral activity against influenza virus strain A/Vietnam/1203/2004 (H5N1). The NIH Molecular Libraries Small Molecule Repository (MLSMR) Molecular Libraries Screening Centers Network (MLSCN) 100,000-compound library was screened at 50 μM. The "hit" rate (>25% inhibition of the viral cytopathic effect) from the single-dose screen was 0.32%. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen yielded 5 active compounds (SI value >3). One compound showed an SI(50) value of greater than 3, 3 compounds had SI values ranging from greater than 14 to 34, and the most active compound displayed an SI value of 94. The active compounds represent 2 different classes of molecules, benzoquinazolinones and thiazoloimidazoles, which have not been previously identified as having antiviral/anti-influenza activity. These molecules were also effective against influenza A/California/04/2009 virus (H1N1) and other H1N1 and H5N1 virus strains in vitro but not H3N2 strains. Real-time qRT-PCR results reveal that these chemotypes significantly reduced M1 RNA levels as compared to the no-drug influenza-infected Madin Darby canine kidney cells.     

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.     

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.     

Induction of a neutralizing immune response to human respiratory syncytial virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against monoclonal antibodies that recognized either F glycoprotein 47F or G glycoprotein 63G, 62G, or 74G of the human respiratory syncytial virus Long strain. Anti-Id sera inhibited the virus binding of the immunizing monoclonal antibodies and in some cases the binding of other antibodies reacting with overlapping epitopes. The anti-Id sera also inhibited virus neutralization mediated by the original monoclonal antibodies. Affinity purified anti-Id antibodies were subsequently used to raise a homologous anti-anti-Id response in rabbits. One of the rabbits, inoculated with anti-Id 63G, generated antibodies that reacted with the G protein of respiratory syncytial virus and neutralized the virus to high titers. The antiviral antibodies induced by anti-Id 63G were broadly cross-reactive with strains of the A and B subtypes. However, the specificities of monoclonal antibody 63G and anti-anti-Id 63G were not exactly the same, as indicated by their reaction with escape mutants to antibody 63G. These results demonstrate for the first time the induction of an anti-respiratory syncytial virus response by anti-Id antibodies.     

High-throughput screening of a 100,000-compound library for inhibitors of influenza A virus (H3N2)

Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-microM compound concentration against influenza strain A/Udorn/72 (H3N2). The "hit" rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-microM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI(50) = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI(50) > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity.     

Clone 13-infected Aedes aegypti salivary components inhibit Rift Valley fever virus pathogenicity

Rift Valley fever virus (RVFV) continues to cause large outbreaks among humans and domestic animals in Africa. RVFV Clone 13, a naturally attenuated clone, is a promising vaccine which was used during the 2009–2010 outbreak in South Africa and played a key role in the control of the disease. In this work, we infected Aedes aegypti mosquitoes with RVFV Clone 13 and prepared salivary gland extracts (SGE). C57BL/6-NRJ male mice were infected with a mixture of SGE infected by Clone 13 and the ZH548 RVFV strain. With the injection of increasing doses of Clone 13-infected SGE, all mice were protected. Our results suggest Clone 13 infected SGE contain unique antiviral components able to counteract the replication of RVFV when injected into vertebrates.     

Regulation of Apobec3F and human immunodeficiency virus type 1 Vif by Vif-Cul5-ElonB/C E3 ubiquitin ligase

The human cytidine deaminase Apobec3F (h-A3F), a protein related to the previously recognized antiviral factor Apobec3G (h-A3G), has antiviral activity against human immunodeficiency virus type 1 (HIV-1) that is suppressed by the viral protein Vif. The mechanism of HIV-1 Vif-mediated suppression of h-A3F is not fully understood. Here, we demonstrate that while h-A3F, like h-A3G, was able to suppress primate lentiviruses other than HIV-1 (simian immunodeficiency virus from African green monkeys [SIVagm] and Rhesus macaques [SIVmac]), the interaction between Vif proteins and h-A3F appeared to differ from that with h-A3G. H-A3F showed no change in its species specificity against HIV-1 or SIVagm Vif when a negatively charged amino acid was replaced with a lysine at position 128, a residue critical for h-A3G recognition by HIV-1 Vif. However, HIV-1 Vif, but not SIVagm Vif, was able to bind h-A3F and induce its polyubiquitination and degradation through the Cul5-containing E3 ubiquitin ligase. Interference with Cul5-E3 ligase function by depletion of Cul5, through RNA interference or overexpression of Cul5 mutants, blocked the ability of HIV-1 Vif to suppress h-A3F. A BC-box mutant of HIV-1 Vif that failed to recruit Cul5-E3 ligase but was still able to interact with h-A3F failed to suppress h-A3F. Interestingly, interference with Cul5-E3 ligase function or overexpression of h-A3F or h-A3G also increased the stability of HIV-1 Vif, suggesting that like the substrate molecules h-A3F and h-A3G, the substrate receptor protein Vif is itself also regulated by Cul5-E3 ligase. Our results indicate that Cul5-E3 ligase appears to be a common pathway hijacked by HIV-1 Vif to defeat both h-A3F and h-A3G. Developing inhibitors to disrupt the interaction between Vif and Cul5-E3 ligase could be therapeutically useful, allowing multiple host antiviral factors to suppress HIV-1.     

Rift Valley fever virus M segment: use of recombinant vaccinia viruses to study Phlebovirus gene expression.

Recombinant vaccinia viruses were constructed and used in conjunction with site-specific antisera to study the coding capacity and detailed expression strategy of the M segment of the Phlebovirus Rift Valley fever virus (RVFV). The M segment could be completely and faithfully expressed in recombinant RVFV-vaccinia virus-infected cells, the gene products apparently being correctly processed and modified in the absence of the RVFV L and S genomic segments. The proteins encoded by the RVFV M segment included, in addition to the viral glycoproteins G2 and G1, two previously uncharacterized polypeptides of 78 and 14 kilodaltons (kDa). By manipulation of RVFV sequences present in the recombinant vaccinia viruses and use of specific antibody reagents, it was found that the 78-kDa protein initiated at the first initiation codon of the open reading frame and encompassed the entire preglycoprotein and glycoprotein G2 coding sequences. The 14-kDa protein appeared to begin from the second in-phase ATG and was composed of only the preglycoprotein sequences. Both viral glycoproteins G2 and G1 could be synthesized and correctly processed in the absence of the 78- and 14-kDa proteins, as well as a large portion of the preglycoprotein sequences. However, the hydrophobic amino acid sequence immediately preceding the mature glycoprotein coding sequences was required for authentic glycoprotein production. The M-segment expression strategy involving aspects of translational initiation and protein processing are discussed. The functional roles of the 78- and 14-kDa proteins remain unclear.     

RhoA-derived peptide dimers share mechanistic properties with other polyanionic inhibitors of respiratory syncytial virus (RSV), including disruption of viral attachment and dependence on RSV G

Large polyanionic molecules, such as sulfated polysaccharides (including soluble heparin and dextran sulfate), synthetic polyanionic polymers, and negatively charged proteins, have been shown to broadly inhibit several enveloped viruses. We recently reported the antiviral activity of a peptide derived from amino acids 77 to 95 of a potential binding partner of respiratory syncytial virus F protein (RSV F), the GTPase RhoA. A subsequent study with a truncated peptide (amino acids 80 to 94) revealed that optimal antiviral activity required dimerization via intermolecular disulfide bonds. We report here that the net negative charge of this peptide is also a determining factor for its antiviral activity and that it, like other polyanions, inhibits virus attachment. In a flow cytometry-based binding assay, peptide 80-94, heparin, and dextran sulfate inhibited the attachment of virus to cells at 4 degrees C at the same effective concentrations at which they prevent viral infectivity. Interestingly, time-of-addition experiments revealed that peptide 80-94 and soluble heparin were also able to inhibit the infectivity of a virus that had been prebound to cells at 4 degrees C, as had previously been shown for dextran sulfate, suggesting a potential role for postattachment effects of polyanions on RSV entry. Neutralization experiments with recombinant viruses showed that the antiviral activities of peptide 80-94 and dextran sulfate were diminished in the absence of the RSV attachment glycoprotein (G). Taken together, these data indicate that the antiviral activity of RhoA-derived peptides is functionally similar to that of other polyanions, is dependent on RSV G, and does not specifically relate to a protein-protein interaction between F and RhoA.