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

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

Identification and evaluation of antivirals for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is the causative agent of Rift Valley fever (RVF) that affects both livestock and humans. There are neither fully licensed RVF vaccines available for human or animal use, nor effective antiviral drugs approved for human use in the U.S. To identify antiviral compounds effective for RVF, we developed and employed a cell-based high-throughput assay using a recombinant RVFV MP-12 strain, which expresses Renilla luciferase in place of the NSs protein, to screen 727 small compounds purchased from the National Institutes of Health. Twenty-three compounds were initially identified using the screening assay. Two compounds, 6-azauridine and mitoxantrone, also inhibited the replication of the parental MP-12 strain encoding the NSs gene, with limited cytotoxic effects. The respective 50% inhibitory concentrations were 29.07 µM and 79.85 µM when tested with the parental MP-12 strain at a multiplicity of infection of 2. The compounds were further evaluated using the STAT-1 KO mouse model. At one hour post intranasal inoculation of MP-12 strain, mice were intranasally treated with each indicated compound twice daily. Mice treated with either placebo or 6-azauridine displayed severe weight loss and reached the threshold for euthanasia with obvious neurologic symptoms. Onset of disease was, however, delayed in mice treated with either ribavirin or mitoxantrone. The results indicated that mitoxantrone can reduce the severity of diseases in RVFV-infected mice. Our studies build the foundation for the initial screening and efficacy studies of RVF antivirals in a BSL-2 environment, avoiding the higher risks of BSL-3 exposure with wild-type virus.

Galidesivir limits Rift Valley fever virus infection and disease in Syrian golden hamsters.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen endemic to sub-Saharan Africa and the Arabian Peninsula. There are no approved antiviral therapies or vaccines available to treat or prevent severe disease associated with RVFV infection in humans. The adenosine analog, galidesivir (BCX4430), is a broad-spectrum antiviral drug candidate with in vitro antiviral potency (EC50 of less than 50 µM) in more than 20 different viruses across eight different virus families. Here we report on the activity of galidesivir in the hamster model of peracute RVFV infection. Intramuscular and intraperitoneal treatments effectively limited systemic RVFV (strain ZH501) infection as demonstrated by significantly improved survival outcomes and the absence of infectious virus in the spleen and the majority of the serum, brain, and liver samples collected from infected animals. Our findings support the further development of galidesivir as an antiviral therapy for use in treating severe RVFV infection, and possibly other related phleboviral diseases.

Anti-Rift Valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir-2, a broad-acting antiviral compound.

Rift Valley fever virus (RVFV) is a mosquito-borne hemorrhagic fever virus affecting both humans and animals with severe morbidity and mortality and is classified as a potential bioterror agent due to the possible aerosol transmission. At present there is no human vaccine or antiviral therapy available. Thus, there is a great need to develop new antivirals for treatment of RVFV infections. Benzavir-2 was previously identified as potent inhibitor of human adenovirus, herpes simplex virus type 1, and type 2. Here we assess the anti-RVFV activity of benzavir-2 together with four structural analogs and determine pre-clinical pharmacokinetic parameters of benzavir-2. In vitro, benzavir-2 efficiently inhibited RVFV infection, viral RNA production and production of progeny viruses. In vitro, benzavir-2 displayed satisfactory solubility, good permeability and metabolic stability. In mice, benzavir-2 displayed oral bioavailability with adequate maximum serum concentration. Oral administration of benzavir-2 formulated in peanut butter pellets gave high systemic exposure without any observed toxicity in mice. To summarize, our data demonstrated potent anti-RVFV activity of benzavir-2 in vitro together with a promising pre-clinical pharmacokinetic profile. This data support further exploration of the antiviral activity of benzavir-2 in in vivo efficacy models that may lead to further drug development for human use.

Sorafenib Impedes Rift Valley Fever Virus Egress by Inhibiting Valosin-Containing Protein Function in the Cellular Secretory Pathway.

There is an urgent need for therapeutic development to combat infections caused by Rift Valley fever virus (RVFV), which causes devastating disease in both humans and animals. In an effort to repurpose drugs for RVFV treatment, our previous studies screened a library of FDA-approved drugs. The most promising candidate identified was the hepatocellular and renal cell carcinoma drug sorafenib. Mechanism-of-action studies indicated that sorafenib targeted a late stage in virus infection and caused a buildup of virions within cells. In addition, small interfering RNA (siRNA) knockdown studies suggested that nonclassical targets of sorafenib are important for the propagation of RVFV. Here we extend our previous findings to identify the mechanism by which sorafenib inhibits the release of RVFV virions from the cell. Confocal microscopy imaging revealed that glycoprotein Gn colocalizes and accumulates within the endoplasmic reticulum (ER) and the transport of Gn from the Golgi complex to the host cell membrane is reduced. Transmission electron microscopy demonstrated that sorafenib caused virions to be present inside large vacuoles inside the cells. p97/valosin-containing protein (VCP), which is involved in membrane remodeling in the secretory pathway and a known target of sorafenib, was found to be important for RVFV egress. Knockdown of VCP resulted in decreased RVFV replication, reduced Gn Golgi complex localization, and increased Gn ER accumulation. The intracellular accumulation of RVFV virions was also observed in cells transfected with siRNA targeting VCP. Collectively, these data indicate that sorafenib causes a disruption in viral egress by targeting VCP and the secretory pathway, resulting in a buildup of virions within dilated ER vesicles.IMPORTANCE In humans, symptoms of RVFV infection mainly include a self-limiting febrile illness. However, in some cases, infected individuals can also experience hemorrhagic fever, neurological disorders, liver failure, and blindness, which could collectively be lethal. The ability of RVFV to expand geographically outside sub-Saharan Africa is of concern, particularly to the Americas, where native mosquito species are capable of virus transmission. Currently, there are no FDA-approved therapeutics to treat RVFV infection, and thus, there is an urgent need to understand the mechanisms by which the virus hijacks the host cell machinery to replicate. The significance of our research is in identifying the cellular target of sorafenib that inhibits RVFV propagation, so that this information can be used as a tool for the further development of therapeutics used to treat RVFV infection.

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

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

The first imported case of Rift Valley fever in China reveals a genetic reassortment of different viral lineages.

We report the first imported case of Rift Valley fever (RVF) in China. The patient returned from Angola, a non-epidemic country, with an infection of a new reassortant from different lineages of Rift Valley fever viruses (RVFVs). The patient developed multiorgan dysfunction and gradually recovered with continuous renal replacement therapy and a short regimen of methylprednisolone treatment. The disordered cytokines and chemokines in the plasma of the patient revealed hypercytokinemia, but the levels of protective cytokines were low upon admission and fluctuated as the disease improved. Whole-genome sequencing and phylogenetic analysis revealed that the imported strain was a reassortant comprising the L and M genes from lineage E and the S gene from lineage A. This case highlights that RVFV had undergone genetic reassortment, which could potentially alter its biological properties, cause large outbreaks and pose a serious threat to global public health as well as the livestock breeding industry.

Protein Phosphatase-1 regulates Rift Valley fever virus replication.

Rift Valley fever virus (RVFV), genus Phlebovirus family Bunyaviridae, is an arthropod-borne virus endemic throughout sub-Saharan Africa. Recent outbreaks have resulted in cyclic epidemics with an increasing geographic footprint, devastating both livestock and human populations. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms and host interactions of RVFV. To date there are no FDA approved therapeutics or vaccines for RVF and there is an urgent need for their development. The Ser/Thr protein phosphatase 1 (PP1) has previously been shown to play a significant role in the replication of several viruses. Here we demonstrate for the first time that PP1 plays a prominent role in RVFV replication early on during the viral life cycle. Both siRNA knockdown of PP1α and a novel PP1-targeting small molecule compound 1E7-03, resulted in decreased viral titers across several cell lines. Deregulation of PP1 was found to inhibit viral RNA production, potentially through the disruption of viral RNA transcript/protein interactions, and indicates a potential link between PP1α and the viral L polymerase and nucleoprotein. These results indicate that PP1 activity is important for RVFV replication early on during the viral life cycle and may prove an attractive therapeutic target.

Favipiravir (T-705) protects against peracute Rift Valley fever virus infection and reduces delayed-onset neurologic disease observed with ribavirin treatment.

Rift Valley fever is a zoonotic, arthropod-borne disease that affects livestock and humans. The etiologic agent, Rift Valley fever virus (RVFV; Bunyaviridae, Phlebovirus) is primarily transmitted through mosquito bites, but can also be transmitted by exposure to infectious aerosols. There are presently no licensed vaccines or therapeutics to prevent or treat severe RVFV infection in humans. We have previously reported on the activity of favipiravir (T-705) against the MP-12 vaccine strain of RVFV and other bunyaviruses in cell culture. In addition, efficacy has also been documented in mouse and hamster models of infection with the related Punta Toro virus. Here, hamsters challenged with the highly pathogenic ZH501 strain of RVFV were used to evaluate the activity of favipiravir against lethal infection. Subcutaneous RVFV challenge resulted in substantial serum and tissue viral loads and caused severe disease and mortality within 2-3 days of infection. Oral favipiravir (200 mg/kg/day) prevented mortality in 60% or greater of hamsters challenged with RVFV when administered within 1 or 6h post-exposure and reduced RVFV titers in serum and tissues relative to the time of treatment initiation. In contrast, although ribavirin (75 mg/kg/day) was effective at protecting animals from the peracute RVFV disease, most ultimately succumbed from a delayed-onset neurologic disease associated with high RVFV burden observed in the brain in moribund animals. When combined, T-705 and ribavirin treatment started 24 h post-infection significantly improved survival outcome and reduced serum and tissue virus titers compared to monotherapy. Our findings demonstrate significant post-RVFV exposure efficacy with favipiravir against both peracute disease and delayed-onset neuroinvasion, and suggest added benefit when combined with ribavirin.

Antiviral autophagy restrictsRift Valley fever virus infection and is conserved from flies to mammals.

Autophagy has been implicated as a component of host defense, but the significance of antimicrobial autophagy in vivo and the mechanism by which it is regulated during infection are poorly defined. Here we found that antiviral autophagy was conserved in flies and mammals during infection with Rift Valley fever virus (RVFV), a mosquito-borne virus that causes disease in humans and livestock. In Drosophila, Toll-7 limited RVFV replication and mortality through activation of autophagy. RVFV infection also elicited autophagy in mouse and human cells, and viral replication was increased in the absence of autophagy genes. The mammalian Toll-like receptor adaptor, MyD88, was required for anti-RVFV autophagy, revealing an evolutionarily conserved requirement for pattern-recognition receptors in antiviral autophagy. Pharmacologic activation of autophagy inhibited RVFV infection in mammalian cells, including primary hepatocytes and neurons. Thus, autophagy modulation might be an effective strategy for treating RVFV infection, which lacks approved vaccines and therapeutics.

A mechanistic paradigm for broad-spectrum antivirals that target virus-cell fusion.

LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50 ≤ 0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen ((1)O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated (1)O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. (1)O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced (1)O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of (1)O2 generation), and (4) 10-100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.

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.

[Experimental and clinicolaboratory evaluation of complex therapy efficacy in arboviral infections].

Search for drugs efficient in prophylaxis and treatment of dangerous infections (especially arboviral ones) is rather actual, since no specific therapy is available. Many-year investigations of interferon inductors showed that they had immunomodulating, antiviral and antiinflammatory effects and were low toxic. The present study demonstrated that the protective effect was the following: Venezuelan equine encephalitis (VEE)--cycloferon > amixin = ridostin, Rift Valley fever (RVF)--cycloferon > amixin > ridostin, predator pox (PP)--cycloferon > amixin = ridostin, that was obvious that cycloferon was the most active agent in the treatment of VEE, RVF and PP, thus making it possible to acknowledge its priority in prophylaxis and therapy of dangerous viral infections (DVI). Ribavirin in combination with cycloferon solution or cycloferon tablets provided shorter periods of the fever, minimized the intoxication syndrome, promoted earlier resolution of hemorrhagic eruption and lowered the frequency of complications, which was in favour of the disease prognosis.

Reverse genetics technology for Rift Valley fever virus: current and future applications for the development of therapeutics and vaccines.

The advent of reverse genetics technology has revolutionized the study of RNA viruses, making it possible to manipulate their genomes and evaluate the effects of these changes on their biology and pathogenesis. The fundamental insights gleaned from reverse genetics-based studies over the last several years provide a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. This review summarizes the successes and stumbling blocks in the development of reverse genetics technologies for Rift Valley fever virus and their application to the further dissection of its pathogenesis and the design of new therapeutics and safe and effective vaccines.

[The antiviral activity of inhibitors of ADP ribosylation in experimental alpha- and bunyavirus infections]. / Protivovirusnaia aktivnost' nekotorykh ingibitorov ADF-ribozilirovaniia pri éksperimental'nykh al'fa- i bun'iavirusnykh infektsiiakh.

The prophylactic and therapeutic effects of ADP-ribosylation inhibitors, 3,N-acetylaminobenzamide and 3,N-butyrylaminobenzamide, were studied in mice inoculated with an alphavirus or a bunyavirus. Both drugs were shown to have high levels of antiviral activity when given as a single subcutaneous injection in a dose of 10 mg/kg while increasing the number of injections did not increase their efficacy or might even decrease it.

[The effect of the peroxidation of ribamidil-containing liposomes on the results of chemotherapy in experimental viral infections]. / Vliianie perekisnogo okisleniia ribamidilsoderzhashchikh liposom na rezul'taty khimioterapii nekotorykh éksperimental'nykh virusnykh infektsii.

A relationship between ribamydil concentration and the intensity of accumulation of peroxidation products was found while storing ribamydil-containing liposomes which indicated a significant pro-oxidant activity of ribamydil. Increasing lethality of guinea pigs and white mice infected with Venezuelan equine encephalomyelitis virus was shown while using liposomal ribamydil containing from 4.78 to 6.82 nmol. ml-1 malonic dialdehyde which appeared to be associated with disordered function of the antioxidant system of the experimental animals.

Enhanced therapeutic efficacy of poly(ICLC) and ribavirin combinations against Rift Valley fever virus infection in mice.

The therapeutic efficacy of polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine and carboxymethyl cellulose [poly(ICLC)] given alone or in combination with ribavirin was evaluated in Swiss Webster mice infected with Rift Valley fever virus. Four or more 20-micrograms doses of poly(ICLC) given at various intervals beginning 24 h after infection protected all mice against death. On the other hand, a treatment regimen consisting of only three doses of poly(ICLC) given 24 h postinfection resulted in a 50% survival rate. When initiated 48 h postinfection, an extended treatment regimen with the same dose was required to yield 40% survivors. Lower doses (5 micrograms) of poly(ICLC) per mouse were only marginally effective even when six injections were given between days 1 and 9 postinfection. The combined administration of ribavirin and poly(ICLC) initiated as late as 48 h postinfection was effective even when treatment consisted of doses that were ineffective when either drug was used alone.

Prophylaxis of Rift Valley fever with antiviral drugs, immune serum, an interferon inducer, and a macrophage activator.

Rift Valley fever virus (RVFV), a member of the family Bunyaviridae, extended its range from sub-Saharan Africa into Egypt in 1977. Its clinical spectrum is recognized to include severe manifestations such as hemorrhagic fever and encephalitis. For these reasons, as well as the limited knowledge of specific therapy for Bunyaviridae infections, we investigated several prophylactic regimens for RVF in a mouse model. Rimantadine, thiosemicarbazone, and inosiplex were ineffective. Pretreatment with glucan was of some use, but the most encouraging results were obtained with the antiviral drug ribavirin, passive antibody, or an interferon inducer polyriboinosinic-polyribocytidylic acid complexed with poly-L-lysine and carboxymethylcellulose (poly[ICLC]). Ribavirin and poly(ICLC) were also shown to be efficacious in preventing disease in hamsters. Ribavirin (loading dose of 50 mg/kg followed by 10 mg/kg at 8-h intervals for 9 days) suppressed viremia in RVF-infected rhesus monkeys. Ribavirin also reduced virus yield in infected cell cultures; sensitivity varied markedly with cell type but not with virus strain. Immune mouse ascitic fluid, with a plaque reduction neutralization titer of 1:1024, was effective in a dose of 4 ml/kg, a volume approximately equivalent to administration of a unit of convalescent plasma to a human. Poly(ICLC) may well have functioned through interferon induction, since RVFV was shown to be sensitive to interferon in cell culture, and since another macrophage activator (glucan) was only marginally effective. These studies suggest that ribavirin, poly(ICLC), and convalescent plasma may have a role in prevention or therapy of human RVF.

Prophylactic and therapeutic efficacy of poly(I,C)-LC against Rift Valley fever virus infection in mice.

The prophylactic and therapeutic efficacy of polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose [poly(I,C)-LC] was evaluated in female Swiss Webster mice against a lethal infection of Rift Valley fever virus (RVFV). Prophylactically, the best effect was obtained with 2-3 doses of 1-20 micrograms poly(I,C)-LC, which fully protected the mice in a schedule-dependent fashion against an LD100 RVFV challenge. Multiple intermittent therapeutic administration of 20 micrograms poly(I,C)-LC 24 or 48 h after infection protected 100% and 50% of the mice, respectively. When given as late as 48 h after infection, 20 micrograms poly(I,C)-LC prevented viremia, and single or multiple doses induced high levels of serum interferon that peaked 24 h after administration of the compound. As late as 48 h postinfection, a high degree of therapeutic synergism was achieved with the combined administration of poly(I,C)-LC and the antiviral compound ribavirin, using doses that were not effective alone.