A novel system for identification of inhibitors of rift valley Fever virus replication (original) (raw)

High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection

Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection.

Structural Elucidation of Rift Valley Fever Virus L Protein towards the Discovery of Its Potential Inhibitors

Pharmaceuticals

Rift valley fever virus (RVFV) is the causative agent of a viral zoonosis that causes a significant clinical burden in domestic and wild ruminants. Major outbreaks of the virus occur in livestock, and contaminated animal products or arthropod vectors can transmit the virus to humans. The viral RNA-dependent RNA polymerase (RdRp; L protein) of the RVFV is responsible for viral replication and is thus an appealing drug target because no effective and specific vaccine against this virus is available. The current study reported the structural elucidation of the RVFV-L protein by in-depth homology modeling since no crystal structure is available yet. The inhibitory binding modes of known potent L protein inhibitors were analyzed. Based on the results, further molecular docking-based virtual screening of Selleckchem Nucleoside Analogue Library (156 compounds) was performed to find potential new inhibitors against the RVFV L protein. ADME (Absorption, Distribution, Metabolism, and Excretio...

Metal Coordinating Inhibitors of Rift Valley Fever virus Replication

2022

Rift Valley Fever Virus (RVFV) is a veterinary and human pathogen and is an agent of bioterrorism concern. Currently, RVFV treatment is limited to supportive care, so new drugs to control RVFV infection are urgently needed. RVFV is a member of the Bunyavirales order, and replication of these viruses depends on the viral endonuclease activity of the viral L protein. Screening for RVFV replication inhibitors among compounds with divalent cation-coordinating motifs similar to known viral nuclease inhibitors identified 31 novel RVFV inhibitors with selective indexes from 5 – 402 and 50% effective concentrations of 0.54 – 56 µM in Vero cells, primarily α-Hydroxytropolones and N-Hydroxypyridinediones. Inhibitor activity and selective index was validated in the human cell line A549. To evaluate specificity, select compounds were tested against another Bunyavirus, La Crosse Virus (LACV). Conservation of the enzymatic activity such as the cap-snatching mechanism among the Bunyavirales implie...

Creation of a Nonspreading Rift Valley Fever Virus

Journal of Virology, 2011

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic bunyavirus of the genus Phlebovirus and a serious human and veterinary pathogen. RVFV contains a three-segmented RNA genome, which is comprised of the large (L), medium (M), and small (S) segments. The proteins that are essential for genome replication are encoded by the L and S segments, whereas the structural glycoproteins are encoded by the M segment. We have produced BHK replicon cell lines (BHK-Rep) that maintain replicating L and S genome segments. Transfection of BHK-Rep cells with a plasmid encoding the structural glycoproteins results in the efficient production of RVFV replicon particles (RRPs). To facilitate monitoring of infection, the NSs gene was replaced with an enhanced green fluorescent protein gene. RRPs are infectious for both mammalian and insect cells but are incapable of autonomous spreading, rendering their application outside biosafety containment completely safe. We demonstrate that a single intramu...

Efficacy and Cytotoxicty of Novel Antiviral Compounds Against Rift Valley Fever Virus

2015

Rift Valley Fever virus (RVFV) is a ss-RNA virus from the Bunyaviridae family found in Africa and the Arabian Peninsula. The virus is usually transmitted by mosquitoes and predominantly affects livestock; however, humans exposed to bodily fluids or tissue from infected animals can also be infected. In humans, Rift Valley Fever is usually characterized by mild febrile illness; however, in rare cases, the disease becomes more severe and can cause liver disease, encephalitis, vision loss, and hemorrhagic fever. Epizootic RVF can lead to abortion storms in which nearly 100% of pregnancies in infected ruminants result in abortion. RVFV is considered a bioterrorism threat and outbreaks have significant socio-economic impacts. Currently, there are no approved vaccines or therapeutics against RVFV for use in humans. Identification of a safe and effective therapeutic is crucial to public health’s success in the prevention, treatment, and control of Rift Valley Fever. Antiviral properties of ...

Quantitative Real-Time PCR Detection of Rift Valley Fever Virus and Its Application to Evaluation of Antiviral Compounds

Journal of Clinical Microbiology, 2001

The Rift Valley fever virus (RVFV), a member of the genus Phlebovirus (family Bunyaviridae) is an enveloped negative-strand RNA virus with a tripartite genome. Until 2000, RVFV circulation was limited to the African continent, but the recent deadly outbreak in the Arabian Peninsula dramatically illustrated the need for rapid diagnostic methods, effective treatments, and prophylaxis. A method for quantifying the small RNA segment by a real-time detection reverse transcription (RT)-PCR using TaqMan technology and targeting the nonstructural protein-coding region was developed, and primers and a probe were designed. After optimization of the amplification reaction and establishment of a calibration curve with synthetic RNA transcribed in vitro from a plasmid containing the gene of interest, real-time RT-PCR was assessed with samples consisting of RVFV from infected Vero cells. The method was found to be specific for RVFV, and it was successfully applied to the detection of the RVFV genome in animal sera infected with RVFV as well as to the assessment of the efficiency of various drugs (ribavirin, alpha interferon, 6-azauridine, and glycyrrhizin) for antiviral activity.

Single-Dose Immunization with Virus Replicon Particles Confers Rapid Robust Protection against Rift Valley Fever Virus Challenge

Journal of Virology, 2012

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 c...

Efficient production of Rift Valley fever virus-like particles: The antiviral protein MxA can inhibit primary transcription of bunyaviruses

Virology, 2009

Rift Valley fever virus (RVFV) is a highly pathogenic member of the family Bunyaviridae that needs to be handled under biosafety level (BSL) 3 conditions. Here, we describe reverse genetics systems to measure RVFV polymerase activity in mammalian cells and to generate virus-like particles (VLPs). Recombinant polymerase (L) and nucleocapsid protein (N), expressed together with a minireplicon RNA, formed transcriptionally active nucleocapsids. These could be packaged into VLPs by additional expression of viral glycoproteins. The VLPs resembled authentic virus particles and were able to infect new cells. After infection, VLP-associated nucleocapsids autonomously performed primary transcription, and co-expression of L and N in VLP-infected cells allowed subsequent replication and secondary transcription. Bunyaviruses are potently inhibited by a human interferon-induced protein, MxA. However, the affected step in the infection cycle is not entirely characterized. Using the VLP system, we demonstrate that MxA inhibits both primary and secondary transcriptions of RVFV. A set of infection assays distinguishing between virus attachment, entry, and subsequent RNA synthesis confirmed that MxA is able to target immediate early RNA synthesis of incoming RVFV particles. Thus, our reverse genetics systems are useful for dissecting individual steps of RVFV infection under non-BSL3 conditions.