Identification and evaluation of antivirals for Rift Valley fever virus

Abstract

Rift Valley fever virus (RVFV) is an enveloped, negative-sense, ssRNA virus with a tripartite genome that causes morbidity and mortality in both livestock and humans. Although RVFV is mainly circulating in mainland Africa, this arthropod-borne virus is a potential threat to the other parts of the world. No fully licensed vaccines for human or animal use in the U.S., and effective antiviral drugs have not been identified. As virulent RVFV strains are only handled in biosafety level (BSL) 3 or higher level facilities in the U.S., few laboratories have access to RVFV which limits antiviral development. However, it is crucial to develop effective antivirals to protect public and animal health. Animal models that reproduce Rift Valley fever are vital to identifying and developing antiviral compounds. The currently available attenuated RVFV strain, MP12, provides a BSL-2 challenge model virus for preliminary investigations of RVFV prior to using the virulent RVFV strains. All strains of RVFV have a highly conserved genome, indicating that antivirals or vaccines effective against any RVFV strain will most likely be effective for all RVFV strains. Therefore, we hypothesize that the MP12 is a suitable model virus that can be used for identification and evaluation of effective RVF antivirals. The first objective of this project was to establish a mouse model susceptible to MP12 infection. Based on the literature, we selected and screened six different strains of mice to test their susceptibilities to MP12. We found the STAT-1 knockout mice are the most susceptible to MP12 infection based on clinical symptoms, mortality, viremia, virus replication, histopathological, and immunochemical analyses. Importantly, these mice displayed acute-onset hepatitis and delayed-onset encephalitis similar to severe cases of human RVFV infection. Our second objective was to identify potential antiviral drugs in vitro. We developed and employed a cell-based assay using the recombinant MP12 virus expressing Renilla luciferase to screen a library of 727 small compounds purchased from National Institutes of Health. Of the compounds, 23 were identified and further tested for their inhibitory activities on the recombinant MP12 virus expressing green fluorescent protein. Further plaque reduction assays confirmed that two compounds inhibited replication of parental RVFV MP12 strain with limited cytotoxic effects. The 50% inhibitory concentrations using an MP12 multiplicity of infection (MOI) of 2 were 211.4 µM and 139.5 µM, respectively. Our third objective was to evaluate these two candidates, 6-azauridine and mitoxantrone, in vivo using our mouse model. After one-hour post MP12 infection via an intranasal route, treatment was given intranasally twice daily. Mice treated with placebo and 6-azauridine displayed severe weight loss and reached the threshold for euthanasia with obvious neurological signs, while mice treated with ribavirin (a known antiviral drug) or mitoxantrone showed delayed onset of disease. This result indicates that the mitoxantro...

Rescue of infectious Arumowot virus from cloned cDNA: Posttranslational degradation of Arumowot virus NSs protein in human cells

“…The rMP12-rLuc, which expresses Renilla luciferase (rLuc) in place of NSs protein, has been used for antiviral screening in past studies [49–52]. Although the rMP12-rLuc is not pathogenic in animals and humans, works in BSL-3 facility will be required to handle MP-12 strain and the variants in most countries other than the U.S.…”

“…The rAMTV-GFP could express GFP along with viral replication, which is useful to detect infected cells real-time. The rMP-12 variants expressing reporter gene (e.g., rMP12-rLuc) have been used for screening of antiviral compounds or host genes affecting viral replication [49–52]. The handling of MP-12 strain, however, still requires BSL-3 facility in many countries other than the U.S. AMTV is classified as a Risk Group 2 pathogen, and there are no known risk of human or animal diseases, which may be suitable for initial screening of broad-acting antivirals effective for both AMTV and RVFV worldwide.…”

Novel ionophores active against La Crosse virus identified through rapid antiviral screening

“…Quininone has activity against enterovirus proteases as well as reverse transcriptases 29,30 . As mentioned previously, mitoxantrone was identified in a screen for anti-RVFV molecules 16 . Additionally, we identified a number of cancer therapeutics, targeting processes such as angiogenesis and topoisomerase, that show significant promise.…”

“…Additionally, Zika virus screens have uncovered known and novel antivirals 13–15 , including mycophenolic acid and daptomycin, among others. More closely related to LACV, a RVFV screen highlighted azauridine and mitoxatrone 16 . The ability to rapidly screen molecules highlights an opportunity to identify both unique virus-specific and broad-spectrum antivirals, as these reports have highlighted.…”

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…The sensitivity of BRET assays has recently been improved by introducing new BRET components: RLuc2 and RLuc8 with improved quantum yields, stability, and brightness [ 143 ] as well as a great variety of acceptors (GFP2, YFP, Venus, mOrange, TagRFP, TurboFP, semiconductor quantum dots or carbon-dots) [ 144 , 145 , 146 ]. However, the main application of RLuc is luciferase genetic reporter assay that has become an invaluable and routine tool for molecular biology research, including identification and characterization of protein functional variants [ 147 , 148 ], investigations of gene expression [ 148 , 149 , 150 ], transcription factors [ 151 , 152 , 153 , 154 , 155 ], receptor activity [ 156 ], miRNA expression [ 157 , 158 , 159 ], monitoring mRNA splicing events in cells [ 160 , 161 ], virus investigations [ 162 , 163 , 164 ], drug discovery [ 165 , 166 ], and identification and evaluation of virus inhibitors [ 167 , 168 ]. Moreover, RLuc is widely used for bioluminescence imaging of different processes in vivo [ 169 , 170 , 171 ] and guiding drug delivery to cells [ 172 , 173 ].…”