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High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Laboratories for Chemical Biology Umeå (LCBU))
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Laboratories for Chemical Biology Umeå (LCBU))
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2016 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 21, no 4, 354-362 p.Article in journal (Refereed) Published
Resource type
Text
Abstract [en]

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 mu M 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 mu M compound concentration and 50% cell viability at 25 mu M 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.

Place, publisher, year, edition, pages
Sage Publications, 2016. Vol. 21, no 4, 354-362 p.
Keyword [en]
high-throughput screening, antiviral, cell-based assay, recombinant virus, Rift Valley fever
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Infectious Medicine
Identifiers
URN: urn:nbn:se:umu:diva-119642DOI: 10.1177/1087057115625184ISI: 000372883200004PubMedID: 26762502OAI: oai:DiVA.org:umu-119642DiVA: diva2:929297
Available from: 2016-05-18 Created: 2016-04-25 Last updated: 2016-10-12Bibliographically approved
In thesis
1. Rift Valley fever: consequences of virus-host interactions
Open this publication in new window or tab >>Rift Valley fever: consequences of virus-host interactions
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Rift Valley fever virus (RVFV) is a mosquito-borne virus which has the ability to infect a large variety of animals including humans in Africa and Arabian Peninsula. The abortion rate among these animals are close to 100%, and young animals develop severe disease which often are lethal.

In humans, Rift Valley fever (RVF) presents in most cases as a mild illness with influenza-like symptoms. However, in about 8% of the cases it progresses into a more severe disease with a high case fatality rate. Since there is such a high abortion rate among infected animals, a link between human miscarriage and RVFV has been suggested, but never proven.

We could in paper I for the first time show an association between acute RVFV infection and miscarriage in humans. We observed an increase in pregnant women arriving at the Port Sudan Hospital with fever of unknown origin, and several of the patients experienced miscarriage. When we analysed their blood samples for several viral diseases we found that many had an acute RVFV infection and of these, 54% experienced a miscarriage. The odds of having a miscarriage was 7 times higher for RVFV patients compared to the RVFV negative women of which only 12% miscarried. These results indicated that RVFV infection could be a contributing factor to miscarriage.

RVFV is an enveloped virus containing the viral glycoproteins n and c (Gn and Gc respectively), where Gn most likely is responsible for the initial cellular contact. The protein DC-SIGN on dendritic cells and the glycosaminoglycan heparan sulfate has been suggested as cellular receptors for RVFV, however other mechanisms are probably also involved in binding and entry. Charge is a driving force for molecular interaction and has been shown to be important for cellular attachment of several viruses, and in paper II we could show that when the charge around the cells was altered, the infection was affected. We also showed that Gn most likely has a positive charge at a physiological pH.

When we added negatively charged molecules to the viral particles before infection, we observed a decreased infection efficiency, which we also observed after removal of carbohydrate structures from the cell surface.

Our results suggested that the cellular interaction partner for initial attachment is a negatively charged carbohydrate. Further investigations into the mechanisms of RVFV cellular interactions has to be undertaken in order to understand, and ultimately prevent, infection and disease.

There is currently no vaccine approved for human use and no specific treatments for RVF, so there is a great need for developing safe effective drugs targeting this virus. We designed a whole-cell based high-throughput screen (HTS) assay which we used to screen libraries of small molecular compounds for anti-RVFV properties. After dose-response and toxicity analysis of the initial hits, we identified six safe and effective inhibitors of RVFV infection that with further testing could become drug candidates for treatment of RVF. This study demonstrated the application of HTS using a whole-cell virus replication reporter gene assay as an effective method to identify novel compounds with potential antiviral activity against RVFV.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2016. 58 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1843
Keyword
Rift Valley fever, Rift Valley fever virus, viral haemorrhagic fever, miscarriage, entry, charge, carbohydrates, high-throughput screening, antiviral, cell-based assay
National Category
Infectious Medicine Microbiology in the medical area
Research subject
Medical Virology
Identifiers
urn:nbn:se:umu:diva-126602 (URN)978-91-7601-558-2 (ISBN)
Public defence
2016-11-04, Hörsal D, Unod T9, 9 trappor, Norrlands universitetssjukhus, Umeå, 09:00 (English)
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Available from: 2016-10-14 Created: 2016-10-12 Last updated: 2016-10-12Bibliographically approved

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Journal of Biomolecular Screening
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)Infectious Medicine

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