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Rift Valley fever: consequences of virus-host interactions
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. (Magnus Evander)ORCID iD: 0000-0002-1269-4770
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 [en]
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: urn:nbn:se:umu:diva-126602ISBN: 978-91-7601-558-2 (print)OAI: oai:DiVA.org:umu-126602DiVA: diva2:1034484
Public defence
2016-11-04, Hörsal D, Unod T9, 9 trappor, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-10-14 Created: 2016-10-12 Last updated: 2016-10-12Bibliographically approved
List of papers
1. Association of Rift Valley fever virus infection with miscarriage in Sudanese women: a cross-sectional study
Open this publication in new window or tab >>Association of Rift Valley fever virus infection with miscarriage in Sudanese women: a cross-sectional study
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2016 (English)In: The Lancet Global Health, ISSN 2352-3026, E-ISSN 2214-109X, Vol. 4, no 11, e864-e871 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Rift Valley fever virus is an emerging mosquito-borne virus that causes infections in animals and human beings in Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever lead to mass abortions in livestock, but such abortions have not been identified in human bezings. Our aim was to investigate the cause of miscarriages in febrile pregnant women in an area endemic for Rift Valley fever.

METHODS: Pregnant women with fever of unknown origin who attended the governmental hospital of Port Sudan, Sudan, between June 30, 2011, and Nov 17, 2012, were sampled at admission and included in this cross-sectional study. Medical records were retrieved and haematological tests were done on patient samples. Presence of viral RNA as well as antibodies against a variety of viruses were analysed. Any association of viral infections, symptoms, and laboratory parameters to pregnancy outcome was investigated using Pearson's χ(2) test.

FINDINGS: Of 130 pregnant women with febrile disease, 28 were infected with Rift Valley fever virus and 31 with chikungunya virus, with typical clinical and laboratory findings for the infection in question. 15 (54%) of 28 women with an acute Rift Valley fever virus infection had miscarriages compared with 12 (12%) of 102 women negative for Rift Valley fever virus (p<0·0001). In a multiple logistic regression analysis, adjusting for age, haemorrhagic disease, and chikungunya virus infection, an acute Rift Valley fever virus infection was an independent predictor of having a miscarriage (odds ratio 7·4, 95% CI 2·7-20·1; p<0·0001).

INTERPRETATION: This study is the first to show an association between infection with Rift Valley fever virus and miscarriage in pregnant women. Further studies are warranted to investigate the possible mechanisms. Our findings have implications for implementation of preventive measures, and evidence-based information to the public in endemic countries should be strongly recommended during Rift Valley fever outbreaks.

FUNDING: Schlumberger Faculty for the Future, CRDF Global (31141), the Swedish International Development Cooperation Agency, the County Council of Västerbotten, and the Faculty of Medicine, Umeå University.

Keyword
Rift Valley fever, Rift Valley fever virus, infectious diseases, virology, miscarriage, Sudan, maternal health
National Category
Infectious Medicine
Research subject
Infectious Diseases; Medical Virology; Obstetrics and Gynaecology
Identifiers
urn:nbn:se:umu:diva-126472 (URN)10.1016/S2214-109X(16)30176-0 (DOI)27692776 (PubMedID)
Available from: 2016-10-07 Created: 2016-10-07 Last updated: 2017-11-30Bibliographically approved
2. Importance of charge interactions in Rift Valley fever virus attachment to host cells
Open this publication in new window or tab >>Importance of charge interactions in Rift Valley fever virus attachment to host cells
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The mosquito-borne Rift Valley fever virus (RVFV) cause disease in both humans and animals and can infect a large range of animals as well as humans. Many different cell types are infected both in vivo and in vitro. To enter a cell the virus needs to attach and enter, and this initial binding to the host cell surface could depend on both general mechanisms, and different specific receptors. Our aim was to characterize determinants for RVFV entry into its host cells.To examine RVFV attachment to host cells we based our experimental assay on RVF virus-like particles containing a reporter gene. The enveloped RVFV uses protruding glycoproteins (Gn and Gc) for attachment and entry and to investigate potential virus-cell surface interactions, the net surface charge of the glycoproteins was first calculated. The RVFV glycoprotein Gn had a predicted isoelectric point (pI) of 7.6 and a net positive charge of +6.9 at pH 7.0, suggesting a charge interaction between the Gn ectodomain and the negatively charged cell surface. RVFV Gc on the other hand, was highly negatively charged, -12.8 at neutral pH, most probably reflecting that Gc is not exposed until after receptor binding. To characterize the general conditions needed for RVFV attachment, cells or virus were treated with various compounds. Both sodium chloride and the negatively charged heparin inhibited RVF virus-like particle infection, strongly indicating that viral binding was charge-dependent. Treatment with sodium periodate pointed to a carbohydrate structure as a cellular interaction partner. Removal of sialic acid or heparan sulfate receptors on the cell surface by enzymatic treatment and blocking of the heparan sulfate receptor did not inhibit virus attachment.In conclusion, RVFV binding to host cells was charge dependent and the results point to a carbohydrate structure with negative charge as a potential attachment factor.

Keyword
Rift Valley fever, receptor, entry, charge, carbohydrates
National Category
Cell and Molecular Biology Microbiology in the medical area
Research subject
Molecular Medicine; Infectious Diseases
Identifiers
urn:nbn:se:umu:diva-126473 (URN)
Available from: 2016-10-07 Created: 2016-10-07 Last updated: 2016-10-12
3. High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection
Open this publication in new window or tab >>High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection
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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
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
Keyword
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:nbn:se:umu:diva-119642 (URN)10.1177/1087057115625184 (DOI)000372883200004 ()26762502 (PubMedID)
Available from: 2016-05-18 Created: 2016-04-25 Last updated: 2017-11-30Bibliographically approved

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