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Desenvolvimento de um modelo experimental de infecção subcutânea por vírus oropouche em hamster / Development of an experimental hamster model of subcutaneous infection by oropouche virusRodrigues, Alcir Humberto 22 October 2004 (has links)
O vírus Oropouche pertence à família Bunyaviridae, gênero Orthobunyavirus, sorogrupo Simbu, e é segunda causa mais freqüente de arbovirose febril no Brasil. Estima-se que mais de meio milhão de casos de febre do Oropouche tenham ocorrido no Brasil nos últimos 30 anos, havendo também ocorrências no Panamá, Peru, Suriname e Trinidad. Epidemias de febre do Oropouche têm sido registradas quase que exclusivamente na Amazônia. Porém, com o aquecimento global do planeta, desmatamentos e conseqüente redistribuição de insetos vetores e animais reservatórios, há risco de disseminação de vírus Oropouche para outras regiões do Brasil e da América do Sul. A patogenia da infecção por Oropouche não é bem entendida. Modelo em roedores, usando inoculação intracerebral, tem sido descrito, mas não com uso da via subcutânea, que mais se assemelha à rota natural da infecção. O objetivo deste trabalho foi estabelecer e caracterizar um modelo experimental de infecção com Oropouche, usando inoculação subcutânea em hamster, a fim de contribuir para o entendimento da patogenia do mesmo. Oropouche da linhagem BeAn19991, passado em cérebro de camundongo recém-nascido, foi inoculado (106,25 TCID50/100?L) por via subcutânea na coxa de hamsters sírios com 2-3 semanas de idade. Em torno de 3 dias alguns animais desenvolveram doença com sinais clínicos brandos caracterizados por: eriçamento dos pêlos e agressividade, outros com características mais graves: perda de peso, tremores sugestivos de calafrios, letargia e paralisia. Os animais foram sacrificados 1, 3, 5, 8 e 11 dias pós-inoculação ou quando eram encontrados em estado agônico. Baço, cérebro, coração, fígado, músculo e sangue foram colhidos para titulação viral, histologia e imunohistoquímica. Infiltrado inflamatório estava presente no cérebro, principalmente em torno dos vasos, meninges e discretamente no coração. O vírus Oropouche foi detectado no tecido cerebral (107,23 TCID50/g), no fígado (106,67TCID50/g) e no sangue (106 TCID50/g). Antígeno de Oropouche foi encontrado, difusamente no fígado, associado com hepatócitos e em neurônios de diversos locais do cérebro. Este modelo reproduz uma infecção sistêmica por Oropouche e pode tornar-se útil no estudo da patogênese, bem como para testar drogas antivirais e possíveis candidatos à vacina. / Oropouche virus belongs to the family Bunyaviridae, genus Orthobunyavirus, serogroup Simbu and is the second most frequent arboviral febrile illness in Brazil. There are estimates of more than half million cases of Oropouche fever in Brazil in the past 30 years, with cases registered in Panama, Peru, Suriname and Trinidad. Oropouche fever has been registered almost exclusively in the Amazon, but with the global warming, deforestation and the consequent redistribution of vectors and reservoir animals, the risk of Oropouche virus dissemination to others areas of Brazil and South America increases. However, the pathogenesis of Oropouche infection is not well understood. Rodent models using intracerebral inoculation have been described, but no attempts to use a route that more closely resembles the natural route of infection have been published. This study was conducted to establish and characterize an experimental model of infection with Oropouche, using subcutaneous inoculation of hamsters, in order to contribute to the understanding of Oropouche pathogenesis. We have established an experimental model through subcutaneous inoculation of hamsters in order to study pathogenesis. Suckling mouse brain passaged Oropouche strain BeAn19991 was inoculated (106,25 TCID50/100?L) subcutaneously in the thigh of syrian hamsters 2-3 weeks old. Around day 3 animals developed disease characterized by lethargy, paralysis, chill-like shaking and ruffled fur. Animals were sacrificed on days 1, 3, 5, 8 and 11 post-inoculation or whenever found to be agonic. Brain, heart, liver, spleen, muscle and blood were harvested for virus titration, histology and Oropouche immunohistochemistry. Inflammatory infiltrate was present in the brain, mostly as perivascular cuffs, meninges and some in the heart. Oropouche titers were 107,23 TCID50/g of tissue in brain 106,67 TCID50/g in liver, and 106 TCID50/g in blood. Oropouche antigen was detected diffusely distributed in the liver in association with hepatocytes, and in neurons in several regions of the brain. This model reproduces systemic Oropouche infection and may become useful in pathogenesis studies, as well as to test antiviral drugs and possible vaccine candidates.
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Desenvolvimento de um modelo experimental de infecção subcutânea por vírus oropouche em hamster / Development of an experimental hamster model of subcutaneous infection by oropouche virusAlcir Humberto Rodrigues 22 October 2004 (has links)
O vírus Oropouche pertence à família Bunyaviridae, gênero Orthobunyavirus, sorogrupo Simbu, e é segunda causa mais freqüente de arbovirose febril no Brasil. Estima-se que mais de meio milhão de casos de febre do Oropouche tenham ocorrido no Brasil nos últimos 30 anos, havendo também ocorrências no Panamá, Peru, Suriname e Trinidad. Epidemias de febre do Oropouche têm sido registradas quase que exclusivamente na Amazônia. Porém, com o aquecimento global do planeta, desmatamentos e conseqüente redistribuição de insetos vetores e animais reservatórios, há risco de disseminação de vírus Oropouche para outras regiões do Brasil e da América do Sul. A patogenia da infecção por Oropouche não é bem entendida. Modelo em roedores, usando inoculação intracerebral, tem sido descrito, mas não com uso da via subcutânea, que mais se assemelha à rota natural da infecção. O objetivo deste trabalho foi estabelecer e caracterizar um modelo experimental de infecção com Oropouche, usando inoculação subcutânea em hamster, a fim de contribuir para o entendimento da patogenia do mesmo. Oropouche da linhagem BeAn19991, passado em cérebro de camundongo recém-nascido, foi inoculado (106,25 TCID50/100?L) por via subcutânea na coxa de hamsters sírios com 2-3 semanas de idade. Em torno de 3 dias alguns animais desenvolveram doença com sinais clínicos brandos caracterizados por: eriçamento dos pêlos e agressividade, outros com características mais graves: perda de peso, tremores sugestivos de calafrios, letargia e paralisia. Os animais foram sacrificados 1, 3, 5, 8 e 11 dias pós-inoculação ou quando eram encontrados em estado agônico. Baço, cérebro, coração, fígado, músculo e sangue foram colhidos para titulação viral, histologia e imunohistoquímica. Infiltrado inflamatório estava presente no cérebro, principalmente em torno dos vasos, meninges e discretamente no coração. O vírus Oropouche foi detectado no tecido cerebral (107,23 TCID50/g), no fígado (106,67TCID50/g) e no sangue (106 TCID50/g). Antígeno de Oropouche foi encontrado, difusamente no fígado, associado com hepatócitos e em neurônios de diversos locais do cérebro. Este modelo reproduz uma infecção sistêmica por Oropouche e pode tornar-se útil no estudo da patogênese, bem como para testar drogas antivirais e possíveis candidatos à vacina. / Oropouche virus belongs to the family Bunyaviridae, genus Orthobunyavirus, serogroup Simbu and is the second most frequent arboviral febrile illness in Brazil. There are estimates of more than half million cases of Oropouche fever in Brazil in the past 30 years, with cases registered in Panama, Peru, Suriname and Trinidad. Oropouche fever has been registered almost exclusively in the Amazon, but with the global warming, deforestation and the consequent redistribution of vectors and reservoir animals, the risk of Oropouche virus dissemination to others areas of Brazil and South America increases. However, the pathogenesis of Oropouche infection is not well understood. Rodent models using intracerebral inoculation have been described, but no attempts to use a route that more closely resembles the natural route of infection have been published. This study was conducted to establish and characterize an experimental model of infection with Oropouche, using subcutaneous inoculation of hamsters, in order to contribute to the understanding of Oropouche pathogenesis. We have established an experimental model through subcutaneous inoculation of hamsters in order to study pathogenesis. Suckling mouse brain passaged Oropouche strain BeAn19991 was inoculated (106,25 TCID50/100?L) subcutaneously in the thigh of syrian hamsters 2-3 weeks old. Around day 3 animals developed disease characterized by lethargy, paralysis, chill-like shaking and ruffled fur. Animals were sacrificed on days 1, 3, 5, 8 and 11 post-inoculation or whenever found to be agonic. Brain, heart, liver, spleen, muscle and blood were harvested for virus titration, histology and Oropouche immunohistochemistry. Inflammatory infiltrate was present in the brain, mostly as perivascular cuffs, meninges and some in the heart. Oropouche titers were 107,23 TCID50/g of tissue in brain 106,67 TCID50/g in liver, and 106 TCID50/g in blood. Oropouche antigen was detected diffusely distributed in the liver in association with hepatocytes, and in neurons in several regions of the brain. This model reproduces systemic Oropouche infection and may become useful in pathogenesis studies, as well as to test antiviral drugs and possible vaccine candidates.
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Caracterización molecular de los virus del grupo C (género Ortobunyavirus), aislados en el PerúCastillo Oré, Roger Melvin January 2018 (has links)
Los virus del grupo C (GRCV) son un complejo que pertenecen al género Orthobunyavirus, de la familia Peribunyaviridae (anteriormente denominado Bunyaviridae). Estos virus están asociados con enfermedades febriles en humanos que generalmente habitan en áreas tropicales y subtropicales de América del Sur y América Central. A pesar de que muchos GRCV han sido aislados de mosquitos, animales y seres humanos, los análisis genéticos de estos virus aún son limitados. En el Perú, el centro de investigaciones de enfermedades tropicales de la marina de los Estados Unidos (NAMRU-6) viene conduciendo desde los años noventa una vigilancia pasiva de las enfermedades febriles. Durante este tiempo, se lograron recuperar e identificar mediante la prueba de inmunofluorescencia 65 aislamientos de GRCV de pacientes febriles habitantes del norte y sur de la Amazonía peruana. Para caracterizar estos aislamientos, se secuenciaron una región de 500 pb del segmento S y una región de 750 pb de los segmentos M y L. El análisis de la secuencia de los aislamientos clínicos mostró identidades de nucleótidos que oscilaban entre el 68% y el 100% y las identidades de secuencia de aminoácidos deducidas que oscilaban entre 72% y 100%. Las secuencias se compararon con las siguientes cepas referenciales: virus Caraparu (CARV), virus Murutucu (MURV), virus Oriboca (ORIV), virus Marituba (MTBV), virus Apeu (APEUV) y virus Madrid (MADV). La comparación de secuencias de los segmentos L y S de las cepas clínicas con cepas referenciales mostró dos clados; clado I formado por aislamientos con alta correlación con CARV-MADV y clado II conformado por aislamientos con alta correlación con MURV, ORIV, APEUV y MTBV. Las secuencias del segmento M contiene algunos aislamientos de GRCV diferentes filogeneticamente a los de segmento L y S y su distribución filogenética está altamente relacionada con la microneutralización serológica. Estos resultados demuestran las relaciones genéticas y serológicas de los GRCV que circula en la Amazonía peruana y la divergencia genética de los GRCV en el norte de la Amazonía. / Tesis
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La structure et la fonction de la polymérase d'orthobunyavirus La Crosse / Structure and function of the La Crosse orthobunyavirus polymeraseGerlach, Piotr 29 June 2015 (has links)
Les virus ne sont rien de plus que des particules composées de lipides et/ou de protéines qui encapsulent de l'information génétique composée d'ARN ou d'ADN. Au cours du cycle viral, les virus entrent dans la cellule hôte où ils dupliquent leur génome, puis forment de nouvelles particules virales qui ressortiront de la cellule pour se diffuser. Alors que pour produire leurs protéines virales les virus détournent la machinerie cellulaire, ils utilisent pour la plupart leur propre polymérase spécifique pour répliquer leur génome.Les Bunyaviridae sont une grande famille des virus à ARN simple brin segmenté de polarité négative. Les Arenaviridae et les Orthomyxoviridae sont les deux autres familles de ce type. Certains bunyavirus provoquent des maladies humaines graves, comme des fièvres hémorragiques, des encéphalites et des méningites. D'autres infectent des plantes et animaux, posant une menace économique sérieuse en agronomie.Les ARN polymérases ARN-dépendante de virus à ARN négatif segmenté sont des machineries multi-fonctionnelles, capables de répliquer le génome viral et de le transcrire en ARNs messagers. La réplication est effectuée de novo, en utilisant un intermédiaire d'ARN complémentaire de polarité positive, alors que la transcription est initiée par vol de coiffe d'ARN cellulaire. Chaque segment du génome viral est recouvert par des nucléoprotéines et fixé à la polymérase par ses extrémités 3' et 5' conservées. Le complexe ARN viral/nucléoprotéines/polymérase forme une ribonucléoprotéine, qui est l'unité fonctionnelle de la réplication/transcription.L'objectif de mon projet de thèse était la caractérisation structurale et fonctionnelle de la polymérase du virus La Crosse, également nommée protéine L. Ce projet était basé sur l'hypothèse que toutes les polymérases de virus à ARN négatif segmenté pourraient partager une organisation et un mode d'action similaire. Lors de la première année de ma thèse, j'ai tenté de caractériser le domaine C-terminal, que nous supposions être responsable de la fixation de coiffe. Au cours de la deuxième année, j'ai étendu mes recherches sur l'étude de l'interaction entre les extrémités de l'ARN viral et la protéine L (protéine entière et construction tronquée en C-terminal). Confronté à des difficultés pour établir des tests de réplication et de transcription in vitro, j'ai poursuivi mes recherches en troisième année avec l'étude d'interactions et de co-cristallisation entre polymérase et ARN viral. Cela a finalement conduit au résultat principal de ma thèse - la détermination de la structure par cristallographie aux rayons X de la polymérase de virus de La Crosse en complexe avec les extrémités 3' et 5' de l‘ARN viral. La structure obtenue constitue une percée dans le domaine de bunyavirus. Elle révèle – à la différence de ce qui avait été initialement proposé – que les extrémités 3' et 5' de l'ARN se lient dans deux sites séparés et conservés. La liaison de l'extrémité 5' de l'ARN viral stabilise de façon allostérique l'un des motifs catalytiques du site actif de la polymérase. La structure révèle l'existence de deux tunnels séparés pour l'ARN produit et l'ARN matrice de sortir, ce qui suggère que le brin d'ARN naissant est séparé de la matrice et quitte la polymérase comme ARN simple brin. La proximité des tunnels d'entrée et de sortie de la matrice explique comment la polymérase peut se déplacer le long de l'ARN génomique avec une perturbation minimale de la ribonucléoprotéine.En parallèle de la structure de la polymérase du virus La Crosse, les structures des polymérases hétérotrimériques de la grippe A et B en complexe avec l'ARN viral ont également été déterminées au sein du groupe du Dr. Stephen Cusack. La comparaison de l'organisation des polymérases des deux familles et de la nature de leur liaison avec l'ARN viral montre que, malgré une homologie de séquence minimale, des similitudes structurelles sont frappantes. Cela suggère fortement la présence d'un ancêtre commun. / Viruses are not more than particles composed of lipids and/or proteins with genetic information – the viral RNA or DNA genome – embedded inside. In order to be efficient, once they enter the host cell they need to multiply this genetic information, package it into new viral particles and spread out from the cell. While in order to produce viral proteins viruses highjack cellular machinery, for replicating their genome most viruses use their own, specialized polymerases.Bunyaviridae is the largest viral family of segmented negative-strand RNA viruses, comprising also Arenaviridae and Orthomyxoviridae families. Some bunyaviruses are causative agents of severe human diseases including heamorrhagic fevers, encephalitis and meningitis. Others infect a variety of plants and animals posing a significant economic threat to the crop cultivation and cattle breeding.RNA-dependent RNA polymerases of segmented negative-strand RNA viruses are multifunctional machines, able to perform both de novo genome replication via positive-strand cRNA intermediate, and viral mRNA transcription using cap-snatched host-derived mRNA primer. Viral RNA genome of bunyaviruses, arenaviruses, and orthomyxoviruses is divided into three, two, and eight segments respectively. Each segment, coated by nucleoproteins and attached through its conserved 3′ and 5′ ends to the polymerase, constitutes an individual ribonucleoprotein particle – an autonomous RNA synthesis unit.The scope of the PhD project described in this thesis was the structural and functional characterization of the La Crosse orthobunyavirus polymerase, also named the L protein. It was based on the hypothesis that all polymerases of segmented negative-strand RNA viruses share a similar domain organization and mode of action. During the 1st year attempts were made to confirm and characterize a putative C-terminal cap-binding domain. During the 2nd year project was extended to study 3′ and 5′ vRNA ends interactions with the full length and C-terminus truncated L protein. Facing difficulties to establish replication and transcription assays in vitro, vRNA binding studies and co-crystallizastion were continued during the 3rd year. This finally led to the main achievement of the thesis – the x-ray structure of La Crosse orthobunyavirus polymerase in complex with vRNA. Obtained structure is a breakthrough in the bunyavirus field. It reveals – unlike it was initially believed – conserved, sequence specific and separate binding sites for 3′ and 5′ vRNA ends located within the polymerase. The 5′ vRNA end binding allosterically structures one of the conserved catalytic motifs within the polymerase active site. The structure sheds also some new light on bunyaviral replication and transcription mechanisms. There exist two distinct product and template exit channels, suggesting that the nascent RNA strand is separated from the template and leaves the polymerase as the single-strand RNA. Close proximity of the template entry and exit channels explains how the polymerase can translocate along the genomic template with minimal disruption of the RNP.In parallel to the La Crosse polymerase structure, structures of Influenza A and B heterotrimeric polymerases in complex with vRNA were also obtained in Stephen Cusack group. This gave a great opportunity to compare the domain organization and the nature of vRNA binding by viral polymerases belonging to Bunyaviriadae and Orthomyxoviridae families, and proved that despite minimal sequence homology the structural similarities are striking. This strongly suggests an evolutionary common ancestor, which can possibly be shared with non-segmented negative-strand RNA viruses as well.
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Orthobunyavirus de la République Centrafricaine détection, séquençage et analyse phylogénétique /Nakouné-Yandoko, Emmanuel. Finance, Chantal. Rihn, Bertrand January 2007 (has links) (PDF)
Thèse de doctorat : Biologie moléculaire : Nancy 1 : 2007. / Titre provenant de l'écran-titre.
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The Pathogenesis of Cache Valley Virus in the Ovine FetusRodrigues, Aline 2011 December 1900 (has links)
Cache Valley virus (CVV) induced malformations have been previously reproduced in ovine fetuses; however, no studies have established the CVV infection sequence of the cells targeted by the virus or the development of the antiviral response of the early, infected fetus that results in viral clearance before development of immunocompetency. To address these questions, ovine fetuses at 35 dg were inoculated in utero with CVV and euthanized at 7, 10, 14, 21 and 28 dpi. On postmortem examination arthrogryposis and oligohydramnios were observed in some infected fetuses. Morphologic studies showed necrosis in the central nervous system (CNS) and skeletal muscle of earlier infected fetuses and hydrocephalus, micromyelia and muscular loss in later infected fetuses. Using immunohistochemistry and in situ hybridization, intense CVV viral antigenic signal was detected in the brain, spinal cord, skeletal muscles and fetal membranes of infected fetuses. Viral signal decreased in targeted and infected tissues with the progression of the infection.
To determine specific cell types targeted by CVV in the CNS, indirect immunofluorescence was applied to sections of the CNS using a double labeling technique with antibodies against CVV together with antibodies against neurons, astrocytes and microglia. CVV viral antigen was shown within the cytoplasm of neurons in the brain and spinal cord. No viral signal was observed in microglial cells; however, infected animals had marked microgliosis.
The antiviral immune response in immature fetuses infected with CVV was evaluated. Gene expression associated with an innate, immune response was quantified by real-time, quantitative PCR. Upregulated genes in infected fetuses included ISG15, Mx1, Mx2, IL-1, IL-6, TNF-?, TLR-7 and TLR-8. The amount of Mx protein, an interferon stimulated GTPase capable of restricting growth of bunyaviruses, was elevated in the allantoic and amniotic fluid in infected fetuses. ISG15 protein expression was significantly increased in target tissues of infected animals. B lymphocytes and immunoglobulin-positive cells were detected in lymphoid tissues and in the meninges of infected animals. This demonstrated that the infected ovine fetus is able to stimulate an innate and adaptive immune response before immunocompetency that presumably contributes to viral clearance in infected animals.
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Uukuniemi virus-like particles : a model system for bunyaviral assembly /Överby, Anna K., January 2007 (has links)
Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.
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Rescue and characterisation of Oropouche virus in mammalian cell linesTilston-Lunel, Natasha Louise January 2016 (has links)
Oropouche virus (OROV) is a medically important orthobunyavirus, which causes frequent outbreaks of a febrile illness in the northern parts of Brazil. However, despite being the cause of an estimated half a million human infections since its first isolation in Trinidad in 1955, details of the molecular biology of this tripartite, negative-sense RNA virus remain limited. The work presented in this thesis has re-determined the nucleotide sequences of OROV strain BeAn19991 (GenBank accession numbers: L, KP052850; M, KP052851 and S, KP052852), and demonstrates that the S segment is significantly longer than the published sequence with an additional 204 nucleotides at the 3' end. Data analysis revealed that there is a critical nucleotide mismatch at position 9 within the base-paired terminal panhandle structure of each genomic segment. Using a combination of deep sequencing and Sanger sequencing the complete genome sequences of 10 field isolates of OROV were also determined for the first time, and led to the identification of a novel OROV reassortant virus. Phylogenetic analysis of these sequences and of published sequences showed that there are two genotypes of OROV, rather than the four genotypes previously proposed. Further work led to the development of a T7-RNA polymerase-driven minigenome and virus-like particle (VLP) production systems for OROV; the information from these was subsequently used to develop a reverse genetics system for OROV. Using reverse genetics, OROV mutants that lack either the non-structural proteins NSm or NSs were generated. In vitro growth properties of the OROV mutant lacking NSm were indistinguishable from the wild-type virus, but the NSs mutant was attenuated in growth, particularly in interferon (IFN) competent cells. Further work demonstrated NSs as a viral IFN antagonist and that it's C-terminus is required for this activity. Interestingly, OROV is more resistant to IFN-α treatment than Bunyamwera virus, but this is not related to its NSs protein. The development of a reverse genetics system for OROV, which is the main human pathogen within the Simbu serogroup of orthobunyaviruses, will prove invaluable for future studies designed to further investigate the molecular pathogenesis of this virus and in the development of attenuated vaccine strains.
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Klonierung der Genomsegmente des Oropouche-Virus und Charakterisierung der Interferon-antagonistischen Aktivität des S-Segment-kodierten NSs-Proteins / Cloning of the genome segments of Oropouche virus and characterization of the interferon-antagonistic activity of the S segment-encoded NSs protein.Keisers, Katharina 04 February 2015 (has links)
No description available.
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Structural studies of bunyavirus interferon antagonist proteinsBarski, Michał S. January 2016 (has links)
Bunyaviridae is one of the biggest known viral families, and includes many viruses of clinical and economic importance. The major virulence factor of most bunyaviruses is the non-structural protein (NSs). NSs is expressed early in infection and inhibits the innate immune response of the host by blocking several steps in the interferon induction and signalling pathways. Hence, NSs significantly contributes to the establishment of a successful viral infection and replication, persistent infection and the zoonotic capacity of bunyaviruses. Although functions and structures of many viral interferon antagonists are known, no structure of a bunyavirus NSs protein has been solved to date. This strongly limits our understanding of the role and the mechanism of interferon antagonism in this large virus family. In this work the first structure for a bunyavirus interferon antagonist, the core domain crystal structure of NSs from the Rift Valley fever virus (RVFV) is presented. RVFV is one of the most clinically significant members of the Bunyaviridae family, causing recurrent epidemics in Africa and Arabia, often featuring high-mortality haemorrhagic fevers. The structure shows a novel all-helical fold. The unique molecular packing of NSs in the crystal creates stable fibrillar networks, which could correspond to the characteristic fibrillation of NSs observed in vivo in the nuclei of RVFV infected cells. This first NSs structure might be a useful template for future structure-aided design of drugs that target the RVFV interferon antagonism. Attempts at characterising other bunyavirus NSs proteins of other genera were made, but were hampered by problems with obtaining sufficient amounts of soluble and folded protein. The approaches that proved unsuccessful for the solubilisation of these NSs proteins, however, should inform future experiments aimed at obtaining recombinant NSs for structural studies.
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