Dissertations / Theses on the topic 'Flaviviru'
To see the other types of publications on this topic, follow the link: Flaviviru.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Flaviviru.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Lequime, Sébastian. "Interactions flavivirus-moustiques : diversité et transmission." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066081/document.
Full textAbstract:
Les flavivirus sont des virus à ARN parmi lesquels certains sont des arbovirus transmis entre hôtes vertébrés par des vecteurs arthropodes, notamment des moustiques. L'interaction avec les moustiques est centrale dans la biologie des flavivirus par son influence sur leur diversité génétique et transmission, mais certains de ses aspects restent méconnus. Au cœur de cette thèse, des approches basées sur les « big data », générées par des technologies modernes ou par compilation de travaux plus anciens, ont éclairé d’un jour nouveau la complexité des relations moustique-flavivirus. En explorant des génomes de moustiques anophèles, nous avons identifié et caractérisé des éléments viraux endogènes d'origine flavivirale chez Anopheles sinensis et An. minimus, suggérant l'existence de flavivirus infectant les anophèles et révélant une facette insoupçonnée de leur diversité. Par ailleurs, nous avons exploré, par séquençage haut-débit, la fine interaction entre le génotype du moustique Aedes aegypti et la diversité intra-hôte du virus de la dengue-1. Nos résultats montrent un fort effet de la dérive génétique lors de l'infection initiale, diminuant l'importance relative de la sélection naturelle, et une modulation de la diversité génétique intra-hôte du virus par le génotype du moustique. Enfin, nous avons compilé la littérature sur la transmission verticale des arbovirus chez les moustiques, c'est-à-dire de la femelle infectée à sa descendance, afin d'identifier des facteurs techniques et biologiques sous-jacents. Nos résultats améliorent la compréhension de ce mode de transmission et des stratégies employées par les arbovirus pour persister dans l’environnementFlaviviruses are RNA virus among which some are arboviruses transmitted between vertebrate hosts and arthropod vectors, like mosquitoes. The interaction with mosquitoes is key in the biology of flaviviruses because it influences their genetic diversity and transmission. However, some aspects however are still poorly understood. At the heart of the work presented in this dissertation, strategies based on ‘big data’, both by taking advantage of modern technologies and by compiling older literature, highlighted new aspects of the complex relationships between flaviviruses and mosquitoes. While exploring Anopheles mosquito genomes, we identified and characterized endogenous viral elements of flaviviral origin in Anopheles sinensis and An. minimus, which supports the existence of flaviviruses infecting Anopheles mosquitoes and highlights new aspected of their diversity. Besides, we explored, by deep sequencing, the fine-tuned interaction between genotypes of the mosquito Aedes aegypti and the intra-host diversity of dengue virus 1. Our results showed a strong effect of genetic drift during initial infection, reducing the relative importance of natural selection, and a modulation of the intra-host viral genetic diversity by the mosquito genotype. Finally, we assembled the litterature on arbovirus vertical transmission in the mosquito vector, i.e. from an infected female to her offspring, in order to identify underlying technical and biological predictors. Our results increase our understanding of this transmission mode and the strategies employed by arboviruses to persist in their environment
2
Khou, Cécile. "Etude du neurotropisme des Flavivirus neuropathogènes." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC305/document.
Full textAbstract:
Les Flavivirus neuropathogènes, tels que le virus de l’encéphalite japonaise (JEV), le virus West Nile (WNV), le virus de la fièvre jaune (YFV) et le virus Zika (ZIKV) causent des maladies neurologiques. Ces maladies sont dues à une infection des cellules du système nerveux central (CNS) par ces virus. Le CNS est un organe privilégié, isolé des agents pathogènes par une barrière entre le sang et le cerveau, appelée barrière hémato-encéphalique (BBB). Les Flavivirus neuropathogènes capables de traverser cette BBB afin d’atteindre leurs cellules cibles, localisées dans le CNS, sont neuroinvasifs. Le but de cette étude est de comprendre les mécanismes cellulaires permettant aux Flavivirus de traverser la BBB et les effets de l’infection par les virus ZIKV et WNV des cellules du CNS sur le développement de celles-ci.Le YFV est un virus hépatotrope, infectant majoritairement le foie et les reins. Deux vaccins vivants atténués dirigés contre le YFV, le vaccin FNV (pour French Neurotropic Virus) et le vaccin 17D, ont été obtenus empiriquement par passages successifs de souches virulentes de YFV sur cerveaux de souriceaux. Ces vaccins ne causent plus de maladies touchant les reins et le foie, mais peuvent parfois causer des encéphalites post-vaccinales. Ces cas d’encéphalites démontrent que ces souches vaccinales sont devenues neurovirulentes mais aussi neuroinvasives car les virus ont pu franchir la BBB. A cause d’une incidence trop élevée d’encéphalites post-vaccinales par rapport au vaccin 17D, le vaccin FNV a été retiré du marché dans les années 1980.Le JEV est un virus neurotrope, causant des encéphalites graves en Asie du Sud-Est. A ce jour, il existe un vaccin vivant atténué, le JEV SA14-14-2, obtenu empiriquement par passages successifs d’une souche virulente sur cellules de hamster. Ce vaccin est moins neurovirulent et moins neuroinvasif que les souches virulentes de JEV en modèle de souris, et protège contre des infections humaines par le JEV. Cependant, des cas d’encéphalites ont été rapportés après injection de ce vaccin. Il apparait donc que, dans certains cas, la souche vaccinale JEV SA14-14-2 est capable de traverser la BBB et d’infecter les cellules neuronales. Les dernières épidémies à virus ZIKV en Polynésie Française et en Amérique du Sud ont induit une augmentation de cas de malformations congénitales dans les zones touchées. Cela a soulevé de nouvelles questions quant à la capacité d’un Flavivirus à provoquer des malformations congénitales du CNS. Dans cette étude, nous avons identifié les mécanismes cellulaires permettant aux Flavivirus de traverser la BBB et les effets de l’infection par les virus ZIKV et WNV des cellules du CNS sur le développement de celles-ci.Nous avons utilisé deux systèmes in vitro permettant d’étudier le développement du CNS et la neuroinvasion des Flavivirus. Un premier système consiste en l’infection de coupes de cerveaux d’embryon de souris. En utilisant ce système, nous avons montré que le ZIKV a un tropisme préférentiel pour les cellules progénitrices de neurones, alors que le WNV a un tropisme préférentiel pour les neurones. Nous avons également montré que l’infection des progéniteurs neuronaux par le ZIKV induit un arrêt de la mitose cellulaire, alors que l’infection par le WNV n’a aucun effet sur la mitose. L’étude sur l’effet apoptotique de l’infection par les deux virus WNV et ZIKV n’a montré aucune différence entre les deux virus à des temps précoces d’infection.Un deuxième système a été mis au point pour l’étude de la neuroinvasion par les Flavivirus neuropathogènes. Ce système est composé de cellules endothéliales hCMEC/D3 pouvant former des jonctions serrées. Ces cellules ont été cultivées sur filtres d’insert de puits de culture cellulaire Transwell, placés au-dessus de cellules neuronales humaines. A l’aide de ce système, nous avons comparé la capacité à traverser la BBB de plusieurs FlavivirusNeuropathogenic Flaviviruses, such as Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV) and Zika virus (ZIKV), cause neurological diseases. These diseases are due to viral infection of central nervous system (CNS) cells. The CNS is a privileged organ, isolated from pathogenic agents by a barrier between the blood and the barrier, called the blood-brain barrier (BBB). Neuropathogenic Flaviviruses which can cross this BBB in order to reach their target cells in the CNS, are neuroinvasive. This study aims at understanding the cellular mechanisms by which YFV and JEV Flaviviruses cross the BBB and the effects of viral infection by WNV and ZIKV of the CNS cells during neocortex development.YFV is a hepatrotopic virus, which mostly infects the liver and the kidneys. The two live-attenuated vaccines against YFV, the FNV (for French Neurotropic Virus) vaccine and the 17D vaccine, were obtained empirically by several passages in suckling mouse brain of YFV virulent strains. These vaccines do not cause any disease targeting the liver or the kidneys, but can sometimes cause post-vaccine encephalitis. These encephalitis cases suggest that the vaccine strains have become neurovirulent and neuroinvasive. Due to high risks of post-vaccine encephalitis, the FNV vaccine use was discontinued in the 1980s.JEV is a neurotropic virus, causing acute encephalitis in South East Asia. To date, there is a live-attenuated vaccine against JEV, the JEV SA14-14-2 vaccine, which was obtained empirically by several passages in primary hamster kidney cells. This vaccine is less neurovirulent and less neuroinvasive than JEV virulent strains in mouse model, and it protects against JEV infections. However, some cases of post-vaccine encephalitis were reported. It thus seems that, in some cases, the vaccine strain JEV SA14-14-2 is able to cross the BBB and infect neuronal cells.The recent ZIKV epidemics in French Polynesia and South America were linked to an increase in the number of congenital malformations, rising questions regarding the capacity of a Flavivirus to induce CNS congenital malformations.In this study, we have identified cellular mechanisms involved in Flavivirus neuroinvasion and studied the effect of ZIKV and WNV infection of neuronal cells under development.To study CNS development, we have infected mouse embryos brain slices. We were able to show that ZIKV has a preferential tropism for neuronal progenitors, whereas WNV has a preferential tropism for neuronal cells. We also show that infection of neuronal progenitors by ZIKV impairs the cell life cycle, whereas no effect on the cell life cycle was observed for WNV-infected cells. Studies on apoptosis induction did not show any difference between both viruses at early time points of infection.To study Flavivirus neuroinvasion, we have used an in vitro model of BBB composed of human endothelial hCMEC/D3 cells that can form tight junctions. These cells were cultivated on Transwell inserts and placed above human neuronal cells. Using this system, we show that YFV FNV cross the BBB more efficiently than YFV 17D, suggesting that YFV FNV is more neuroinvasive than YFV 17D. This observation can explain the higher post-vaccine encephalitis risks associated with YFV FNV vaccine compared to YFV 17D vaccine. We also confirmed that JEV SA14-14-2 vaccine strain is less neuroinvasive than JEV RP9.We also examined how JEV crosses the BBB and the endothelial cell response following JEV treatment. We show that both JEV RP9 and SA14-14-2 are able to cross the BBB without infecting its endothelial cells and without disrupting the BBB. Preliminary results suggest that JEV RP9, but not JEV SA14-14-2, crosses the BBB by dynamin-dependant transcytosis. Transcriptomic analysis of endothelial cells treated by either virus show slight, but significant, differences in regulation of genes implicated in several pathways associated with CNS diseases
3
Silveira, Roberta Maraninchi. "Localização subcelular do vírus da Zika durante a infecção em células humanas." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/17/17136/tde-13092018-105525/.
Full textAbstract:
O vírus da Zika (ZIKV) é um arbovírus emergente da família Flaviviridae, do gênero Flavivirus transmitido por mosquitos Aedes. Apesar da sua importância emergente na saúde pública, ainda pouco se conhece sobre os mecanismos moleculares envolvidos no ciclo replicativo do ZIKV em célula humanas. Assim, o objetivo geral deste estudo foi caracterizar a distribuição subcelular do ZIKV na célula hospedeira e elucidar fatores celulares que regulam o tráfego intracelular de proteínas envolvidos nesses processos. Mais especificamente, determinar os compartimentos celulares que servem de plataforma de montagem para o ZIKV. Além disso, também verificar se o funcionamento da maquinaria Endosomal Sorting Complexes Required for Transport (ESCRT) é requerido no ciclo replicativo de ZIKV. Para identificar a localização subcelular do ZIKV, foram utilizados diferentes marcadores celulares, e, de acordo com os resultados, foi demonstrado que com 3 horas pós infecção (h. p. i.) ocorre colocalização de proteínas do ZIKV com um marcador de endossomo primário, enquanto que com 15h p.i. já é possível detectar proteínas virais no Retículo Endoplasmático (RE). Subsequentemente, com 27h p.i. o ZIKV direciona-se para o complexo de Golgi. Juntos, esses resultados indicam o direcionamento do ZIKV através da via secretória ao longo do tempo. Além disso, foi testado o envolvimento da maquinaria dos ESCRTs por meio do silenciamento da expressão da proteína TSG101 de ESCRT-I em células infectadas com ZIKV. Os resultados obtidos, sugerem que ESCRT-I tem participação importante na replicação do ZIKV, ocorrendo a diminuição dos títulos virais quando TSG101 é depletada da célula. Em conjunto, os resultados permitem concluir que ao longo da infecção o ZIKV encontrase associado aos compartimentos da via secretória inicial (RE e complexo de Golgi), e que a proteína TSG101 de ESCRT-I exerce papel importante na replicação viral. Sendo assim, esse estudo possibilitou um melhor entendimento sobre a dinâmica de replicação do ZIKV em células humanas.Zika virus (ZIKV) is an arbovirus of the Flaviviridae family, of the genus Flavivirus that is transmitted by Aedes mosquitoes. Despite its emerging importance in public health, little is known about the molecular mechanisms involved in the replicative cycle of ZIKV in human cells. Thus, the general objective of this study was to characterize the subcellular distribution of the ZIKV in the host cell and to elucidate cellular factors that regulate the intracellular trafficking of proteins involved in these processes. More specifically, to determine the cellular compartments that serve as assembly platforms for the ZIKV. In addition, the study aimed to verify if the functioning of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is required in the replicative cycle of ZIKV. In order to identify the subcellular localization of ZIKV, different intracellular markers were used, and, according to the results, it was demonstrated that at 3 hours post infection (h. p. i.) ZIKV proteins colocalize with an early endosome marker, whereas within 15h p.i. it is already possible to detect newlysynthesized viral proteins in the endoplasmic reticulum (ER). Subsequently, within 27h p.i., the ZIKV is directed to the Golgi complex. Together, these results delineate the targeting of ZIKV proteins through the secretory pathway over time. In addition, the involvement of the ESCRT machinery was tested by knocking down the expression of ESCRT-I protein TSG101 in ZIKV-infected cells. The results obtained suggest that ESCRT-I plays an important role in ZIKV replication, with viral titers decreasing when TSG101 levels are depleted in the cell. Together, the results allow us to conclude that ZIKV is associated with the initial secretory pathways (RE and Golgi complex) throughout the infection, and that the ESCRT-I TSG101 protein plays an important role in viral replication. Thus, this study contributes to a better understanding of the dynamics of ZIKV replication in human cells.
4
Grard, Gilda. "Génomique et évolution des flavivirus transmis par les tiques et découverte d'un nouveau lignage du genre flavivirus." Aix-Marseille 2, 2006. http://www.theses.fr/2006AIX20679.
Full text
5
Gollins, S. W. "Mechanisms of flavivirus neutralization and cellular infection." Thesis, University of Oxford, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355752.
Full text
6
Dayaraj, Cecilia. "Molecular and immunological studies on flavivirus virulence." Thesis, University of Oxford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.279888.
Full text
7
Carney, Jennifer. "Viral Determinants of Flavivirus Neurotropism in Humans." Thesis, University of Liverpool, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526956.
Full text
8
Pacca, Carolina Colombelli. "Screening de novos antivirais inibidores de flavivirus." Faculdade de Medicina de São José do Rio Preto, 2013. http://bdtd.famerp.br/handle/tede/201.
Full textAbstract:
Made available in DSpace on 2016-01-26T12:51:48Z (GMT). No. of bitstreams: 1
carolinacolombellipacca_tese.pdf: 2227429 bytes, checksum: 4bcc12b8c06f6322170e32bfccfc8fa1 (MD5)
Previous issue date: 2013-11-01Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Introduction. Arboviruses, arthropod-borne viruses, are frequently associated with human outbreaks and represent a serious health problem. The genus Flavivirus, which includes both the Yellow Fever Virus (YFV) and Saint Louis Encephalitis Virus (SLEV), are important pathogens that result in high morbidity and mortality rates worldwide. In Brazil, YFV has a sylvatic cycle and occurs annually, despite the efficiency of the vaccine. Saint Louis Encephalitis is an infectious illness that can cause acute fever caused by SLEV, which is widely distributed in the Americas. The emergence of SLEV became a serious concern after the first related outbreak in Brazil in 2006, in the city of Sao Jose do Rio Preto. There is no specific antiviral drug for these viruses, only supporting treatment that can alleviate the symptoms and prevent complications. The need to develop effective and safe antiviral drugs is indispensable for the treatment of these infections. Objective. The aim of this work was to identify new possible antiviral drugs against the arboviruses that can cause acute fever and encephalitis (YFV and SLEV) and to evaluate the capacity of inhibition of these compounds in ABR mice. Material and Methods. Plaque reduction assay, flow citometry, immunofluorescence and cellular viability were used to test the compounds in vitro. ABR mice were inoculated with YFV, and the biological samples were tested for the presence of the virus through the use of plaque reduction assay and qPCR. Neutralization assay was also performed. Results. Treated cells showed efficient inhibition of viral replication at concentrations that presented minimal toxicity to the cells. The assays showed that ftalyl-tiazole and fenoxytiosemicarbazone were more effective, and that they reduced viral replication by 60% and 75% for YFV and SLEV, respectively. The analysis also revealed that the ABR mice inoculated with YFV had histopathological alterations in the liver; however, the samples did not present viral title. Neutralization assay showed a high concentration of antibodies in the serum. Conclusion. The inhibitions of viral replication were confirmed through the use of some assays in vitro, and the effectiveness of the selected compounds show that they are an option in the treatment of these viruses. More detailed studies are needed to determine the mechanism of action of these molecules. The mice were found to have histopathological alterations, which indicates viral infection; however, they also presented with high concentrations of antibodies. More studies about animal models are necessary to make in vivo experiments.
Introdução: Os arbovírus, vírus transmitidos por artrópodes, são freqüentemente associadas a surtos em seres humanos e representam um problema sério de saúde pública. Os vírus pertencentes ao gênero Flavivirus, tais como vírus da Febre Amarela (YFV) e vírus da Encefalite de Saint Louis (SLEV), são importantes patógenos que podem causar alta taxa de morbidade e mortalidade no mundo. No Brasil, YFV é mantido em ciclo silvestre notificados anualmente, a despeito da segurança e eficiência da vacina. A encefalite de Saint Louis é uma doença infecciosa febril aguda causada pelo SLEV amplamente distribuída nas Américas. A emergência do SLEV passou a ser um fato preocupante no Brasil a partir da constatação do primeiro surto no país em 2006, na cidade de São Jose do Rio Preto. Não existe tratamento específico para estas viroses, somente tratamento de suporte para ajudar a aliviar os sintomas e prevenir complicações. Desta forma, há uma grande necessidade de que sejam desenvolvidos antivirais efetivos e seguros para o tratamento destas infecções. Objetivos: O objetivo deste trabalho foi identificar potenciais compostos antivirais contra os arbovírus causadores de doença febril aguda e encefalites (YFV e SLEV) in vitro e avaliar a capacidade de inibição da replicação viral dos compostos in vivo em camundongos ABR. Materiais e Métodos: Para tanto, foram realizados ensaios de redução de placas, citometria de fluxo, imunofluorescencia, bem como testes de viabilidade celular para as analises in vitro. Além disto, camundongos ABR foram inoculados com YFV e seus materiais biológicos testados para a presença de partículas virais por ensaio de redução de placas e qPCR. Adicionalmente, foi realizado ensaio de neutralização do soro dos animais. Resultados: Celulas tratadas com os compostos mostraram eficiente inibição da replicação viral em concentrações que apresentam baixa citotoxicidade. Os ensaios mostraram que derivados de ftalyl-tiazole e fenoxytiosemicarbazone foram os mais eficazes na ação antiviral, apresentando redução de 60% e 75% para YFV e SLEV, respectivamente. Camundongos ABR inoculados com YFV apresentaram alterações histológicas no fígado, entretanto, não foi constatado título viral nas amostras testadas. O ensaio de neutralização mostra altas concentrações de anticorpos no soro dos animais. Conclusões: A inibição da replicação foi comprovada por vários ensaios in vitro evidenciando as moléculas como potentes alternativas para o tratamento dos vírus. Mais estudos são necessários para a determinação do mecanismo de ação destas moléculas. Os camundongos apresentaram alterações histopatológicas sendo um indicativo de infecção, entretanto, apresentam altas taxas de anticorpos. Mais estudos sobre modelo animal são necessários para a realização de ensaios in vivo.
9
Singethan, Katrin. "Untersuchungen zur Inhibition Paramyxo- und Flavivirus-induzierter Membranfusion." kostenfrei, 2009. http://www.opus-bayern.de/uni-wuerzburg/volltexte/2009/3634/.
Full text
10
Cunha, Mariana Sequetin. "Validação e uso de transcrição reversa seguida da reação em cadeia pela polimerase em tempo real (RT-qPCR) para a vigilância e diagnóstico de flavivírus transmitidos por mosquitos circulantes no Brasil." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/10/10133/tde-16102018-113026/.
Full textAbstract:
Os flavivírus são considerados uma séria ameaça à saúde pública em diversas partes do mundo, pois muitos são agentes altamente patogênicos a seres humanos e animais, tais como os vírus da febre amarela, vírus do Oeste do Nilo, vírus da encefalite japonesa e vírus da dengue, capazes de causar encefalites ou febres hemorrágicas em seus hospedeiros. Muitos deles têm avançado a diferentes regiões geográficas onde sua circulação não havia sido detectada previamente, causando novos surtos. O diagnóstico clínico destas infecções é, muitas vezes, difícil, devido ao grande número de sintomas apresentados, que podem se confundir com outras enfermidades de diferentes causas etiológicas. Os principais métodos diretos utilizados atualmente no Brasil para detecção destes vírus são a inoculação intracerebral em camundongos neonatos, inoculação em culturas de células e RTPCR específica. O presente trabalho tem como objetivos avaliar a sensibilidade e validar a detecção dos vírus pertencentes ao gênero Flavivirus circulantes no Brasil por meio de uma reação single de RT-PCR em tempo real e implementá-la, tanto na rotina diagnóstica de casos com suspeita de arbovirose como na pesquisa de amostras de campo para monitoramento viral. Amostras dos flavivírus padrões da Febre Amarela, Bussuquara, Iguape, Ilheus, Encefalite de Saint Louis, Cacipacore e Zika foram quantificados por titulação em unidades formadoras de placa (UFP) ou TCID50 para se avaliar os limites de detecção para cada um deles por RT-qPCR que detecta o gênero Flavivirus. Os limites encontrados variaram de 0,01 UFP, para o vírus Ilheus, a 1 UFP, para os vírus da Febre Amarela e Iguape, e 1x101,6 TCID50/100µL para o vírus Bussuquara. Além disso, o presente trabalho foi capaz de identificar, após sequenciamento de cDNA gerado, os vírus Zika, isolado de um paciente febril, e os vírus Ilheus e Iguape, isolados a partir de diferentes espécies de Culicídeos, após uma única reação de RT-qPCR, e um possível novo flavivírus específico de insetos, isolado de mosquitos Aedes coletados em Guapiaçu, São Paulo. Não houve sinal de amplificação para os Alphavirus Mayaro e Chikungunya. O presente protocolo mostrou-se com alta sensibilidade e especificidade, podendo dessa forma ser utilizado para o diagnóstico diferencial dos diferentes flavivírus que ocorrem no Brasil, bem como para estudos de monitoramento viral em animais sentinelas e vetores, colaborando dessa forma com a saúde pública. Pode-se, ainda, detectar possíveis novos vírus específicos de artrópodesFlaviviruses are considered a serious threat to public health in many parts of the world, as many are highly pathogenic to humans and animals, such as Yellow Fever virus, West Nile virus, Japanese encephalitis virus and dengue virus, which are capable of causing encephalitis or hemorrhagic fever in their hosts. Many of them have spread to different geographic regions where their circulation had not been detected previously, causing new outbreaks. Diagnosis of these infections is often difficult, due to the large number of symptoms presented, which can be confused with other diseases of different etiological causes. The main direct methods currently used in Brazil for detecting these viruses are intracerebral inoculation in neonatal mice, inoculation in cell cultures and specific RT-PCR. The present work aims to evaluate the sensitivity and validate the detection of viruses belonging to the genus Flavivirus circulating in Brazil through a single real-time RT-PCR reaction and to implement it, both in the diagnostic routine of cases with arbovirus suspicions and in field samples for viral monitoring. Samples of the standard flaviviruses Yellow Fever, Bussuquara, Iguape, Ilheus, Saint Louis Encephalitis, Cacipacore and Zika were quantified by titration by plaque forming units (UFP) or TCID50 to evaluate the detection limits for each of them by RT- qPCR that detects genus Flavivirus. The limits found ranged from 0.01 PFU for Ilheus virus to 1 PFU for Yellow Fever and Iguape viruses and 1x101.6 TCID50 / 100L for the Bussuquara virus. In addition, the present work was able to identify, after cDNA sequencing Zika virus, isolated from a febrile patient, and both Ilheus and Iguape viruses, isolated from different species of Culicidae, and a possible new insect-specific flavivirus, isolated from Aedes mosquitoes collected in Guapiaçu, São Paulo. The Alphaviruses Mayaro and Chikungunya were not amplified. The present protocol shoed high sensitivity and specificity, and therefore it may may be used for the differential diagnosis of the different flaviviruses that occur in Brazil, as well as for viral monitoring studies in sentinel animals and vectors, thus collaborating with public health. It is also possible to detect new flavivirus that are arthopode-specific.
11
Silva, Jaqueline Raymondi. "Pesquisa de infecções por Flavivírus da encefalite de Saint Louis, Rocio e Oeste do Nilo em cavalos, por inquérito sorológico e isolamento viral." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/17/17147/tde-23062010-150025/.
Full textAbstract:
Arboviroses são grave problema de saúde pública no Brasil e destas destacam-se aquelas causadas por Flavivírus, dos quais onze já foram descritos no Brasil. Destes, dois importantes em saúde pública, e que pertencem ao sorocomplexo da Encefalite Japonesa, são o vírus da encefalite de Saint Louis (SLEV) e o Rocio (ROCV). O vírus Oeste do Nilo (WNV), introduzido no continente americano em 1999, ainda não foi detectado no Brasil, contudo sua introdução é muito provável. Neste estudo, avaliou-se a circulação de SLEV, ROCV e WNV em cavalos, por tentativas de isolamento viral e inquérito soro-epidemiológico. As tentativas de isolamento viral, em 11 tecidos cerebrais de cavalos do estado da Paraíba, resultaram negativas. O inquérito sorológico, por IgG-ELISA tendo como antígeno peptídeos recombinantes do domínio III da proteína de envelope de SLEV, WNV e ROCV, foi utilizado em 753 soros de animais dos estados de São Paulo, Mato Grosso do Sul, Minas Gerais, Rio de Janeiro e Paraíba. Soros de 271 cavalos foram positivos para SLEV (35,98%), 254 para WNV (33,73%) e 144 para ROCV (19,12%). Portanto, o ELISA mostrou-se adequado, diagnosticando infecções prévias por estes Flavivírus. Também, observou-se intensa circulação destes vírus infectando cavalos nos locais de estudo. Ainda, obteve-se, pela primeira vez, evidencia de que WNV foi introduzido no Brasil e encontra-se a infectar cavalos nos estados pesquisados exceto Minas Gerais. Finalmente, o inquérito sorológico em cavalos mostrou-se uma abordagem adequada à vigilância das flaviviroses por SLEV, ROCV e WNV no Brasil.Arboviruses are a serious public health problem in Brazil and, from these, the most important are caused by Flavivirus. Eleven Flavivirus have been described in Brazil. Of these, Saint Louis Encephalitis Virus (SLEV) and Rocio Virus (ROCV) are major public health problems and belongs to the Japanese Encephalitis Serocomplex. West Nile Virus (WNV), introduced in the American continent in 1999, has not yet been detected in Brazil. In this study, it was evaluated the circulation of SLEV, WNV and ROCV in horses, by viral isolation attempts and a serosurvey. Viral isolation attempts were performed in 11 brain tissues of horses from Paraíba state with negative results. It was used for the serosurvey, an IgG-ELISA with recombinant peptides of domain III of SLEV, WNV and ROCV envelope protein as antigens. Sera from 753 animals from São Paulo, Mato Grosso do Sul, Minas Gerais, Rio de Janeiro and Paraíba states were tested, and 271 of them were positive for SLEV (35,98%), 254 for WNV (33,73%) and 144 for ROCV (19,12%). Therefore, this ELISA has been a suitable approach for diagnosis of ancient infections by these viruses. An intense circulation of flaviviruses infecting horses was observed in the study sites. Besides, it was found, for the first time, the presence of WNV in Brazil, infecting horses from all the studied states with the only exception of Minas Gerais. Finally, serosurvey in horses proved to be an appropriate approach for surveillance of Flavivirus infections by SLEV, WNV and ROCV.
12
Araujo, Marina Reus Tassi de. "Expressão de proteínas recombinantes de vírus do gênero Flavivirus." reponame:Repositório Institucional da UFPR, 2010. http://hdl.handle.net/1884/22380.
Full text
13
Gaunt, Michael W. "The epidemiology of louping ill virus and flavivirus evolution." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363978.
Full text
14
Lopes, Samuel Franco. "Identificação de Flavivirus em aves silvestres da Amazônia Central." Universidade Federal do Amazonas, 2011. http://tede.ufam.edu.br/handle/tede/4594.
Full textAbstract:
Submitted by Geyciane Santos (geyciane_thamires@hotmail.com) on 2015-08-24T14:40:04Z
No. of bitstreams: 1
Dissertação - Samuel Franco Lopes.pdf: 9746386 bytes, checksum: 9949bc4c8abaac65b768f7a3f4108131 (MD5)Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-08-26T20:25:24Z (GMT) No. of bitstreams: 1 Dissertação - Samuel Franco Lopes.pdf: 9746386 bytes, checksum: 9949bc4c8abaac65b768f7a3f4108131 (MD5)
Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-08-26T20:28:31Z (GMT) No. of bitstreams: 1 Dissertação - Samuel Franco Lopes.pdf: 9746386 bytes, checksum: 9949bc4c8abaac65b768f7a3f4108131 (MD5)
Made available in DSpace on 2015-08-26T20:28:31Z (GMT). No. of bitstreams: 1 Dissertação - Samuel Franco Lopes.pdf: 9746386 bytes, checksum: 9949bc4c8abaac65b768f7a3f4108131 (MD5) Previous issue date: 2011-11-24
CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The Amazon region has the highest biodiversity on the planet, as well as the largest number of arboviruses isolated, mainly due to the great diversity of species of wild vertebrates and hematophagous arthropods. Among the arboviruses, Flavivirus stand out both for producing the highest number of infections and human diseases, such as the severity of these diseases. Moreover, it is known that birds act as reservoir of some poorly studied Flavivirus, such as: Ilheus, Saint Louis Encephalits, Rocio, Cacipacoré and Bussuquara. Despite intensive studies in the brazilian amazon, especially in certain areas of the state of Para, few epidemiological information about most of these viruses were obtained. In this study, a specie of Flavivirus has been identified in whole blood samples from wild birds, captured in-situ (Alter do Chão/PA) and ex-situ (Manaus) by polymerase chain reaction preceded by reverse transcription (RT- PCR) followed by Multiplex-Nested PCR (MN-PCR) tests for species-specific identification. Among the 189 samples, 7(4,23%) were suggestive of Ilheus virus. The diagnostic technique used was effective in identifying the genus Flavivirus in samples of wild birds presenting itself as practical, fast and secure for the identification of brazilian arboviruses. The circulation of enzootic viruses both in captive and free-living birds, increase the role of birds as host in the cycles of transmission of zoonoses.
A Amazônia apresenta os maiores índices de biodiversidade do planeta, assim como o maior número de arbovírus isolados, principalmente em função da grande diversidade de espécies de artrópodes hematófagos e vertebrados silvestres. Entre os arbovírus, destacam-se os Flavivírus tanto por produzirem o maior número de infecções e doenças humanas, como pela gravidade destas doenças. Além disso, sabe-se que as aves atuam como reservatório de alguns Flavivirus pouco estudados como: Ilhéus, Saint Louis, Rocio, Cacipacoré e Bussuquara. Apesar de estudos intensivos realizados na Amazônia brasileira, sobretudo em certas áreas do Estado do Pará, poucas informações epidemiológicas a respeito da maioria desses vírus foram obtidas. Nesse estudo, foi identificada uma espécie de Flavivirus em amostras de sangue total de aves silvestres, capturadas in-situ (Alter do chão/PA) e ex-situ (Manaus/AM) através da reação em cadeia de polimerase conjugada a transcrição reversa (RT-PCR) seguida por multiplex nested-PCR (MN-PCR) para testes de identificação espécie-específicos. Entre as 189 amostras analisadas, foram encontrados 7 (4,23%) amplicons sugestivos do vírus Ilhéus entre as amostras positivas. A técnica de diagnóstico utilizada mostrou-se eficaz para a identificação do gênero Flavivírus em amostras de aves silvestres apresentando-se como uma alternativa prática, rápida e segura para a identificação de arboviroses brasileiras. A circulação enzoótica dos vírus tanto em aves de cativeiro como de vida livre reforçam o papel das aves como hospedeiro nos ciclos de transmissão destas zoonoses.
15
Moureau, Grégory. "Génomique des Flavivirus : Contribution à l'analyse taxonomique et phylogénique." Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM5002.
Full textAbstract:
Les virus à ARN - à l'exception des rétrovirus - représentent plus de 200 pathogènes humains et/ou vétérinaires majeurs. Ils sont pour la plupart considérés comme émergents, durant les dernières années ils ont été retrouvés au-delà de leur territoire d'origine, dans de nouvelles régions du monde. Les plus connus de ces virus sont le virus du West Nile, le virus chikungunya, la grippe, le coronavirus SRAS, l'entérovirus 71 (agent responsable de la fièvre aphteuse), les virus de la dengue, l'hépatite C virus, le virus de la fièvre hémorragique de Crimée Congo, le virus de la vallée du Rift, l'encéphalite japonaise et plusieurs entérovirus humains. En terme de mortalité et morbidité ils sont à l'origine de plus 100 millions de cas par an et la menace a tendance à augmenter. Ces statistiques sont démoralisantes quand on considère les immenses progrès de la médecine et de la science durant ces dernières dizaines d'années. Les recherches présentées dans ma thèse portent sur le genre Flavivirus, au sein duquel on retrouve le virus de la fièvre jaune, un pathogène humain responsable d'épidémies majeures en Afrique et en Amérique latine durant les 300 à 400 dernières années. Ce virus est toujours responsable d'épidémies majeures en Afrique malgré un vaccin très bien toléré et des plus efficaces. On assiste au même scénario avec le virus de l'encéphalite japonaise en IndeWithout including the retroviruses, there are in excess of 200 RNA viruses that are recognised human and/or animal pathogens, many of which are considered to be emerging viruses because during recent years they have dispersed beyond their original territories causing epidemics in new regions of the World. The more well known of these emerging viruses include, West Nile virus, chikungunya virus, influenza virus, SARS coronavirus, EV71 virus (the aetiological agent of hand foot and mouth disease), dengue virus, hepatitis C virus, Crimean Congo haemorrhagic fever virus, Rift Valley fever virus, Japanese encephalitis virus and many human enteroviruses from a variety of genera. The combined global morbidity and mortality figures for these viruses add up to 100s of millions per year and the current trend appears to be upwards. This is a depressing statistic when one considers the amazing medical and scientific achievements that we have witnessed during the past decades. The studies described in my thesis were focused on the genus Flavivirus the type species of which is yellow fever virus, another terrifyingly virulent human pathogen that has caused so much suffering in Africa and Latin America during the past 300 to 400 years. This virus continues to cause major epidemics in Africa despite the availability of one of the safest and most effective vaccines with which to control infections due to yellow fever virus. Indeed similar comments can be made in the context of the flavivirus Japanese encephalitis virus in India
16
Bos, Sandra. "Undestanding the viral molecular factors involved in Zika virus pathogenicity in humans." Thesis, La Réunion, 2019. http://www.theses.fr/2019LARE0005.
Full textAbstract:
Le virus Zika (ZIKV) est un phénomène épidémiologique sans précédent qui surprit le monde entier. Pendant de nombreuses années, il fut considéré comme un virus anodin responsable d’une poignée d’infections humaines, auto-limitées et bénignes, en Afrique et en Asie du Sud-est. Mais, après des décennies de propagation silencieuse, une première épidémie éclata en Micronésie en 2007 - tel un signal d'alarme. Quelques années plus tard, une soudaine épidémie de ZIKV de plus grande ampleur se déclara dans les îles du Pacifique avant d'atteindre le Brésil en 2015. Au cours de cette période, Zika fut associé à de graves complications neurologiques, mettant en évidence son fort potentiel pathogène pour l'homme. Depuis son émergence, plus de 80 pays et territoires ont été touchés par la pandémie de ZIKV, désormais reconnu comme un virus neurotrope et tératogène. L'association des souches contemporaines de ZIKV à des formes graves de maladie chez l'homme, qui n'ont jamais été signalées auparavant, a soulevé l'hypothèse d'une pathogénicité nouvellement acquise. Ainsi, mes travaux de doctorat visaient à déterminer si l'ampleur de l'épidémie actuelle pouvait en partie avoir été facilitée par des facteurs viraux qui auraient renforcé la fitness du ZIKV. À cette fin, mon projet de recherche s'est concentré sur l'identification des facteurs moléculaires viraux impliqués dans la pathogénicité du virus Zika chez l’homme à partir du développement de clones moléculairesZika virus (ZIKV) is an unprecedented epidemiological phenomenon which surprised the world. For many years, it was considered a trivial virus responsible for only a handful of human infections, self-limited and benign, in Africa and Southeast Asia. But then, after decades of silent spread, a first epidemic broke out in Micronesia in 2007 – like a warning signal. A few years later, a sudden Zika outbreak of larger scale occurred in the Pacific islands before reaching Brazil in 2015. During this period, Zika was associated with severe neurological complications, highlighting its serious pathogenic potential for humans. Since its emergence, more than 80 countries and territories have been affected by the ZIKV pandemic, which is now recognized as a neurotropic and teratogenic virus. The association of contemporary ZIKV strains with severe forms of disease in humans, that have never been reported before, has raised the hypothesis of newly acquired pathogenicity. In this regard, my doctoral research aimed to determine whether the scope of the current epidemic was partly facilitated by viral factors that improved ZIKV fitness. To this end, my research project focused on the identification of the viral molecular factors involved in Zika virus pathogenicity in humans based on the development of molecular clones
17
Dutra, Lilia Mara Mesquita. "Perfil da imunidade humoral para o vírus da febre amarela em duas populações assintomáticas da zona rural de região de Mata Atlântica do estado de São Paulo, Brasil." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-29012010-110424/.
Full textAbstract:
A Febre Amarela (FA) é uma doença viral infecciosa, não contagiosa que pode se manifestar desde um quadro febril até a forma clássica íctero-hemorrágica, cujo agente etiológico é o vírus da febre amarela (VFA). A doença tem avançando para as regiões Sudeste e Sul com um aumento de 4 vezes no número de vítimas fatais. O presente trabalho teve como objetivos avaliar a possível circulação do vírus da febre amarela silvestre, através da detecção de anticorpos IgM por meio da técnica de MAC-ELISA, e anticorpos IgG, pelas técnicas de Inibição da hemaglutinação e Neutralização; detecção do genoma viral utilizando a técnica de RT-PCR no soro de indivíduos assintomáticos; bem como avaliar a proteção vacinal, por meio da técnica de neutralização em soros de indivíduos das zonas rurais do município de Jacupiranga e Teodoro Sampaio. Um total de 238 soros foram coletados, 152 (Jacupiranga) e 86 (Teodoro Sampaio). Foram detectados um total 15,9% (38/238) de anticorpos IH contra o vírus selvagem e/ou vacinal. Destes 13,16% (5/38) anticorpos IH foram provenientes dos soros de Jacupiranga e 86,84 (33/38) de Teodoro Sampaio. Anticorpos neutralizantes foram detectados em 34% (13N/38IH) dos IH reativos, destes 15,38% (2/13) foram oriundos deJacupiranga e 84,62% (11/13). A detecçção de anticorpos neutralizantes nos indivíduos de Jacupiranga levantam a necessidade de pesquisas futuras na busca do vírus da febre amarela nos seus reservatórios selvagens e vetores. Não houve a detecção do genoma viral por RTPCR nas duas populações, bem como a detecção de anticorpos IgM específicos, por meio das reações de MACELISA, o que evidencia a não circulação recente deste vírus na população estudada. O grau de imunizaçao na população de Teodoro Sampaio, com histórico de vacinação, por meio das reações de IH e Neutralização foi baixo, uma vez que somente 42,30% da população apresentou anticorpos inibidores da hemaglutinação e o grau de proteção pela reação de neutralização foi de apenas 33,33%. Este trabalho aponta não só para a necessidade de monitoramento pós-vacinal em áreas endêmicas de febre amarela, com indicação de vacinação, como também a necessidade de estudos epidemiológicos, uma vez que se detectou reação monotípica no teste do IH, além de anticorpos neutralizantes para o vírus selvagem da febre amarela em um indivíduo residente em município de risco para a febre amarela.Yellow fever (YF) is an non-contagious infection disease. The clinical signs can be a nonspecific fever or the classical ictero-hemorrhagic form as a result of the Yellow Fever virus (YFV) infection. Considering the recent spread of the disease to the South and Southeast of Brazilian regions and the increased number of fatal cases, the aim of this study was to evaluate the possible circulation of the YFV, using the IgM enzyme-linked immnunosorbent assay (MACELISA) and Reverse transcriptase polymerase chain reaction (RT-PCR), as well as, to conduct a serological inquire and evaluate the protective vaccine response, by the hemagglutination inhibition test (HI) and serum neutralization test (SN), in the rural population of Jacupiranga and Teodoro Sampaio, located in the southern region of Brazil. A total of 238 serum samples were tested, 152 from Jacupirang and 86 from Teodoro Sampaio. Of the serum collected, 15,9% (38/238) were positive by HI, using wild and vaccine strains of YF, and out of these 13,16% (5/38) were from Jacupiranga and 86,84% (33/38) were from Teodoro Sampaio. Neutralizing antibodies were detected in 34% of the HI positive samples (13SN/38HI) and out of these 15,38% (2/13) were from Jacupiranga and 84,62% (11/13) were from Teodoro Sampaio. None of the samples were positive by MACELISA and RT-PCR indicating no evidences of recent virus circulation in the population analyzed in this study. However, the YF neutralizing antibodies detection in samples from Jacupiranga indicates the necessity of further researches in order to detect the YFV in its wild reservoirs and vectors. On the other hand, considering that 42,30% of the Teodoro Sampaio samples were positive by HI and out of these only 33,33% had neutralizing antibodies to the YFV, our results also pointed out to the importance of pos-vaccination protective immunity surveys, in order to evaluate the efficiency of the vaccines in endemic areas.
18
Fulop, Lynda Dorothy. "Molecular analysis of flavivirus genome sequences : implications for virus classification." Thesis, University of Surrey, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308496.
Full text
19
Dejarnac, Ophélie. "Molecular and cellular basis of phosphatidylserine receptors mediated flavivirus infection." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC297/document.
Full textAbstract:
Le virus de la dengue (DENV) et le virus Zika (ZIKV) sont deux virus transmis par le moustique et responsables de maladies importantes chez l’Homme. En absence de vaccin et de traitements antiviraux efficaces, ces pathogènes représentent des problèmes de santé publique majeurs. Les bases moléculaires des interactions qu’établissent le DENV et ZIKV et la cellule hôte lors de l’entrée virale sont peu connues. Notre laboratoire a récemment identifié, les protéines TIM (TIM-1 et TIM-4) et TAM (Tyro3 et Axl), deux familles de récepteurs à la phosphatidylsérine (PtdSer) impliqués dans la reconnaissance et l’élimination des cellules apoptotiques par phagocytose, comme de nouveaux facteurs d’entrée du DENV. Les récepteurs TIM et TAM permettent l’infection par le DENV en interagissant avec la PtdSer associée aux virions selon un mécanisme similaire à la reconnaissance des cellules apoptotiques (mimétisme apoptotique). L’objectif général de mon travail de thèse a été d’explorer les mécanismes moléculaires et cellulaires par lesquels TIM-1 et Axl médient l’entrée des flavivirus. A l’aide de techniques d’imagerie en temps réel nous avons montré que TIM-1 et DENV sont co-internalisés et que TIM-1 joue un rôle actif dans l’entrée du DENV. Notamment, nous avons montré que deux résidus lysine présentes dans le domaine cytoplasmique de TIM-1 sont importantes pour l’ubiquitination du récepteur et pour l’endocytose du virus. La recherche de partenaires de TIM-1 par des études de spectrométrie de masse a permis d’identifier STAM, un membre du complexe ESCRT-0 impliqué dans le trafic des récepteurs ubiquitinés, comme facteur important pour l’infection. Collectivement, nos résultats suggèrent très fortement que TIM-1 est le premier récepteur bona fide caractérisé pour le DENV.Identifier les facteurs d’entrée du ZIKV représente un enjeu majeur dans la compréhension du tropisme et de la pathogénèse associée à ce virus. Nous avons montré que le récepteur Axl est essentiel pour l’entrée du ZIKV dans les cellules microgliales, les astrocytes du cerveau humain en développement ainsi que dans les fibroblastes de la peau. Nos études ont démontré un double rôle du récepteur Axl dans l’infection par ZIKV. Axl lie et permet l’internalisation des particules virales, mais aussi, contribue à l’établissement d’un environnement favorable à la réplication virale en inhibant la réponse immunitaire innée. En conclusion, ce travail a contribué à améliorer notre compréhension des mécanismes d’entrée des virus DENV et ZIKV. Nos résultats indiquent que ces deux virus exploitent plusieurs récepteurs aux phospholipides pour initier leur cycle infectieux, ce qui pourrait contribuer à l’élargissement de leur tropismeDengue virus (DENV) and ZIKA virus (ZIKV) are two mosquito-borne viruses responsible for important diseases in humans. Since there is currently no vaccine neither antiviral treatment available against these human pathogens, they are two major health concerns. The molecular basis of DENV and ZIKV host cell interactions leading to virus entry are poorly understood, hampering the discovery of new targets for antiviral intervention. Our laboratory recently discovered that TIM (TIM-1 and TIM-4) and TAM (Tyro3 and Axl) proteins, two receptor families that contribute to the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, are DENV entry factors. TIM and TAM receptors mediate DENV infection by interacting with virion-associated PtdSer through a mechanism similar to the recognition and engulfment of apoptotic cells by phagocytes (viral apoptotic mimicry). The general objective of my PhD was to establish a detailed understanding of the molecular mechanisms by which TIM-1 and Axl mediated infection. Using live imaging, we demonstrated that TIM-1 and DENV are co-internalised and TIM-1 play an active role during DENV endocytosis. We showed that TIM-1 cytoplasmic domain is essential for DENV internalization, especially, we identified two lysine residues that are essential for TIM-1 ubiquitination and DENV endocytosis. Proteomic analysis of TIM-1 interacting partners identified STAM, a member of the ESCRT-0 complex involved in intracellular sorting of ubiquitinated cargos, as an essential host factor for DENV infection. Collectively our results establish TIM-1 as the first identified DENV bona fide receptor.Identifying ZIKV entry factors represents a major challenge in the understanding of ZIKV tropism and pathogenesis. We showed that Axl is responsible for ZIKV infection of microglial cells and astrocytes in the human developing brain and primary fibroblasts in human skin, suggesting an important role of this receptor during ZIKV life cycle. We also highlighted the dual role of the Axl receptor in ZIKV infection, which simultaneously promotes viral entry and dampens the innate immune response to facilitate a post entry step of the ZIKV life cycle. In conclusion, this work provided new insights in our understanding of the DENV and ZIKV entry program. Both viruses engage phospholipid receptors for their infectious entry, providing a rational to ascertain therapeutic strategies targeting virion-associated phospholipids
20
Defrocourt, Christophe. "L'encéphalite japonaise en 2003 : épidémiologie et nouvelles perspectives de vaccin." Rouen, 2004. http://www.theses.fr/2004ROUEP008.
Full textAbstract:
L'encéphalite japonaise est une maladie croissante en Asie du Sud-Est et dans les pays du Pacifique occidental puisqu'elle s'étend aujourd'hui à l'Australie et à la Russie. Elle est actuellement la première cause d'encéphalite virale en Asie et se rencontre surtout en zone rurale. Cette maladie est provoquée par un virus de la famille des Flavivirus. Elle est véhiculée par une piqûre de moustique de la famille des Culex. Les porcs et les oiseaux aquatiques ont un rôle de réservoirs dans la transmission de la maladie et seule leur augmentation explique la transmission à l'homme, puisqu'e celui-ci est un hôte accidentel. L'encéphalite japonaise est une maladie souvent bénigne mais peut cependant être fatale et laisser d'importantes séquelles chez les survivants. Les personnes les plus touchées sont les enfants et les personnes âgées ou immunodéficientes. Actuellement, aucun médicament efficace n'existe, mais la vaccination et une protection mécanique permettenet d'empêcher la maladie. De nouveaux vaccins sont en cours de recherche et la terminologie du "Chimerivax" va apporter dans peu de temps une vaccination à immunisation rapide et aussi efficace que celle utilisée contre le virus de la fièvre jaune. De plus, un accord entre professionnels pharmaceutiques et instituts de recherches donne l'espoir de la fabrication d'un traitement curatif.
21
Scaramozzino, Natale. "Flavivirus : étude d'une cible diagnostique, la région NS codant pour la polymérase et d'une cible thérapeutique, la protéase NS3 du virus Langat." Université Joseph Fourier (Grenoble), 2002. http://www.theses.fr/2002GRE19004.
Full textAbstract:
Les flavivirus sont des virus responsables d'une considérable morbidité et mortalité dans le monde en causant des encéphalites sévères, des fièvres hémorragiques, ainsi que des symptômes fébriles chez l'homme. L'absence de symptômes cliniques spécifiques pour une infection liée à un virus donné, et la présence simultanée de différents arbovirus dans une même région impose la nécessité de disposer d'un diagnostic de genre par PCR. Parmi les amorces proposées dans la littérature, une seule paire a permis la détection des différents flavivirus testés avec une sensibilité limite de 105 doses infectieuses. ML-1. La PCR semi-nichée développée au laboratoire utilise 2 nouvelles amorces et permet la détection des différents flavivirus avec une sensibilité de moins de 200 doses infectieuses. ML-1. Après le séquençage des produits amplifiés, la construction d'un dendrogramme permet d'orienter le diagnostic vers les principales espèces de flavivirus. Actuellement, il n'existe pas de traitement spécifique des infections à flavivirus. La protéase virale est une cible privilégiée pour ce type de thérapie. La protéase NS3 du virus Langat, virus utilisé comme modèle du virus de l'encéphalite à tiques, a été exprimée, purifiée et son activité enzymatique, sous sa forme recombinante, a été caractérisée. L'activité protéasique a été réalisée par hydrolyse de différents substrats peptidiques chromogéniques. Cette protéase virale clive in vitro les substrats comportant en position P1 un acide aminé basique tel l'arginine ou la lysine. Ces observations sont confirmées par l'étude comparative des sites de clivage naturels au sein de la polyprotéine des flavivirus. Cette protéase recombinante devrait pouvoir être utilisée pour le criblage de molécules antiprotéases utilisables dans le traitement des infections à flavivirusFlaviviruses are responsible for considerable morbidity and mortality and may cause severe encephalitis, hemorrhagic fever, hepatitis, and febrile symptoms in vertebrates, including humans (. . . ). Different p-nitroanilide substrates, defined on canonic sequences for their susceptibility to Ser-protease, were applied to the proteolytic assays of the protein. The highest values were obtained from substrates containing an Arg or Lys (amino acid) residue at the P1 position. This purification method will facilitate the future development of reliable testing procedures for anti-proteases directed to NS3 proteins
22
Ludolfs, Diana. "Charakterisierung typenspezifischer B-Zell-Epitope der Flaviviren." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=966135768.
Full text
23
Figueiredo, Mario Luis Garcia de. "Identificação de flavivirus infectando culicídeos de 1999 a 2007 no Brasil." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-19052010-153321/.
Full textAbstract:
Introdução: Arbovírus são vírus transmitidos por artrópodos, pertencendo, principalmente, aos gêneros Flavivirus (Flaviviridae), Alphavirus (Togaviridae) e Orthobunyavirus (Bunyavirus). Os Flavivirus, em sua maioria, são associados a zoonoses, causando doenças humanas febrís, febres hemorrágicas e encefalites. Inclusive, causam epidemias que são sério problema de saúde pública. Este estudo mostra uma pesquisa de Flavivirus em culicídeos, de diferentes regiões do país, utilizando uma técnica para identificação viral por RT-PCR com primers Flavivirusespecíficos e uma Multiplex-nested-PCR com primers espécie-específicos. Métodos: Culicídeos foram capturados, quantificados, identificados, agrupados em lotes por espécie e congelados. No laboratório, os animais foram macerados e tiveram o RNA extraído. Estes extratos foram submetidos a RT-PCR gênero-específica e à Multiplex-nested-PCR, para detecção e identificação dos vírus a nível de espécie. Resultado: De 3317 culicídios adultos e 571 larvas coletados em 4 diferentes regiões do Brasil, Sul, Sudeste, Norte e Nordeste, fez-se 246 lotes de mosquitos e desses foi possível obter amplicon sugestivo de Flavivirus em 16 (6,5%). Em 3 lotes contendo larvas de Aedes albopictus obteve-se amplicon sugestivo de vírus do dengue tipo 3. Também, em 13 lotes contendo Haemagogus leucocelaenus, Aedes aegypti e Aedes albopictus foi possível obter amplicons sugestivos de vírus do dengue tipos 1 e 2. Dos amplicons obtidos, 4 tiveram nucleotídios seqüenciados o que permitiu confirmar a presença dos vírus do dengue tipo 3 e Cacipacoré. Conclusão: O trabalho permitiu concluir que: a metodologia de RT-PCR para Flavivirus seguida de Multiplex-nested-PCR espécie-específica foi adequada para detecção e identificação destes vírus em culicídios; amplificaram-se genomas de Flavivirus em 6,5% dos lotes de culicídios estudados; vírus do dengue tipo 1 e tipo 2 foram encontrados infectando Aedes aegypti de Santos em 1999, Manaus em 2005-2006 e Foz do Iguaçu; vírus do dengue tipos 2 e 3 foram encontrados em Aedes albopictus de Santos em 1999 e Manaus em 2005-2006, sugerindo que este mosquito participe na transmissão de dengue; vírus do dengue tipo 3 foi encontrado em larvas de Aedes albopictus mostrando transmissão vertical do vírus; vírus do dengue tipo 1 foi encontrado infectando Haemagogus sp. sugerindo existência de ciclo silvático deste vírus; Aedes aegypti do Amazonas estavam infectados com o vírus Cacipacoré.Introduction: Arbovirus are rodent-borne viruses mostly from Flavivirus (Flaviviridae), Alphavirus (Togaviridae) e Orthobunyavirus (Bunyavirus) genus. Flavivirus, are commonly zoonotic and can cause febrile illness, haemorrhagic fever and encephalitis. Flavivirus outbreaks occur in Brazil and are a major public health problem. We show here a research looking for Flavivirus infections in Culicidae by a RT-PCR using Flavivirus-especific primers and a Multiplex-nested-PCR using specie-specific primers for virus identification. Methods: Culicidae were captured, quantified, identified, pooled based on the specie and frozzen. In the laboratory, the animals were crushed and had the RNA extracted. These extracts were tested by a Flavivirus genus-specific RT-PCR followed by a specie-specific Multiplex-nested-PCR. Results: From 3317 captured adult Culicidae and 571 collected larvae in 4 different regions of Brazil, 246 pools were obtained and from these, Flavivirus indicative amplicons were obtained in 16 (6.5%). Amplicons of dengue type 3 were obtained from 3 pools of Aedes albopictus larvae. It was also possible to obtain indicative amplicons of dengue types 1 and 2 in 13 pools of Haemagogus leucocelaenus, Aedes aegypti and Aedes albopictus. Besides, 4 amplicons had the nucleotides sequenced, confirming the mosquito infection by dengue type 3 and Cacipacoré viruses. Conclusion: The technique combining a Flavivirus genus-specific RT-PCR followed by a specie-specific Multiplex-nested-PCR was suitable for detection of these viruses in the mosquitoes; Flavivirus infecting Culicidae were detected in 6.5% of the analyzed mosquito pools; dengue virus type 1 and type 2 were found infecting Aedes aegypti from Santos (1999), Manaus (2005-2006) and Foz do Iguaçu cities; dengue type 2 virus was found in Aedes albopictus from Santos city (1999) and Manaus city (2005-2006), suggesting that this mosquito could be participating on dengue transmition; dengue type 3 virus was found in Aedes albopictus larvae showing the vertical transmission of this virus; dengue type 1 virus was found infecting Haemagogus sp. what suggests on the existence of a sylvatic maintenance cycle of this virus; Aedes aegypti from Amazonas state were found infeted by Cacipacoré virus.
24
Nguyen, Jennifer B. "Molecular Mechanisms of Host-Pathogen Interactions in Flavivirus and Hookworm Infection." Thesis, Yale University, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=3580786.
Full textAbstract:
Microbial pathogens and their hosts have evolved complex adaptations to ensure their individual survival, resulting in a so-called "molecular arms race." While hosts may have acquired diverse mechanisms to protect themselves from the microbial invader, pathogens have developed elaborate strategies to evade and subvert these defenses. Viruses and hookworms are important pathogens which have evolved to successfully invade and infect their human hosts. Although structural biology has provided significant mechanistic insight into these processes of invasion, many specific host-pathogen interactions and their dynamics have not been well studied or characterized.
The work presented in this dissertation clarifies the mechanisms of cellular entry of one particular family of viruses, the flaviviruses, and discusses strategies for viral clearance by host cells. Additional insight into the role of a cytoplasmic DNA sensor, LRRFIP1, in mediating an innate immune response to non-flavivirus microbial infection is presented. Finally, strategies for the development of small-molecule or peptide inhibitors of virus entry and hookworm infection are proposed.
25
Caleiro, Giovana Santos. "Investigação da presença do retrovírus da Reticuloendoteliose aviária (REV) e do anticorpo IgG do vírus Oeste do Nilo (WNV) em aves." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/99/99131/tde-04092018-090320/.
Full textAbstract:
As aves podem carregar um grande número de patógenos. As aves migratórias, por viajarem longas distâncias, são as principais responsáveis pela disseminação de agentes infecciosos. Entre os agentes, destacam-se os vírus, como por exemplo o retrovírus da Reticuloendoteliose aviária (REV), amplamente distribuído; e o vírus da febre do Oeste do Nilo (WNV), uma virose reemergente, com caráter zoonótico. Os principais sintomas da Reticuloendoteliose aviária incluem anemia, doença de Runting e síndrome não neoplásica aguda. Já o agente etiológico da Febre do Nilo Ocidental, é o Flavivirus West Nile (WNV).. As aves são seus hospedeiros definitivos e os humanos são hospedeiros acidentais, podendo manifestar quadro febril, e em menor porcentagem, meningite e encefalite. Mosquitos dos gêneros Culex e Aedes spp são os principais transmissores do vírus. Ao contrário do REV que não dispõe de evidências de sua circulação no Brasil, há evidências do WNV em aves e equinos e mais recentemente, em humanos. O objetivo desse trabalho foi investigar a presença do REV e do WNV em aves silvestres e de cativeiro da cidade de São Paulo e do Norte do estado do Pará. Sangue, soro e swab de cloaca foram coletados, totalizando mais de 1000 amostras. Através de técnicas moleculares foi possível detectar a presença do REV em 74 amostras (16%), todas do estado do Pará. O sequenciamento parcial dessas amostras e sua filogenia sugeriu que a migração de aves EUA-Brasil possa ter sido a rota utilizada. Através de ELISA anti-IgG de WNV, 4 amostras de São Paulo foram positivas. Apresentamos a primeira evidência do REV no país e sugerimos a presença do WNV no estado de São Paulo.Birds can carry a large number of pathogens. The migratory birds are most responsible for the spread of infectious agents due to long distance travels. Among these pathogens, the most notable are viruses, such as the avian Reticuloendotheliosis retrovirus (REV), widely distributed; and the West Nile virus (WNV), a reemerging zoonotic disease. The main symptoms of avian reticuloendotheliosis include anemia, Runting\'s disease and acute nonneoplastic syndrome. The etiological agent of West Nile fever is Flavivirus West Nile (WNV). Birds are their definitive hosts and humans are accidental hosts, which generaly present febrile symptoms, but at less proportion,, meningitis and encephalitis. Mosquitoes of the genus Culex and Aedes spp are the main vectors of the virus. Differently from the REV that has no evidence of its circulation in Brazil, there is evidence of WNV in birds and horses and more recently in humans. The objective of this work was to investigate the presence of REV and WNV in wild birds and captive birds from the city of São Paulo and Northern from Pará State. Blood, serum and cloacal swab were collected, resulting in more than 1000 samples. Through molecular techniques it was possible to detect the presence of REV in 74 samples (16%), all from the State of Pará. The partial sequencing of these samples and their phylogeny suggested that the migration of US-Brazil may have been the route for the virus entry. Through anti-WNV IgG ELISA, 4 samples from São Paulo were positive. We present the first evidence of REV in the country and suggest the presence of WNV in the state of São Paulo.
26
Cruz, Ana Cecilia Ribeiro. "Caracterização Molecular e Biológica do vírus dengue circulante no Brasil." reponame:Repositório Institucional da FIOCRUZ, 2005. https://www.arca.fiocruz.br/handle/icict/4111.
Full textAbstract:
Submitted by Tatiana Oliveira (tsilva@icict.fiocruz.br) on 2012-05-31T16:28:56Z
No. of bitstreams: 1
ana_cecilia_r_cruz_ioc_bp_0001_2005.pdf: 4732352 bytes, checksum: ea13b3487709eb14b3bda0c2d649e513 (MD5)Made available in DSpace on 2012-05-31T16:28:56Z (GMT). No. of bitstreams: 1 ana_cecilia_r_cruz_ioc_bp_0001_2005.pdf: 4732352 bytes, checksum: ea13b3487709eb14b3bda0c2d649e513 (MD5) Previous issue date: 2005
Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil
A febre do dengue está amplamente distribuída por todas as áreas tropicais do mundo. Em certas áreas endêmicas, as formas graves da doença, febre hemorrágica do dengue (FHD) e síndrome de choque do dengue (SCD), ocorrem com freqüência. Outras manifestações clínicas, como, por exemplo, encefalopatias, encefalites, mielite transversa, também tem sido observadas. O nosso objetivo foi a caracterização genética e biológica de amostras de vírus VDEN2 e 3, com ênfase no caráter de neurovirulência do vírus VDEN2. As amostras de VDEN 2 e 3 utilizadas foram isoladas na Serviço de Arbovírus do Instituto Evandro Chagas a partir de soro de pacientes com quadro clínico de dengue (FD e FHD) e manifestações neurológicas. Os vírus padrão foram o VDEN2 Nova Guiné C e 44-2, VDEN3 H87. As 11 seqüências do vírus VDEN2 quando comparados entre si e com 16 outras seqüências do VDEN2 apresentaram percentual médio de identidade nucleotídica e aminoácido de 89,5 e 99,8% respectivamente. Os estudos filogenéticos realizados mostram os isolados brasileiros pertencem ao genótipo III de origem Asiática. As 10 seqüências do vírus VDEN3 quando comparados entre si e com 16 outras seqüências do VDEN3 apresentaram percentual médio identidade nucleotídica e aminoácido de 89,6 e 100% respectivamente.Os estudos filogenéticos realizados mostram que estão agrupados ao genótipo III também de origem Asiática. O virus VDEN2 isolado a partir de caso mielite transversa (Bel61082) foi caracterizado pelo sequenciamento nucleotidico da região estrutural assim como em testes de neurovirulência em camundongos recém-nascidos. A susceptibilidade de culturas primárias de neurônios e astrócitos, células de glioma humano, também foi estudada para fins de caracterização do caráter neurovirulento do vírus.O estudo de neurovirulência em infecção de camundongo mostrou sinais aparentes de doença apenas após a inoculação da cepa NGC, mas a replicação do vírus Bel 61082 pode ser detectada por RT-PCR em suspensão de cérebro de camundongo. Obtivemos evidências experimentais de apoptose nas células neuronais infectadas com VDEN2 NGC e Bel61082. No sequenciamento nucleotídico, a principal alteração observada está localizada na proteína M 28, região esta conhecida como pró-apoptótica.
The dengue fever is widely distributed in all tropical areas of the world. In certain endemic areas, severe disease, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), occur frequently. Other clinical manifestations such as, for example, encephalopathies, encephalitis, transverse myelitis, has also been observed. Our objective was to characterize genetic and biological samples of viruses VDEN2 and 3, with emphasis on the neurovirulence of the virus VDEN2. The samples of DENV 2 and 3 used were isolated in the Service of the Instituto Evandro Chagas Arboviruses from the serum of patients with clinical dengue (DF and DHF) and neurological manifestations. Viruses were standard VDEN2 New Guinea C and 44-2, VDEN3 H87. The 11 sequences of the virus VDEN2 compared among themselves and with 16 other sequences showed VDEN2 average percentage of nucleotide and amino acid identity of 89.5 and 99.8% respectively. Phylogenetic studies performed show the Brazilian isolates belonged to genotype III of Asian origin. The 10 sequences of the virus VDEN3 compared among themselves and with 16 other sequences showed VDEN3 average percentage nucleotide and amino acid identity of 89.6 and 100% respectively.The phylogenetic studies carried out show that they are grouped to genotype III also of Asian origin. The virus isolated from VDEN2 case transverse myelitis (Bel61082) was characterized by nucleotide sequencing of the framework region as well as tests neurovirulence in mice newborn. The susceptibility of primary cultures of neurons and astrocytes, human glioma cells was also investigated for the characterization of the character neurovirulent vírus.O study of infection in mouse neurovirulence showed no apparent signs of disease after inoculation only NGC strain, but virus replication 61,082 Bel can be detected by RT-PCR on mouse brain suspension. We obtained experimental evidence of apoptosis in neuronal cells infected with NGC and VDEN2 Bel61082. In nucleotide sequencing, the main change observed is located at 28 M protein, an area known as pro-apoptotic.
27
COLE, E. R. "Estudo Fitoquímico do Óleo Essencial dos Frutos da Aroeira (Schinus terebinthifolius RADDI) e Sua Eficácia no Combate ao Dengue." Universidade Federal do Espírito Santo, 2008. http://repositorio.ufes.br/handle/10/4634.
Full textAbstract:
Made available in DSpace on 2016-08-29T15:35:20Z (GMT). No. of bitstreams: 1
tese_2440_Eduardo Roberto Cole.pdf: 824296 bytes, checksum: 4e07ddc64143283e79dda5885925fe74 (MD5)
Previous issue date: 2008-03-07O dengue é considerado a mais importante das arboviroses humanas, constituindo atualmente um dos principais problemas de Saúde Pública no mundo. A doença é causada por um vírus do gênero Flavivirus, com quatro diferentes sorotipos, transmitidos por mosquitos do gênero Aedes, sendo o Aedes aegypti seu principal vetor. O controle do vetor por meio de inseticidas sintéticos figura como a principal medida adotada no combate a doença, uma vez que não existem vacinas efetivas contra os diferentes sorotipos do vírus. Os inseticidas, apesar de eficazes, apresentam sérios problemas relacionados ao seu uso, dentre os quais podem ser destacados os danos ambientais, a toxicidade para uso humano e o risco de desenvolvimento de larvas e adultos resistentes.
28
Machado, Daiane Cristina. "Sequenciamento do genoma completo do vírus Culex flavivirus (Flaviviridae) isolado no Brasil /." São José do Rio Preto, 2016. http://hdl.handle.net/11449/154714.
Full textAbstract:
Orientador: Maurício Lacerda NogueiraBanca: Marilia de Freitas Calmon
Banca: Aripuanã Sakurada Aranha Watanabe
Resumo: No Brasil, a presença de ecossistemas com uma grande riqueza e diversidade de animais e cidades grandes e densamente povoadas oferece condições para a ocorrência de diversas arboviroses. Vários arbovírus transmitidos por mosquitos do gênero Culex pertencem ao gênero Flavivirus. Vírus desse gênero são conhecidos por causar doenças em humanos e animais, no entanto, flavivírus que não infectam ou causam doença em vertebrados, replicando somente em artrópodes têm sido descritos. O vírus Culex flavivirus é um exemplo. Ele foi isolado de espécies de mosquitos do gênero Culex na América do Norte, América Central, América do Sul, África e Ásia. Apesar dos estudos realizados em outras partes do mundo, no Brasil há ainda pouca informação sobre esse vírus. Nesse trabalho, realizamos o seqüenciamento do genoma completo do vírus Culex flavivirus (CxFV) isolado de mosquitos Culex de São José do Rio Preto (SP), por meio das técnicas de RT-PCR e seqüenciamento nucleotídico. O genoma viral do isolado CxFV_RPBR07_2007, o qual foi obtido no presente estudo apresentou 10.706 nucleotídeos com uma ORF de 10.092 nucleotídeos (3.364 aminoácidos) flanqueada por uma 5'UTR de 32 nucleotídeos e uma 3'UTR de 582 nucleotídeos. Análises filogenéticas revelaram que o CxFV_RPBR07_2007 agrupa-se com outros flavivírus de insetos, como o Cell fusing agent virus (CFAV) e o Kamiti River virus (KRV), e relaciona-se intimamente com isolados de CxFV do México (América Latina) e de Uganda (África); mantendo relação também com isolados dos Estados Unidos (América do Norte) e do Japão (Ásia). Esses agrupamentos foram também observados em outros estudos filogenéticos
Abstract: In Brazil, the presence of ecosystems with a wealth and diversity of animals, and cities large and densely populated offers conditions for the occurrence of many arboviruses. Several arboviruses transmitted by mosquitoes of the genus Culex belong to the genus Flavivirus. Viruses of this genus are known to cause disease in humans and animals, however, flaviviruses do not infect or cause disease in vertebrates, replicating only in arthropods have been described. Culex flavivirus virus is an example. He was isolated from species of mosquitoes of the genus Culex in North America, Central America, South America, Africa and Asia. Although studies conducted in other parts of the world, in Brazil there is still little information about this virus. In this work we performed the sequencing of the complete genome of Culex flavivirus virus (CxFV) isolated from Culex mosquitoes in São José do Rio Preto (SP) using the techniques of RT-PCR and nucleotide sequencing. The viral genome of the isolated (CxFV_RPBR07_2007) obtained in the present study presented 10.706 nucleotides with an ORF of 10.092 nucleotides (3.364 amino acids) flanked by a 5'UTR of 32 nucleotides and a 3'UTR of 582 nucleotides. Phylogenetic analyzes revealed that the CxFV_RPBR07_2007 groups with other insect flaviviruses such as the Cell fusing agent virus (CFAV) and Kamiti River virus (KRV), and is closely related to CxFV isolates from Mexico (Latin America) and Uganda (Africa); also maintaining relationship with isolates from the United States of America (North America) and Japan (Asia). These groupings were also observed in other phylogenetic studies
Mestre
29
Machado, Daiane Cristina [UNESP]. "Sequenciamento do genoma completo do vírus Culex flavivirus (Flaviviridae) isolado no Brasil." Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/154714.
Full textAbstract:
Made available in DSpace on 2018-07-27T18:26:11Z (GMT). No. of bitstreams: 0
Previous issue date: 2014-10-24. Added 1 bitstream(s) on 2018-07-27T18:30:32Z : No. of bitstreams: 1
000869213.pdf: 1151866 bytes, checksum: 95a9e9bbe8d1c2b6b9784eae9f7abb27 (MD5)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
No Brasil, a presença de ecossistemas com uma grande riqueza e diversidade de animais e cidades grandes e densamente povoadas oferece condições para a ocorrência de diversas arboviroses. Vários arbovírus transmitidos por mosquitos do gênero Culex pertencem ao gênero Flavivirus. Vírus desse gênero são conhecidos por causar doenças em humanos e animais, no entanto, flavivírus que não infectam ou causam doença em vertebrados, replicando somente em artrópodes têm sido descritos. O vírus Culex flavivirus é um exemplo. Ele foi isolado de espécies de mosquitos do gênero Culex na América do Norte, América Central, América do Sul, África e Ásia. Apesar dos estudos realizados em outras partes do mundo, no Brasil há ainda pouca informação sobre esse vírus. Nesse trabalho, realizamos o seqüenciamento do genoma completo do vírus Culex flavivirus (CxFV) isolado de mosquitos Culex de São José do Rio Preto (SP), por meio das técnicas de RT-PCR e seqüenciamento nucleotídico. O genoma viral do isolado CxFV_RPBR07_2007, o qual foi obtido no presente estudo apresentou 10.706 nucleotídeos com uma ORF de 10.092 nucleotídeos (3.364 aminoácidos) flanqueada por uma 5'UTR de 32 nucleotídeos e uma 3'UTR de 582 nucleotídeos. Análises filogenéticas revelaram que o CxFV_RPBR07_2007 agrupa-se com outros flavivírus de insetos, como o Cell fusing agent virus (CFAV) e o Kamiti River virus (KRV), e relaciona-se intimamente com isolados de CxFV do México (América Latina) e de Uganda (África); mantendo relação também com isolados dos Estados Unidos (América do Norte) e do Japão (Ásia). Esses agrupamentos foram também observados em outros estudos filogenéticos
In Brazil, the presence of ecosystems with a wealth and diversity of animals, and cities large and densely populated offers conditions for the occurrence of many arboviruses. Several arboviruses transmitted by mosquitoes of the genus Culex belong to the genus Flavivirus. Viruses of this genus are known to cause disease in humans and animals, however, flaviviruses do not infect or cause disease in vertebrates, replicating only in arthropods have been described. Culex flavivirus virus is an example. He was isolated from species of mosquitoes of the genus Culex in North America, Central America, South America, Africa and Asia. Although studies conducted in other parts of the world, in Brazil there is still little information about this virus. In this work we performed the sequencing of the complete genome of Culex flavivirus virus (CxFV) isolated from Culex mosquitoes in São José do Rio Preto (SP) using the techniques of RT-PCR and nucleotide sequencing. The viral genome of the isolated (CxFV_RPBR07_2007) obtained in the present study presented 10.706 nucleotides with an ORF of 10.092 nucleotides (3.364 amino acids) flanked by a 5'UTR of 32 nucleotides and a 3'UTR of 582 nucleotides. Phylogenetic analyzes revealed that the CxFV_RPBR07_2007 groups with other insect flaviviruses such as the Cell fusing agent virus (CFAV) and Kamiti River virus (KRV), and is closely related to CxFV isolates from Mexico (Latin America) and Uganda (Africa); also maintaining relationship with isolates from the United States of America (North America) and Japan (Asia). These groupings were also observed in other phylogenetic studies
30
Beck, Cécile. "Nouvelles stratégies diagnostiques et thérapeutiques contre les flavivirus neurotropes en médecine vétérinaire." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS083/document.
Full textAbstract:
Les flavivirus ayant un impact en médecine vétérinaire sont largement distribués dans le monde (à l’exemple de la fièvre West Nile (WNF) présente sur les cinq continents ou de l’Encéphalite japonaise (EJ) en Asie du Sud-Est) et sont responsables de maladies à dominante neurologique chez l’homme et/ou le cheval.La virémie étant généralement brève lors de ces infections virales, les méthodes de diagnostic utilisées sont essentiellement sérologiques. Or le chevauchement fréquent des aires de répartition des flavivirus complique le diagnostic sérologique. En effet, des réactions sérologiques croisées entre flavivirus sont observées lors de l’utilisation de méthodes de diagnostic usuelles telles que l’ELISA et l’immunofluorescence (IF). Les résultats sérologiques doivent donc être confirmés par la méthode fastidieuse de séroneutralisation (SNT) virale avec les différents flavivirus existants dans la région. De plus, le risque d’émergence sur un territoire donné de nouveaux flavivirus comme le virus Zika au Brésil ou en Amérique du Nord ne peut être exclu.Nous avons donc développé dans la première partie de ce travail une nouvelle stratégie de diagnostic sérologique multiplexe à l’aide de la méthode d’immuno-essai (MIA) sur billes. Sachant que la glycoprotéine d’enveloppe soluble (sE) des flavivirus est divisée en 3 domaines (D) structuraux, DI, DII et DIII et que les épitopes du DIII sont spécifiques de chaque flavivirus, des protéines EDIII recombinantes de différents flavivirus d’intérêt ont été synthétisées en système d’expression Drosophila S2 et leur antigénicité a été testée sur sérums équins et ovins. Nous avons obtenu des résultats très encourageants en démontrant que l’utilisation des EDIII associée avec la capacité de multiplexage de la méthode MIA apparaît comme une réponse adaptée au défi du diagnostic sérologique des infections à flavivirus.Nous avons en outre utilisé la même méthode de multiplexage mais avec des antigènes NS1 du WNV pour mettre en place un test DIVA (Differentiating Infected from Vaccinated Animals) permettant de différencier les chevaux infectés par le WNV des chevaux vaccinés avec un vaccin recombinant WNV.Un autre écueil en médecine vétérinaire est le traitement des affections à flavivirus. En effet l’arsenal thérapeutique est limité et le traitement est avant tout symptomatique. Nous avons dans la seconde partie de ce travail testé in vitro une molécule antivirale à large spectre, le sr1057 sur nos virus d’intérêt (WNV et JEV). Cette molécule qui provient d’une stratégie de criblage développée par l’Institut Pasteur a probablement pour cible la cellule de l’hôte car elle est capable d’inhiber des virus très différents, à génome à ARN de polarité positive ou négative et ADN.Les résultats que nous avons obtenus avec ce composé sont en demi-teinte pour les flavivirus testés avec une efficacité démontrée pour le JEV mais plus modeste pour le WNV. Ils n’excluent cependant pas une possible utilisation de cet antiviral en médecine vétérinaire équine car une activité inhibitrice in vitro sur les virus de l’Herpes équin de type I et de l’artérite virale a été confirmée par d’autres collaborateursFlaviviruses with a major impact in veterinary medicine are widely distributed (e.g. West Nile fever (WNF) has spread across the five continents and Japanese Encephalitis (JE) is reported in South-East Asia) and are mainly responsible for neurological diseases in humans and/or horses.After flavivirus infection, viremia in mammal hosts is generally short and consequently indirect methods are mostly used to diagnose flavivirus infections. However, frequent spatial overlapping in their circulation areas renders the interpretation of serological assays difficult. Indeed, cross-reactions between flaviviruses are observed in rapid serological tests such as in ELISA and immunofluorescence assays (IFA). Serological assay results should thus be confirmed by the tedious comparative virus neutralization test (VNT) using a panel of viruses known to circulate in the area. Moreover, the risk of emergence of new flaviviruses such as reported recently with the Zika virus in Brazil or in North America should be considered when studying flavivirus epidemiology.In the first section, a new strategy aiming at improving the serological diagnosis of flavivirus infections was developed using the multiplexing capacity of microsphere immunoassays (MIA). The flavivirus soluble envelope (sE) glycoprotein ectodomain is composed of three domains (D), e.g. DI, DII and DIII, with EDIII containing virus-specific epitopes. Recombinant EDIIIs of different flaviviruses were synthesized in the Drosophila S2 expression system. The microspheres coupled with rEDIIIs were assayed with equine and ovine sera from natural and experimental flavivirus infections or non-immune samples. Very encouraging results have been obtained and this innovative multiplex immunoassay based on flavivirus rEDIIIs appears to be a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.MIA with WNV nonstructural 1 protein were also implemented to differentiate Infected from Vaccinated Animals (DIVA). Such a DIVA approach was only successful when horses had been immunized with a recombinant canarypox vaccine, while horses receiving inactivated WNV vaccine developed immune responses close to the ones induced after natural infection.Another pitfall in veterinary medicine is the lack of therapeutics for viral diseases and specifically for flaviviruses. The therapeutic arsenal is indeed rather limited and animals are generally administered supportive treatments only. In the second part, the results of the in vitro testing of a broad spectrum antiviral named sr1057 on WNV and JEV replication are presented. This chemical, identified from a unique screening strategy developed by Pasteur Institute, is probably targeting the host cell and was found to inhibit the replication of varied RNA and DNA viruses belonging to different virus families. The sr1057 compound was not as efficient at inhibiting the replication of flaviviruses as for other RNA+ viruses, with a modest antiviral effect demonstrated for WNV and a higher efficacy on JEV. This antiviral presents however potentials for applications in equine veterinary medicine because it efficiently inhibited equine herpes virus-1 and equine arteritis virus in vitro, as clearly shown by other collaborators
31
Ottendorfer, Christy L. "Impact of West Nile virus on the natural history of St. Louis encephalitis virus in Florida." [Tampa, Fla] : University of South Florida, 2008. http://purl.fcla.edu/usf/dc/et/SFE0002452.
Full text
32
Fernandes, Lícia Natal. "Identificação de infecção por Flavivirus em mosquitos (Diptera: Culicidae) coletados em áreas verdes da cidade de São Paulo." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/99/99131/tde-03082015-102328/.
Full textAbstract:
Introdução: Dos arbovírus transmitidos no Brasil, os Flavivirus apresentam destaque por causarem o maior número de infecções e doenças no homem e pela gravidade das doenças que provocam. A cidade de São Paulo possui áreas verdes, representadas por parques municipais em que existem lagos, aves e mamíferos. Esses locais são frequentados pela população e podem servir de refúgio para mosquitos que infestam a área urbana. Alguns trabalhos revelam a ocorrência de espécies de mosquitos conhecidas como vetoras de Flavivirus em tal cidade, o que pode favorecer o aparecimento de casos e transmissão de doenças causadas por este tipo de vírus. No entanto, a presença de Flavivirus infectando tais artrópodes não é conhecida. Objetivo: Identificar presença de Flavivirus em mosquitos (Diptera: Culicidae) provenientes de áreas verdes da cidade de São Paulo. Método: Sete parques municipais localizados em diferentes regiões da cidade foram selecionados para o estudo. Foram realizadas coletas de mosquitos mensais em cada parque no período de março de 2011 e fevereiro de 2012. As armadilhas utilizadas foram: aspirador, Shannon e CDC-CO2. Os mosquitos foram transportados para o laboratório em gelo seco, identificados em mesa fria e agrupados em \"pools\" de até 10 fêmeas não ingurgitadas, segundo espécie, data e local de coleta. Fêmeas ingurgitadas foram armazenadas individualmente. As amostras foram submetidas à extração de ácidos nucleicos, seguida de RT-PCR em tempo real para amplificação de fragmento de aproximadamente 200pb do gene NS5 de Flavivirus. Amostras positivas foram encaminhadas para sequenciamento e análises filogenéticas. Resultados: Dentre as 5.213 fêmeas não ingurgitadas (818 pools) e as 778 fêmeas ingurgitadas coletadas, ocorreu presença de onze gêneros de Culicídeos, sendo mais frequentes Culex e Aedes. Dezenove pools de fêmeas não ingurgitadas obtiveram resultado positivo para Flavivirus. Análises filogenéticas mostraram presença de RNA relacionado à Aedes flavivirus (AEFV) em dois pools de Aedes e à Culex flavivirus (CxFV) nos demais pools, todos de Culex. Conclusões: CxFV e AEFV estão presentes em mosquitos dos gêneros Culex e Aedes, respectivamente, coletados em parques municipais da cidade de São Paulo. Embora flavivírus patogênicos ao homem não tenham sido encontrados no presente trabalho, recomenda-se vigilância de flavivírus nas áreas estudas, visto que elas possuem presença de mosquitos vetores e hospedeiros vertebrados, ou seja, elementos necessários para a transmissão destes vírus.Introduction: Among the arboviruses transmitted in Brazil, Flavivirus are noteworthy once they cause the largest number of infections and diseases in humans and also because they can cause serious illnesses. In the city of São Paulo there are green spaces, represented by city parks in which lakes, birds and mammals are found. The population visits those places, which can also be a refuge for mosquitoes that infect the urban area. Some studies reveal the occurrence of mosquitoes species known to be vectors of Flavivirus in this city, what can contribute to the emergence of cases and transmission of diseases caused by this kind of virus. However, the presence of Flavivirus infecting those arthropods is not known. Objective: To Identify infection by Flavivirus in mosquitoes (Diptera: Culicidae) collected in São Paulo\'s city parks. Method: Seven city parks located in different places of the city were selected for the study. Monthly mosquito collections were carried out in each park from March 2011 to February 2012. The traps employed were aspirator, Shannon and CDC-CO2. The mosquitoes were transported to the laboratory on dry ice. Identification was performed in a chill table. Up to 10 non-engorged females were pooled according to specie, date and place of collection. Engorged females were stored individually. Nucleic acids were extracted and submitted to real time RT-PCR that amplifies a 200pb fragment of NS5 gene of Flavivirus. Positive samples were sequenced and phylogenetic analyses were performed. Results: Eleven genus of Culicidae were found among the 5,213 non engorged females (818 pools) and the 778 engorged females. Culex and Aedes were the most frequent collected genus. Nineteen non engorged female pools had positive results for the presence of Flavivirus RNA. Phylogenetic analyses showed the RNA found was related to Aedes flavivirus (AEFV) in two pools of Aedes and to Culex flavivirus (CxFV) in the other pools, all of them consisting of Culex. Conclusion: CxFV and AEFV are present in mosquitoes Culex and Aedes, respectively, collected in São Paulo\'s city parks. Flavivirus of medical importance were not detected. Although, once there are species of mosquitoes that can act as vectors of pathogenic Flavivirus and vertebrate hosts, which are required for the transmission cycle of those virus, surveillance is recommended in the studied areas.
33
Cespedes, Graziely Ferreira [UNESP]. "Sínteses e estudos dos diferentes peptídeos de fusão dos flavivirus causadores da dengue." Universidade Estadual Paulista (UNESP), 2013. http://hdl.handle.net/11449/108473.
Full textAbstract:
Made available in DSpace on 2014-08-13T14:50:39Z (GMT). No. of bitstreams: 0
Previous issue date: 2013-09-16Bitstream added on 2014-08-13T18:01:31Z : No. of bitstreams: 1
000746985_20150927.pdf: 1074107 bytes, checksum: 736b109d2ccde3c59ca3e85ff6da7aea (MD5) Bitstreams deleted on 2015-09-28T16:42:56Z: 000746985_20150927.pdf,. Added 1 bitstream(s) on 2015-09-28T16:43:44Z : No. of bitstreams: 1
000746985.pdf: 3182272 bytes, checksum: 2f1f8eea88932fcd1181df434e0bea11 (MD5)O vírus da dengue afeta milhões de pessoas a cada ano no mundo todo. O processo de infecção viral pela via endossomal é intermediada pela sequência 98 a 112 (DRGWGNGCGLFGKGG), conhecido como peptídeo de fusão, este segmento faz parte da glicoproteína E, localizado no envelope do vírus. Todos os flavivirus, incluindo os quatro sorotipos da dengue, apresentam esta sequência em regiões próximas, além de apresentarem alta identidade. Apesar de intensas pesquisas e o aumento da incidência de casos da dengue, não existem drogas antivirais específicas, até este momento, para o combate desta doença. Deste modo, a compreensão da interação do vírus com as células hospedeiras é importante na busca de novos fármacos. Neste sentido, este trabalho visou entender melhor o modo de ação do peptídeo de fusão da dengue e a importância de suas regiões laterais. O objetivo deste projeto teve por finalidade avaliar as contribuições das regiões laterais ao peptídeo de fusão, isto é, 88-97 e 113-123, em sua atividade, para os quatro sorotipos da dengue. A obtenção dos peptídeos foi realizada por meio da síntese em fase sólida. Os modelos de membrana utilizados foram micelas zwitteriônicas (LPC) e vesículas (PC ovo e POPG). Sabendo-se que a exposição desta sequência e, consequente, fusão da membrana é dependente do pH do meio, os estudos foram realizados em pHs 5,5 e 7,1. Nestes estudos, foram utilizadas as técnicas de Transferência de Energia de Ressonância de Föster (FRET), Dicroísmo Circular, Fluorescência, Monocamadas de Langmuir e BAM. O aminoácido triptofano foi usado como sonda para analisar a profundidade da inserção em miméticos de membrana. Os resultados mostraram que a SPFS foi útil, obtendo os materiais desejados. Avaliando a habilidade fusogênica dos 14 peptídeos estudados, observamos que cada sorotipo apresenta atividade diferente um do outro, sendo que os peptídeos do sorotipo 2 foram os que...
Dengue virus affects millions of people worldwide every year. The process of viral infection via endosomal is mediated by the sequence 98-112 (DRGWGNGCGLFGKGG), known as the fusion peptide. This segment is part of the glycoprotein envelope of the virus. All flavivirus, including all four dengue serotypes, contain this fusion sequence and all serotypes present high sequence identity. Although intense researches, the dengue cases have increased and, up to date, there are no specific antiviral drugs to combat this disease. Thus, understanding the virus interaction with host cells is essential for the search and development of new drugs against the dengue virus. Therefore, this study craved a better understanding of the mode of action of the dengue fusion peptide sequence and the importance of its lateral regions. This project aimed to assess the contributions of the lateral regions of the fusion peptide (88-97 and 113-123 sequences) on its activity, for the four dengue serotypes. The synthesis of peptides was performed by solid phase peptide synthesis (SPPS). The membrane models used were zwitterionic micelles (LPC) and vesicles (egg PC and POPG). Since the exposure of this sequence and the resulting membrane fusion is dependent on pH, studies were performed in pH 5.5 and 7.1. In these studies, the techniques of Resonance Energy Transfer Föster (FRET), circular dichroism, fluorescence, Langmuir monolayers and BAM (Brewster angle microscopy) were used. The amino acid and natural fluorophore tryptophan was used as a probe to analyze the insertion depth of the fusion peptide in the membrane mimetic by fluorescence. The results showed that the SPPS was feasible, obtaining the desired materials. Assessing the fusogenic ability of 14 peptides studied, the data indicated that each serotype has different activity and the serotype 2 peptides presented the major interaction. The studies of interaction peptide/membrane mimetic indicated...
34
Pettersson, John H. O. "The Origin of the Genus Flavivirus and the Ecology of Tick-Borne Pathogens." Doctoral thesis, Uppsala universitet, Systematisk biologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-211090.
Full textAbstract:
The present thesis examines questions related to the temporal origin of the Flavivirus genus and the ecology of tick-borne pathogens. In the first study, we date the origin and divergence time of the Flavivirus genus. It has been argued that the first flaviviruses originated after the last glacial maximum. This has been contradicted by recent analyses estimating that the tick-borne flaviviruses emerged at least before 16,000 years ago. It has also been argued that the Powassan virus was introduced into North America at the time between the opening and splitting of the Beringian land bridge. Supported by tip date and biogeographical calibration, our results suggest that this genus originated circa 120,000 (156,100–322,700) years ago if the Tamana bat virus is included in the genus, or circa 85,000 (63,700–109,600) years ago excluding the Tamana bat virus. In the second study we estimate the prevalence of tick-borne encephalitis virus (TBEV) in host-seeking Ixodes ricinus from 29 localities in Sweden and compare our data with those of neighbouring countries. Nymphs and adult ticks were screened for TBEV using a real-time PCR assay. The mean TBEV prevalence for all tick stages combined was 0.26% for Sweden and 0.28% for all Scandinavian countries, excluding Iceland. The low prevalence of TBEV in nature may partly be explained by the fact that TBEV occurs in spatially small foci and that the inclusion of ticks from non-infected foci will reduce the prevalence estimate. In the third and fourth study, we conducted the first large-scale investigations to estimate the prevalence and geographical distribution of Anaplasma spp. and Rickettsia spp. in host-seeking larvae, nymphs and adults of I. ricinus ticks in Sweden. Ticks were collected from several localities in central and southern Sweden and were subsequently screened for the presence of Anaplasma spp. and Rickettsia spp. using a real-time PCR assay. For all active tick stages combined, the mean prevalence of Anaplasma spp. and Rickettsia spp. in I. ricinus in Sweden was estimated to 1.1% and 4.8%, respectively. It was also shown that A. phagocytophilum and R. helvetica are the main Anaplasma and Rickettsia species occurring in Sweden.
35
Mather, Stuart Thomas. "Development of pseudotyped virus assays for the serological study of Japanese encephalitis flavivirus." Thesis, University of Kent, 2017. https://kar.kent.ac.uk/62936/.
Full textAbstract:
Japanese encephalitis virus (JEV) is one of the primary global causes of viral encephalitis, with approximately 68,000 clinical cases and 20,000 deaths attributed to the virus annually. Between 30% and 50% of survivors suffer from debilitating neurological sequelae. Despite being a vaccine-preventable disease, no antiviral treatments are licensed and commercially available to counteract JEV infection. In order to quantify the neutralising antibody response raised against antigenic epitopes on flavivirus prME glycoproteins, conventional serological assays such as the plaque reduction neutralisation test (PRNT) can be employed. However, these assays often necessitate the handling of pathogenic wild-type virus in expensive high-biosafety laboratories, restricting the scope of their application, particularly in resource-deprived areas. Chimeric, replication-deficient pseudotype viruses can offer a solution to this problem, as they mimic wild-type virus entry mechanisms, enabling their use in pseudotype virus neutralisation assays (PVNAs). PVNAs bypass high biosafety requirements and permit vaccine evaluation and serosurveillance studies with no risk of inadvertent infection. This project focuses on the production of functional pseudotype viruses displaying the prM and E surface glycoproteins of the JEV flavivirus, for utilisation in serological neutralisation assays. Subcloning of the prME gene into an appropriate eukaryotic expression vector and insertion mutagenesis to produce prME with 15- and 24-residue upstream signal peptides are shown, before production of JEVpp with either HIV or MLV cores is attempted, via the conventional multi-plasmid co-transfection approach or the utilisation of constitutive gag-pol expressing cell lines. The impact of additional plasmid-derived furin protease expression and low glucose culture medium, as well as the construction of JEV/VSV chimeric prME glycoproteins and the introduction of Kozak consensus sequences upstream of the prME gene, to enhance the efficiency of JEVpp generation is also explored. Finally, the infectivity of lentiviral pseudotype viruses following lyophilisation, storage and reconstitution is confirmed, thus enabling their affordable global distribution.
36
Evangelista, Villavicencio Julio Antonio. "Caracterización de un nuevo flavivirus aislado de Culex (melanoconion) ocossa de Iquitos, Perú." Master's thesis, Universidad Nacional Mayor de San Marcos, 2016. https://hdl.handle.net/20.500.12672/4885.
Full textAbstract:
Describe el aislamiento y caracterización molecular de un nuevo flavivirus únicamente de mosquito, aislado de Culex (Melanoconion) occossa colectados en 2009 de un área urbana de Iquitos, Perú, ubicada en la cuenca del Amazonas, en la región noreste del Perú. La evidencia del Flavivirus es detectada mediante ensayo de inmunofluorescencia indirecta (IFI) en sobrenadante de cultivo celular de las células infectadas C6/36 usando anticuerpos policlonales grupo de Flavivirus y confirmada por RT-PCR. En comparación por pares de las secuencias de la región ENV, la más alta de nucleótidos (47,4%) y de aminoácidos (39,8%) la identidad se observa en el virus Nounané (NOUV). En comparación por pares de la región NS5, la identidad de nucleótidos más alta se observa en el virus Spondweni (65,9%), el virus de Iguape (IGUV; 65,7%) y el virus de Kedougou (65,6%); sin embargo, en el nivel de aminoácidos, la más alta identidad en pares de bases se observa en IGUV (69,8%), virus Naranjal (69,6%) y el virus Bussuquara (69,3%). El análisis filogenético utilizando ENV parcial y secuencias de aminoácidos NS5 revela que este Flavivirus forma un clado con NOUV. Para investigar la gama de huéspedes del nuevo Flavivirus, se inoculan una variedad de células de mamíferos (Vero 76, Vero E6, BHK, LLCMK y MDCK) con el tercer pasaje del aislamiento C6/36 y monitorea el efecto citopático (EC). No se detecta EC, y todas las líneas de células de mamífero son negativas para el antígeno Flavivirus por IFI y ARN Flavivirus por RT-PCR luego de catorce días de incubación. Propone que este Flavivirus genéticamente diferente sea llamado Nanay Virus (NANV), debido a la zona de Iquitos, Perú, donde fue detectado por primera vez.Tesis
37
Cespedes, Graziely Ferreira. "Sínteses e estudos dos diferentes peptídeos de fusão dos flavivirus causadores da dengue /." Araraquara, 2013. http://hdl.handle.net/11449/108473.
Full textAbstract:
Orientador: Eduardo Maffud CilliBanca: Reinaldo Marchetto
Banca: Saulo Santesso Garrido
Banca: Pietro Ciancaglini
Banca: Luciana Malavolta Quaglio
Resumo: O vírus da dengue afeta milhões de pessoas a cada ano no mundo todo. O processo de infecção viral pela via endossomal é intermediada pela sequência 98 a 112 (DRGWGNGCGLFGKGG), conhecido como peptídeo de fusão, este segmento faz parte da glicoproteína E, localizado no envelope do vírus. Todos os flavivirus, incluindo os quatro sorotipos da dengue, apresentam esta sequência em regiões próximas, além de apresentarem alta identidade. Apesar de intensas pesquisas e o aumento da incidência de casos da dengue, não existem drogas antivirais específicas, até este momento, para o combate desta doença. Deste modo, a compreensão da interação do vírus com as células hospedeiras é importante na busca de novos fármacos. Neste sentido, este trabalho visou entender melhor o modo de ação do peptídeo de fusão da dengue e a importância de suas regiões laterais. O objetivo deste projeto teve por finalidade avaliar as contribuições das regiões laterais ao peptídeo de fusão, isto é, 88-97 e 113-123, em sua atividade, para os quatro sorotipos da dengue. A obtenção dos peptídeos foi realizada por meio da síntese em fase sólida. Os modelos de membrana utilizados foram micelas zwitteriônicas (LPC) e vesículas (PC ovo e POPG). Sabendo-se que a exposição desta sequência e, consequente, fusão da membrana é dependente do pH do meio, os estudos foram realizados em pHs 5,5 e 7,1. Nestes estudos, foram utilizadas as técnicas de Transferência de Energia de Ressonância de Föster (FRET), Dicroísmo Circular, Fluorescência, Monocamadas de Langmuir e BAM. O aminoácido triptofano foi usado como sonda para analisar a profundidade da inserção em miméticos de membrana. Os resultados mostraram que a SPFS foi útil, obtendo os materiais desejados. Avaliando a habilidade fusogênica dos 14 peptídeos estudados, observamos que cada sorotipo apresenta atividade diferente um do outro, sendo que os peptídeos do sorotipo 2 foram os que...
Abstract: Dengue virus affects millions of people worldwide every year. The process of viral infection via endosomal is mediated by the sequence 98-112 (DRGWGNGCGLFGKGG), known as the fusion peptide. This segment is part of the glycoprotein envelope of the virus. All flavivirus, including all four dengue serotypes, contain this fusion sequence and all serotypes present high sequence identity. Although intense researches, the dengue cases have increased and, up to date, there are no specific antiviral drugs to combat this disease. Thus, understanding the virus interaction with host cells is essential for the search and development of new drugs against the dengue virus. Therefore, this study craved a better understanding of the mode of action of the dengue fusion peptide sequence and the importance of its lateral regions. This project aimed to assess the contributions of the lateral regions of the fusion peptide (88-97 and 113-123 sequences) on its activity, for the four dengue serotypes. The synthesis of peptides was performed by solid phase peptide synthesis (SPPS). The membrane models used were zwitterionic micelles (LPC) and vesicles (egg PC and POPG). Since the exposure of this sequence and the resulting membrane fusion is dependent on pH, studies were performed in pH 5.5 and 7.1. In these studies, the techniques of Resonance Energy Transfer Föster (FRET), circular dichroism, fluorescence, Langmuir monolayers and BAM (Brewster angle microscopy) were used. The amino acid and natural fluorophore tryptophan was used as a probe to analyze the insertion depth of the fusion peptide in the membrane mimetic by fluorescence. The results showed that the SPPS was feasible, obtaining the desired materials. Assessing the fusogenic ability of 14 peptides studied, the data indicated that each serotype has different activity and the serotype 2 peptides presented the major interaction. The studies of interaction peptide/membrane mimetic indicated...
Doutor
38
Serra, Otacília Pereira. "Arbovírus dos gêneros Flavivirus e Alphavirus em culicídeos capturados em Cuiabá, Mato Grosso." Universidade Federal de Mato Grosso, 2015. http://ri.ufmt.br/handle/1/694.
Full textAbstract:
Submitted by Valquíria Barbieri (kikibarbi@hotmail.com) on 2018-04-20T17:14:55Z
No. of bitstreams: 1
DISS_2015_Otacília Pereira Serra.pdf: 1299760 bytes, checksum: adf22b742c77d45b935dd95c6e46cbda (MD5)Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2018-04-27T17:52:07Z (GMT) No. of bitstreams: 1 DISS_2015_Otacília Pereira Serra.pdf: 1299760 bytes, checksum: adf22b742c77d45b935dd95c6e46cbda (MD5)
Made available in DSpace on 2018-04-27T17:52:07Z (GMT). No. of bitstreams: 1 DISS_2015_Otacília Pereira Serra.pdf: 1299760 bytes, checksum: adf22b742c77d45b935dd95c6e46cbda (MD5) Previous issue date: 2015-04-20
CAPES
FAPEMAT
Arbovírus são transmitidos por artrópodes hematófagos, representando um problema de saúde pública em áreas tropicais. O objetivo deste estudo foi investigar a diversidade de espécies de culicídeos e sua frequência de infecção por Alphavirus e Flavivirus em Cuiabá, MT. Foram realizadas capturas com aspirador de Nasci e puçá entre janeiro e abril de 2013 em três locais de 200 setores censitários, definidos aleatoriamente. Os culicídeos foram identificados com chave dicotômica de Forattini, alocados em pools (1-20 mosquitos) segundo sexo, espécie, data, local de coleta e armazenados a -80˚C. Pools de fêmeas foram submetidos à extração de RNA e DNA total, à multiplex semi-nested-RT-PCR para cinco alphavírus e 11 flavivírus e Nested- PCR para identificação de Culex (Cx.) quinquefasciatus. Amostras positivas para SLEV, DENV-1, -4 e MAYV foram submetidas a single RT-PCR e sequenciamento nucleotídico. Pools positivos para o MAYV foram inoculados em células Vero e submetidos a RT-PCR para o gene de envelope E1 dos alphavirus. Pools positivos para flavivirus foram inoculados em células C6/36 (Flavivirus). Calculou-se a taxa de infecção mínima (MIR). Foram capturados 11.090 mosquitos, 4.556 fêmeas de 14 espécies, perfazendo 610 pools. Foram analisados 171 pools de Aedes (Ae.) aegypti; 1 Ae. albopictus; 1 Aedes sp.; 5 Cx. bidens/interfor; 1 Cx. spinosus; 403 Cx. quinquefasciatus; 1 Galindomyia sp; 6 Limatus sp; 2 Mansonia wilsoni; 5 Psorophora (Ps.) sp; 1 Ps. ciliata; 11 Ps. varipes/albigenu; 1 Sabethes chloropterus e 1 Uranotaenia sp. Encontrou-se 1/171 (MIR=0,92) pool de Ae. aegypti positivo para DENV-1; 1/403 (MIR= 0,2) Cx. quinquefasciatus para SLEV genótipo V-A, 12/403 (MIR=3,5) de Cx. quinquefasciatus, 4/171 (MIR=3,67) de Ae. aegypti para MAYV; destes, cinco pools apresentaram co-infecção com DENV-4. Um produto de 1,3 kb obtido com o protocolo para o gene de envelope de três pools positivos para o MAYV resultou em sequências nucleotidicas inespecíficas. O MAYV foi isolado de dois pools contendo duas fêmeas não ingurgitadas de Ae. aegypti (#958) e duas de Cx. quinquefasciatus (#489). Positividade para DENV-4 foi identificada em 58/171 (MIR=53,35) Ae. aegytpi, 105/403 (MIR=30,65) Cx. quinquefasciatus, 2/5 (MIR=400) Psorophora sp, 2/11 (MIR=142,85) Ps. varipes/albigenu, 1/1 (MIR= 1000) Sabethes chloropterus, 2/5 (MIR=285.7) Cx. bidens/interfor e 1/1 (MIR=1000) Aedes sp. O DENV-4 foi isolado de dois pools contendo três (#329) e 16 (#806) fêmeas de Cx. quinquefasciatus não ingurgitadas. O SLEV, MAYV e os sorotipos do DENV foram identificados em pacientes com suspeita de dengue na cidade de Cuiabá em estudos prévios do Laboratório de Virologia. Experimentalmente, Culex e Aedes spp. são vetores competentes do MAYV. A identificação do vírus em fêmeas não ingurgitadas sugere que estas espécies podem estar envolvidas no ciclo urbano do MAYV em Cuiabá. Dentre os sorotipos do DENV, somente o DENV-1 e o DENV-4 foram identificados em culicídeos. O DENV-4 tem produzido importantes epidemias em MT desde 2012. A positividade para DENV-4 em diferentes espécies de culicídeos pode ser decorrente de infecção natural ou da hematofagia em humanos, pois muitos destes pools apresentavam fêmeas ingurgitadas. Cuiabá possui ecossistema favorável para a ocorrência de arboviroses e proliferação de vetores. Estudos envolvendo vigilância entomológica e virológica são importantes para estimar a situação epidemiológica dos arbovírus no estado.
Arbovirus are transmitted by hematophagous arthropods, posing a public health issue in tropical areas. The aim of this study was to investigate the diversity of culicidae and their frequency of infection by arboviruses from Alphavirus and Flavivirus genus in Cuiabá, MT. To achieve that, culicids were captured with Nasci aspirators and hand net between January and April 2013 in three locations of 200 censitary sectors randomly defined. The specimens were identified using the Forattini dichotomy key, allocated in pools (1-20 mosquitoes), according to sex, species, day and place of capture, and stored at -80˚C. Female pools were subjected to total RNA and DNA extraction and to multiplex semi-nested-RT-PCR for five alphaviruses and 11 flaviviruses and Nested-PCR for Culex (Cx.) quinquefasciatus identification. The pools positive for SLEV, DENV-1, -4 and MAYV were subjected to single RT-PCR and nucleotide sequencing. MAYVpositive pools were inoculated in Vero cells and subjected to RT-PCR for the E1 envelope gene of alphaviruses. Pools positive for flaviviruses were inoculated in C6/36 cells. The minimum infection rate (MIR) was calculated. 11,090 mosquitoes were captured, 4,556 females belonging to 14 species, comprising 610 pools, 171 pools of Aedes (Ae.) aegypti specimens; 1 Ae. albopictus; 1 Aedes sp.; 5 Cx. bidens/interfor; 1 Cx. spinosus; 403 Cx. quinquefasciatus; 1 Galindomyia sp.; 6 Limatus sp.; 2 Mansonia wilsoni; 5 Psorophora sp.; 1 Ps. ciliata; 11 Ps. varipes/albigenu; 1 Sabethes chloropterus e 1 Uranotaenia sp. Among them, 1/171 (MIR=0.92) Ae. aegypti pool was positive for DENV-1 and 1/403 (MIR=0.3) Cx. quinquefasciatus for SLEV genotype V-A. For MAYV, 12/403 (MIR=3.5) Cx. quinquefasciatus, 4/171 (MIR=3.67) Ae. aegypti were positive, five of them were also infected by DENV-4. A DNA product with 1.3 kb obtained from three pools positive for MAYV with the protocol for the envelope gene resulted in unspecific nucleotide sequences. MAYV was isolated from two pools, both containing two non-engorged females of Ae. aegypti (#958) and Cx. quinquefasciatus (#489). DENV-4 was detected in 58/171 (MIR=53.35) Ae. aegytpi, 105/403 (MIR=30,65) Cx. quinquefasciatus, 2/5 (MIR=400) Psorophora sp., 2/11 (MIR=142.85) Ps. varipes/albigenu, 1/1 (MIR= 1000) Sabethes chloropterus, 2/5 (MIR=285.7) Cx. bidens/interfor and 1/1 (MIR=1000) Aedes sp. DENV-4 was isolated from two pools containing three (#329) and 16 (#806) non-engorged females of Cx. quinquefasciatus. The SLEV, MAYV and the four DENV serotypes were identified in patients suspected of harboring dengue infection in Cuiabá in previous studies of the Virology Laboratory. Experimentally, Culex and Aedes spp. are competent vectors for MAYV. The identification of the virus in nonengorged females suggests these species may be involved in the urban cycle of MAYV in Cuiabá. Among the DENV serotypes, only DENV-1 and DENV-4 were identified in culicids captured in the city. DENV-4 has been responsible for major outbreaks in MT since 2012. The identification of DENV-4 in several mosquito species might be resultant either from natural infection or hematophagy in humans, since several of these pools presented engorged females. Cuiabá presents a favorable ecosystem for the occurrence of arboviruses and vector proliferation. Studies involving entomological and virological surveillance are important to estimate the epidemiological situation of arboviruses in the state.
39
Desole, Giovanna. "Comparative analysis of Zika virus and other Flavivirus infection in human neural cells." Doctoral thesis, Università degli studi di Padova, 2018. http://hdl.handle.net/11577/3424985.
Full textAbstract:
Background: Zika virus (ZIKV), West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that generally cause mild or asymptomatic disease in humans. However, ZIKV infection has been associated with fetal microcephaly and Guillain-Barrè syndrome in adults; WNV infection may evolve to severe neuroinvasive disease in the elderly and immunocompromised subjects; DENV may rarely cause neurological complications in infected individuals. In addition, another emerging mosquito-borne flavivirus, Usutu virus (USUV), which may cause fatal neuroinvasive disease in different bird species, has been recently shown to infect humans but its pathogenicity is unknown.
Aim of the study: In the context of the recent outbreak of ZIKV in the Americas and the increasing evidences of an association between ZIKV infection and the occurrence of fetal microcephaly, aim of this study was to investigate the effect of ZIKV infection in human neural cells in comparison with other flavivirus infections. To this aim, ZIKV infectivity, replication kinetics, cytopathic effect (CPE), and induction of innate antiviral responses were investigated in human induced pluripotent stem cells (hiPSCs), hiPSCs-derived neural stem cells (NSCs) and neurons and compared with other flaviviruses, i.e., WNV, DENV and USUV.
Methods: NSCs and neurons were differentiated from hiPSCs. hiPSCs, NSCs, and neurons were infected with isolates of ZIKV Asian lineage (KU853013), WNV lineage 2 (KF179640), DENV serotype 2, and USUV Europe lineage 1 (AY453411). Time course experiments were performed to evaluate viral load by qRT-PCR and TCID50, expression of host genes involved in antiviral innate immunity by qRT-PCR, expression of cell differentiation markers by IF and qRT-PCR, cell viability and cell death by flow cytometry. The impact of ZIKV on embryogenesis and neurogenesis was evaluated by infection of hiPSCs and NSCs during neural differentiation and embryo body formation.
Results: ZIKV infected and replicated efficiently in NSCs, neurons and hiPSCs. Infection led to typical CPE and cell death by apoptosis. ZIKV infection of hiPSCs, NSCs, and neurons induced the expression of innate immune response genes, especially the cellular pattern recognition receptor (PRR) IFH1 gene (MDA5), IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) and 2 (IFIT2). Infected embryoid bodies were massively destroyed by ZIKV infection and infected hiPSCs and NSCs died before ending the neural differentiation process. ZIKV replication efficiency in NSCs was significantly higher than that of DENV-2 and USUV, but lower than that of WNV. In particular, WNV replicated more efficiently, induced more cell death and higher levels of antiviral gene expression than ZIKV in NSCs, neurons and hiPSCs. The induction of innate immune response genes in NSCs after infection with ZIKV and DENV-2 infection was milder than after infection with WNV and USUV, in agreement with the adaptation of these viruses to the human host and their ability to shut down the antiviral response.
Conclusion: ZIKV infects and replicates efficiently in NSCs and induces cell death abrogating neural development, although less efficiently than WNV. Because of the similarities between flaviviruses in their interactions with host neural cells, it is conceivable that infection of other human cells, such as those involved in the extablishment of the blood-placenta barrier, are crucial for ZIKV-induced damage of the fetal brain.Presupposti dello studio: Zika virus (ZIKV), West Nile virus (WNV), dengue virus (DENV) e Usutu virus (USUV) sono trasmessi da zanzare ed appartengono al genere Flavivirus della famiglia Flaviviridae. L’infezione da ZIKV è associata a microcefalia fetale e sindrome di Guillan-Barrè; WNV può causare una grave sindrome neuroinvasiva nell’anziano e nei soggetti immunocompromessi; l’infezione da DENV raramente si associa a complicazioni neurologiche; USUV può causare una sindrome neuroinvasiva fatale in diverse specie di uccelli, è stato dimostrato che può infettare pure l’uomo, ma la sua patogenicità resta ancora da chiarire. Scopo: Alla luce della recente epidemia di ZIKV in America e di una probabile associazione tra l’infezione da ZIKV e lo sviluppo di microcefalia fetale, lo scopo di questo studio è stato confrontare l’infezione da ZIKV sulle cellule neurali umane con l’infezione da WNV, DENV e USUV. A tal fine, la cinetica di replicazione, l’effetto citopatico e l’immunità innata indotta dall’infezione virale sono state analizzate in cellule staminali pluripotenti indotte (hiPSCs), cellule staminali neurali derivate da iPSCs e neuroni. Materiali e metodi: Le NSCs ed i neuroni sono stati differenziati da hiPSCs. I diversi tipi cellulari sono stati infettati con l’isolato di ZIKV lignaggio asiatico (KU853013), WNV lignaggio 2 (KF179640), DENV sierotipo 2 e USUV lignaggio 1 europeo (AY453411). La carica virale è stata valutata a diversi tempi dall’infezione mediante qRT-PCR e TCID50, il livello di espressione dei geni coinvolti nell’immunità innata è stato analizzato mediante qRT-PCR e l’espressione dei markers di differenziamento cellulare mediante IF e qRT-PCR, la sopravvivenza cellulare e l’apoptosi mediante il saggio MTT e analisi dell’attivazione di caspasi-3. L’impatto dell’infezione da ZIKV sull’embriogenesi e la neurogenesi è stato valutato infettando le hiPSCs e le NSCs durante il differenziamento neurale e durante la formazione dei corpi embrioidi. Risultati: ZIKV era in grado di infettare e replicare efficientemente nelle NSCs, nei neuroni e nelle hiPSCs, causando un tipico effetto citopatico e morte cellulare per apoptosi. L’infezione ha indotto un significativo aumento dell’espressione dei geni dell’immunità innata, in particolare dei geni MDA5 (the cellular pattern recognition receptor (PRR) IFH1 gene), IFIT1 (IFN-induced protein with tetratricopeptide repeats 1) e IFIT2. I corpi embrioidi sono stati distrutti dal virus e le hiPSCs e le NSCs infettate sono morte prima di completare il differenziamento neurale. L’efficienza di replicazione di ZIKV nelle NSCs era maggiore rispetto a quella di DENV-2 e USUV, ma minore rispetto al WNV. Infatti, WNV replicava in modo più efficiente, induceva una maggiore morte cellulare e stimolava una più elevata risposta antivirale rispetto a ZIKV nei diversi tipi cellulari. Conclusione: ZIKV infetta e replica nelle NSCs, inducendo morte cellulare e impedendo lo sviluppo neurale, ma in modo meno efficiente rispetto al WNV. E’ probabile quindi che l’infezione di altri tipi cellulari sia determinante per il danno al sistema nervoso fetale indotto in modo specifico da ZIKV.
40
Shomiad, Shueb Rafidah Hanim. "Contribution of different components of innate and adaptive immunity to severity of flavivirus-induced encephalitis in susceptible and resistant hosts." University of Western Australia. School of Biomedical, Biomolecular and Chemical Sciences, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0199.
Full textAbstract:
[Truncate abstract] Flaviviruses are small, positive-stranded RNA viruses belonging to the family Flaviviridae. Flavivirus infection in humans could cause diseases ranging from febrile illnesses to fatal encephalitis. Mice provide a useful small animal model to study flavivirus-induced encephalitis in humans since mice also develop encephalitis during flavivirus infection. Some strains of mice have been shown to be resistant to flavivirus challenge and this resistance is conferred by a single autosomal dominant gene, designated as Flvr. Recently, OAS1b gene has been identified to be a gene candidate for Flvr. Several congenic resistant mouse strains have been developed by introducing resistance genes from outbred or wild mice onto the genetic background of susceptible C3H mice. These new resistant strains that carry different allelic variants at the Flv locus include C3H/PRI-Flvr (RV), C3H.MOLD-Flvmr (MOLD) and C3H.M.domesticus-Flvr-like (DUB), the latter two being developed in the same laboratory in which the work described in this thesis was accomplished. Preliminary studies in this laboratory found that flavivirus resistant mice are vulnerable to certain flavivirus infections, particularly when challenged by intracerebral (i.c.) route. Intracerebral (i.c.) challenge with flaviviruses such as West Nile virus (WNV) Sarafend strain and Kunjin virus (KUNV) MRM16 strain were found to induce high mortality in flavivirus resistant mice while infection with Murray Valley encephalitis virus (MVEV) OR2 strain did not cause any apparent disease in the same mice. ... Thus, it can be concluded that CD8+ T cells exerted harmful effect to resistant DUB mice during KUNV i.c. infection by producing excessive IFN[gamma] that could be toxic, causing functional loss of the CNS cells. It was shown from in vitro studies that WNV had the highest tropism for macrophages and dendritic cells, followed by KUNV. MVEV however did not replicate well in these cells. This combined with the data from the in vivo studies indicates that macrophages might be involved in the pathogenesis of intraperitoneal (i.p.) infection of WNV but not KUNV and MVEV. The reason for this could be that the production of KUNV in macrophages may not be high enough to induce viraemia and subsequent fatal encephalitis in mice. In contrast, MVEV appears to use different mechanism or cells for virus dissemination. Although macrophages may not be involved in KUNV pathogenesis after i.p. infection, the fact that macrophages support KUNV replication in vitro may indicate the possibility that blood-borne macrophages were recruited to the brain where they can get infected with KUNV during i.c. infection and therefore could participate in KUNV pathogenesis in DUB mice. This study provides evidence for the first time on the detrimental effect of host antiviral immunity and inflammatory mediators during flavivirus i.c. infection in resistant mice. However, it also launches a new question on the selective cell tropism of KUNV versus MVEV responsible for inducing different pattern of immune responses and consequently leading to different outcomes of infection in resistant mice.
41
Pastorino, Boris. "Etude du complexe protéasique des flavivirus et des alphavirus : caractérisations enzymatiques et recherche de cibles cellulaires." Aix-Marseille 2, 2008. http://www.theses.fr/2008AIX20671.
Full text
42
Rechoum, Yassine. "Détection de séquences d'ARN du VHC dans des extraits de moustiques du genre Aedes : étude cinétique après infections expérimentales." Grenoble 1, 2009. http://www.theses.fr/2009GRE10205.
Full textAbstract:
Dans le but d'explorer la capacité des moustiques à répliquer le VHC, nous avons réalisé une série d'infections expérimentales sur Aede caspius, Aedes vexans et genre Cu/ex pipiens sauvage et d'élevage. Après collecte des moustiques, les femelles ont été nourries par un repas sanguin virémique (VHC 1 b positif). Les moustiques ont ensuite été maintenus en élevage pendant plusieurs jours. Après mise au point des conditions de détection, nous avons réussi à apporter la preuve de la réplication du VHC dans les moustiques du genre Aedesvexans. Dans la première infection expérimentale réalisée sur Ae. Vexans , nous avons détecté de l'ARN du VHC dans -50% des moustiques du 21,ème jours post-infection (JPI), par PCR en point final. La seconde expérience d'infection, réalisée sur les deux genres dE moustiques: Aedes Sp, et Cu/ex pipiens, nous a permis de : (i) confirmer par qRT-PCR le résultat de la première expérience avec un tau: d'infection de -33% ; (ii) démontrer que seuls les moustiques du genre Aedes contenaient de l'ARN du VHC, les moustiques du genre Cu/ex étaient tous négatif; (iii) montrer que l'ARN détecté était bien issu de la réplication, dans la mesure où des mutations d'adaptation ont été identifiées dans la séquence de la RdRp (ARN Polymérase ARN dépendante), avec une spécificité pour le genre Aedes, puisque tous les moustiques Cu/ex à 21 JPI étaient ARN VHC-négatifs. Un autre moyen de distinguer l'ARN issu de la réplication de l'A RN résiduel a été de montrer l'existence d'une dissémination au sein du moustique. Pour ce faire, nous avons réalisé une troisième expérience d'infections (genres Ae. Caspius et Ae. Vexans), sur une durée de 15 jours et avons analysé la présence de l'ARN du VHC distinctement dans les têtes et les corps. En effectuant des prélèvements à différentes dates après infection (0, 4, 8 et 15 jours), nous avons réussi à obtenir une cinétique d'infection, séparément, dans les têtes et dans les corps, détection réalisée parWTA (Whole Transcriptome Amplification) suivie de qPCR. Au jour de l'infection (JO), le taux d'infection dans les têtes et les corps des moustiques éta de 9,1 % et 100% respectivement, ce taux est passé à 57,1 % et 85,7% respectivement à J15 après une négativation huit jours après infection. D'autre part, nous avons identifié des mutations d'adaptation dans la séquence de l'IRES des moustiques à J15 comparés à ceux à JO. Il est intéressant de noter que les moustiques ARN VHC-positifs appartenaient tous à l'espèce vexans, montrant ainsi que la réplication est spécifique d'espèce. Différentes régions du génome du VHC ont, ensuite, étaient amplifiées. Enfin, nous avons eu recoun à des moustiques d'élevage. L'infection de cette souche a également montré, en utilisant différentes approches de détection (qRT-PCR, WTA-qPCR, HCV-GA notamment), qu'à 30 JPI, l'ARN du VHC était présent dans -33% des têtes et -66% des corps, alors que les taux d'infection à JO étaient de -28% et -71 % dans les têtes et les corps respectivement. Les résultats se sont appuyés sur des séquençage effectués sur les régions amplifiées du génome du VHC. L'ensemble de ces résultats montrent que le VHC est capable de se répliquer dans les moustiques du genre Ae. Vexans, ce qui suggère que le VHC, à l'image d'autres F/avivirus, puisse alterner entre deux hôtes rimates et moustiQues) et Qu'il ait perdu son potentiel de réplication chez le moustiQue au fil du tempsLn order to explore the ability of mosquitoes to replicate HCV, we conducted a series of experimental infections caspius on Aedes, Aedes vexans and Culex pipiens wild and farmed. After collection of mosquitoes, the females were fed a blood meal viremic (HCV 1b positive). The mosquitoes were th en maintained in breeding for several days. After development of detection conditions, we were able to demonstrate HCV repli cation in the mosquito Aedes vexans. Ln the first experimental infection performed on Ae. Vexans, we detected HC' RNA in - 50% of mosquitoes 21st days post-infection (JPI), PCR end point. The second infection experiment, conducted on Iwo types of mosquitoes: Aedes SP, and Culex pipiens, has allowed us to: (i) confirmation by qRT-PCR results of the first experience with an infection rate of - 33%, (ii) demonstrate that only the mosquito Aedes contained RNA HCV, the Culex mosquitoes were ail negative, (iii) show that the RNA detected was good of replication, the extent of adaptive mutations were identified in the sequence of the RdRp (RNA polymerasi RNA dependent), with specificity for the genus Aedes, since ail Culex mosquitoes at 21 YPI were HCV RNA-negative. Another way to distinguish RNA from the replication of RNA remaining was to show the existence of a release within the mosquito. To do this, we conducted a third experiment infections (gender Ae. Caspius and Ae. Vexans), over a'period of 15 days and analyzed the presence of HCV RNA in distinct heads and bodies. Ln conducting samples at different times after infection (0, 4, 8 and 15 days), we managed to obtain a kinetic infection separately in heads and bodies, detection performed by WTA (Whole transcriptomics Amplification) followed by qPCR. On the day of infection (DO), the infection rate in the heads and bodies of mosquitoes was 9. 1 % and 100% respectively, the rate rose to 57. 1% and 85. 7% respectively J15 negativity after eight days after infection. On the other hand, we have identified mutations in th adaptation of the IRES sequence of mosquito D15 compared to those on day O. It is interesting to note that mosquito-positive HCV RNA ail belonged to the species vexans, showing that replication is species specific. Different regions of the HCV genome, then, were amplified. Finally, we made use of mosquito breeding. Infection of this strain has also shown, using different approaches to detection (qRT-PCR, qPCR-WTA, including HCV-GA), only 30 JPI, HCV RNA was present in - 33% heads and - 66% of the body, while infection rates were JO - 28% - 71 % in the head and body respectively. The results were based on sequencing performed on the amplified region! of the HCV genome. Ali these results show that HCV is able to replicate in mosquitoes of the genus Ae. Vexans, suggesting that HCV, likt other flaviviruses, can alternate belween Iwo hosts (primates and mosquitoes) and has lost its potential for replication in the mosquito ove time
43
Orico, Lilian Dias. "Pesquisa de infecções por Flavivirus sp. em aves silvestres provenientes das áreas verdes do município de São Paulo." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/6/6132/tde-11112013-142955/.
Full textAbstract:
INTRODUÇÃO: Em grandes cidades, como São Paulo, a pequena porcentagem de matas existentes é representada pelos parques municipais. Estas áreas, além de representarem ambientes de lazer para as pessoas, albergam uma enorme diversidade biológica, desde mosquitos até aves e mamíferos. A interação destas espécies favorece a circulação de Flavivirus, causadores de importantes doenças humanas, que têm nas aves um importante reservatório. Atribui-se às aves migratórias o papel de carreadoras dos vírus, já que as mesmas percorrem longas distâncias para completar seu ciclo biológico. A chegada de aves do Hemisfério Norte ocorre em alguns Parques, o que favoreceria a dispersão de alguns vírus, como o Vírus do Nilo Ocidental, já que nestas áreas estão presentes mosquitos potencialmente vetores. OBJETIVOS: identificar infecção por Flavivírus nas aves dos parques municipais de São Paulo. MÉTODOS: De Março de 2012 a Janeiro de 2013, foram coletadas amostras de swab de cloaca, orofaringe e sangue de aves capturadas em redes de neblina de duas áreas do município de São Paulo: Parque Anhanguera e Fazenda Castanheiras/APA Bororé Colônia. As aves foram anilhadas e liberadas após a coleta do material. Foi realizada técnica de RT-PCR em tempo real, utilizando iniciadores genéricos que amplificam fragmento do gene NS5 de Flavivirus. Amostras positivas foram encaminhadas para sequenciamento. RESULTADOS: Foi capturado um total de 231 aves, em sua maioria da Ordem Passeriformes. De um total de 463 amostras, nenhuma amostra apresentou presença de RNA viral. DISCUSSÃO: Em se tratando de alguns Flavivírus, os passeriformes são considerados os reservatórios mais competentes no ciclo de transmissão, pois atingem altos níveis de viremia. Cabe ressaltar que algumas espécies desta ordem, de ocorrência na cidade de São Paulo, já foram identificadas como portadoras dos vírus Rocio e Ilhéus. A ausência de positividade é esperada, pois embora altamente sensível, a técnica de PCR depende do estado de viremia das aves, que é curta. CONCLUSÃO: Os parques municipais são áreas que aproximam aves, mosquitos e humanos, pelo papel ambiental e de lazer que os mesmos representam. Este fato classifica estas áreas como locais com potencial de transmissão de Flavivirus, o que torna importante a continuação este estudo, aumentando as áreas de abrangência, para conhecer os Flavivírus circulantes e realizar vigilância para vírus que podem ocasionar problemas de Saúde PúblicaINTRODUCTION: In big cities, as São Paulo, the little percentage of existing forests is represented by the municipal parks. These áreas, besides acting as entertainment environments to the users, promote a huge biodiversity, including mosquitoes, birds and mammals. These species interaction promotes Flavivirus circulation, viruses responsible for important human diseases. The avian species are important reservoirs for these viruses, specially the migrating birds that can fly for long distances, carrying these viruses to several areas. In some parks, the arrival of migrating birds from the North Hemisphere is documented, fact that can support some viruses dispersion, for example, the West Nile Vírus, considering that in these areas potencial vector for this virus can be found. OBJECTIVES: detect Flavivirus infection in birds captured in municipal parks of São Paulo city. METHODS: from March, 2012 to January, 2013, oropharyngeal and cloacal swabs, and blood samples were collected from birds captured in mist-net webs located in two municipal areas: Anhanguera Park and Castanheiras Farm/APA Bororé Colônia. The birds were ringed and released after samples collection. The real time RT-PCR was performed, using generic primers which amplify NS5 gene fragment. Positive samples were forwarded to sequence analysis. RESULTS: a total of 231 birds were capture, in which the majority belongs to Passeriforms order. Of 463 samples collected, all samples were negative for the viral RNA presence. DISCUSSION: when talking about Flaviviruses, the passeriforms are considered the most competent reservoirs in the transmission cycle, because they can achieve great viremia levels. Some passeriforms species, endemic in São Paulo city, were identified as Rocio and Ilhéus viruses carriers in previous studies. The negativity is expected, once the Real Time RT-PCR, although highly sensitive, depends on the viremia duration, which is short in avian species. CONCLUSION: the public parks are areas which encloses birds, mosquitoes and the human being, for the environmental and entertainment role they play. This fact classifies these parks as areas with great potencial of Flavivirus transmission, ressalting the great importance to continue this study, increasing the number of areas, to detect the circulating Flavivirus and to perform surveillance for viruses which can cause public health problems
44
Bouvet, Mickaël. "Etude d'enzymes de modification d'ARN impliquées dans la réplication des flavivirus et des coronavirus." Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX20714.
Full textAbstract:
Ce travail de thèse a porté sur l’étude d’activités enzymatiques virales impliquées dans la réplication de deux genres viraux : les Flavivirus et les Coronavirus. Dans un premier temps, nous avons étudié des activités enzymatiques impliquées dans la formation de la structure coiffe des ARNm viraux. En effet, du fait de leur cycle réplicatif cytoplasmique, ces virus n’ont pas accès à la machinerie de formation de la coiffe cellulaire et expriment donc une machinerie dédiée. Le processus canonique de formation de la coiffe fait appel à quatre activités enzymatiques, une ARN 5’-triphosphatase, une guanylyltransférase et deux méthyltransférases.Chez les flavivirus, nous avons développé des outils permettant d’identifier l’activité guanylyltransférase ainsi que des essais enzymatiques nécessaires à la caractérisation des activités méthyltransférases. Ces outils nous ont notamment permis d’évaluer l’effet inhibiteur de molécules choisies par des méthodes de criblages virtuels sur les deux activités méthyltransférases de la protéine NS5 nécessaires à la formation de la coiffe.Chez les coronavirus, nous nous sommes intéressés à une activité méthyltransférase impliquée dans la formation de la coiffe et notamment à sa régulation par un partenaire viral. Nous avons démontré que le processus de méthylation de la coiffe suit un ordre obligatoire, initié par la méthylation de la position N7 par la protéine nsp14. Dans une seconde étape, les structures coiffe-0 (7MeGpppA) sont converties en coiffe-1 (7MeGpppA2’OMe) par la protéine nsp16 en complexe avec nsp10. Nous avons démontré que l’activité 2’O-méthyltransférase portée par la protéine nsp16 nécessite une interaction spécifique avec la protéine nsp10 qui joue probablement un rôle d’échafaudage.Dans un second temps, nous avons démontré que l’activité exoribonucléase portée par la protéine nsp14 est également régulée par la protéine nsp10. La stimulation de l’activité passe par une interaction directe entre les deux protéines et il semble que les surfaces d’interaction de nsp10 avec nsp14 et nsp16 soient chevauchantes. Enfin, la caractérisation de l’activité exoribonucléase confirme la possibilité de son implication dans un mécanisme de réparation des erreurs incorporées lors de la synthèse d’ARN par la polymérase viraleThis work focused on enzymatic activities of two RNA virus genera, Flavivirus and Coronavirus.We first studied the mRNA cap synthesis machinery of these viruses. Indeed, as they replicate in the cytoplasm of the infected cell, these viruses encode their own mRNA cap-forming enzymes. The canonical mechanism of cap synthesis uses four enzymatic activities, a RNA 5’-triphosphatase, a guanylyltransferase and two methyltransferases.We tried to identify the guanylyltransferase activity involved in this process for flaviviruses and we developed enzymatic assays to characterize both guanylyltransferase and methyltransferase activities. We used the methyltransferase assay in order to test the inhibitor effect of molecules, selected by virtual screening, on the methyltransferase activities of the NS5 protein involved in the capping process.Concerning coronaviruses, we first focused on the methyltransferase activities of the nsp14 and nsp16 proteins. We have reconstituted the complete SARS-CoV mRNA cap methylation in vitro. We showed that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 7MeGpppA-RNAs. The latter are then selectively 2′O-methylated by the 2′O-methyltransferase nsp16 in complex with its activator nsp10 to give rise to cap-1 7MeGpppA2′OMe-RNAs. Then, we took interest in the exoribonuclease activity of the nsp14 protein and found that this activity is also regulated by the same cofactor, the nsp10 protein. The interaction between the proteins is required to observe the stimulatory effect and it seems that the surface areas of nsp10 interacting with nsp14 and nsp16 overlap. The in vitro characterization of the nuclease activity of nsp14 is according with its potential implication in RNA proofreading mechanism
45
CARVALHO, Amanda Gomes de Oliveira. "Utilização do vírus da febre amarela no desenvolvimento de ferramentas para o uso em diagnóstico diferencial e descoberta de novos antivirais contra flavivírus." reponame:Repositório Institucional da UFPE, 2015. https://repositorio.ufpe.br/handle/123456789/14114.
Full textAbstract:
Submitted by Daniella Sodre (daniella.sodre@ufpe.br) on 2015-07-03T12:30:18Z
No. of bitstreams: 2
license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5)
Tese Amanda Oliveira Genética_biblioteca 2015.1.pdf: 4426810 bytes, checksum: 6cb96a980de7c8479debfd9b3835f0e1 (MD5)Made available in DSpace on 2015-07-03T12:30:18Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Amanda Oliveira Genética_biblioteca 2015.1.pdf: 4426810 bytes, checksum: 6cb96a980de7c8479debfd9b3835f0e1 (MD5) Previous issue date: 2015-03-02
FACEPE; CNPq
Os Flavivírus estão entre os vírus emergentes mais importantes do mundo. Ainda não há tratamento específico para nenhum membro da família Flaviviridae e novos casos são continuamente anunciados, com centenas de mortes. Logo, é de prioridade o desenvolvimento de vacinas, antivirais e métodos de diagnóstico, precisos e rápidos, principalmente de baixo custo para este gênero. O objetivo deste trabalho foi desenvolver ferramentas para o uso em diagnóstico sorológico diferencial de flavivírus, em NB2, através do uso de técnicas de genética reversa e triagem em larga escala de extratos naturais com atividade antiviral contra flavivírus. Para o diagnóstico sorológico diferencial, foram construídas três quimeras substituindo as glicoproteínas estruturais prM/E do vírus da febre amarela (YFV) pelas dos vírus Ilhéus (ILHV), vírus rocio (ROCV) e vírus encefalite de São Luís (SLEV). As quimeras construídas apresentaram replicação atenuada em cultivo celular, quando comparadas ao vírus parental. A quimera YFV-WNV (vírus Oeste do Nilo), construída anteriormente, foi utilizada para o estabelecimento de um protocolo de diagnóstico sorológico por ELISA de captura. Analisou-se 164 amostras de soros de equinos do Estado de Pernambuco para o WNV por ELISA, resultando em 62 amostras positivas. Todos os quatro vírus quiméricos citados foram utilizados na validação dos resultados por PRNT50. No diagnóstico diferencial por PRNT50, de um total de 62 amostras positivas no ELISA, observaram-se três amostras positivas para WNV, 27 positivas para ILHV, quatro para o ROCV, quatro para o SLEV e uma para YFV17DD. No segundo enfoque do trabalho utilizou-se a linhagem celular BHK-21- rep-YFV17D-LucNeoIRES, contendo o replicon bicistrônico repórter do YFV e o vírus repórter YFV-GLuc para triar 5200 extratos, onde foram identificados 73 extratos naturais com possível atividade antiviral contra o YFV, confirmados por ensaio utilizando o YFV17DD. Essa técnica inovadora de triagem de drogas, em larga escala, deve auxiliar o desenvolvimento de antivirais contra flaviviroses, assim como o protocolo de diagnóstico diferencial deverá auxiliar no correto diagnóstico e no monitoramento de flavivírus emergentes no Brasil.
46
Rückert, Claudia. "Alphavirus and flavivirus infection of Ixodes tick cell lines : an insight into tick antiviral immunity." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/10063.
Full textAbstract:
Arthropod-borne viruses, arboviruses, have the ability to replicate in both vertebrates and invertebrates and are transmitted to susceptible vertebrate hosts by vectors such as mosquitoes and ticks. Ticks are important vectors of many highly pathogenic arboviruses, including the flavivirus tick-borne encephalitis virus (TBEV) and the nairovirus Crimean-Congo haemorrhagic fever virus. In contrast, alphaviruses are principally mosquito-borne and have been isolated only rarely from ticks; ticks have not been implicated as their vectors. Nevertheless, the alphavirus Semliki Forest virus (SFV) replicates in cell lines derived from many different tick species, including those of the genus Ixodes, which includes vectors of TBEV and its lesspathogenic relative Langat virus (LGTV). In vertebrate cells, arboviruses generally cause cytopathic effects; however, arbovirus infection of arthropod cells usually results in a persistent low-level infection without cell death. While little is known about antiviral immunity in tick cells, the immune system of other arbovirus vectors such as mosquitoes has been studied extensively over the last decade. In insects, pathways such as RNA interference (RNAi), JAK/STAT, Toll, Imd and melanisation have been implicated in controlling arbovirus infection, with RNAi being considered the most important antiviral mechanism. In tick cells, RNAi has been shown to have an antiviral effect, but current knowledge of other immunity pathways is limited and none have been implicated in the antiviral response. In the present study, SFV and LGTV replication in selected Ixodes spp. tick cell lines was characterised and the Ixodes scapularis-derived cell line IDE8 was identified as a suitable cell line for this project. Potential antiviral innate immunity pathways were investigated; putative components of the tick JAK/STAT, Toll and Imd pathways were identified by BLAST search using available sequences from well-studied arthropods including the fruit fly Drosophila melanogaster. Using gene silencing, an attempt was made to determine whether these pathways play a role in controlling SFV and LGTV infection in tick cell lines. Selected genes were silenced in IDE8 cells using long target-specific dsRNA and cells were subsequently infected with either SFV or LGTV. Effects of gene silencing on virus replication were assessed by quantitative real time PCR (qPCR) or luciferase reporter assay. Effects on infectious virus production were measured by plaque assay. Replication of the orbivirus St Croix River virus (SCRV), which chronically infects IDE8 cells, was also quantified by qPCR after silencing of selected genes. Interestingly, SFV or LGTV infection of IDE8 cells resulted in a significant increase in SCRV replication, possibly as a result of interference with antiviral pathways by SFV and LGTV or possibly due to diversion of cellular responses from sole control of SCRV. No evidence for an antiviral role for the JAK/STAT or Toll pathways was found in IDE8 cells. However, an antiviral effect was observed for protein orthologues putatively involved in the RNAi response. Argonaute proteins play an important role in translation inhibition and target degradation mediated by RNAi, and silencing of selected Argonaute proteins resulted in a significant increase in SFV and SCRV replication. The carboxypeptidase CG4572 is essential for an efficient antiviral response in D. melanogaster, and supposedly involved in the systemic RNAi response. A putative tick orthologue of CG4572 was identified and this appeared to be involved in the antiviral response in IDE8 tick cells. When expression of CG4572 was silenced and cells subsequently infected with SFV or LGTV, replication of both viruses was significantly increased. In addition, it was shown that three mosquito orthologues of CG4572 also had an antiviral role against SFV in Aedes mosquito cells. In conclusion, of the tick cell lines investigated, IDE8 provided a suitable model system for investigating tick cell responses against arboviruses and new insight into the nature of the tick cell antiviral response was gained.
47
Youseff, Brian. "The Role of Tumor Necrosis Factor Receptor-Associated Factor 6 in Tick-Borne Flavivirus Infection." University of Toledo Health Science Campus / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=mco155691388498993.
Full text
48
Bessaud, Maël. "Etude in vitro du complexe protéasique [NS2B/NS3] des flavivirus, cible potentielle de molécules antivirales." Aix-Marseille 2, 2005. http://theses.univ-amu.fr.lama.univ-amu.fr/2005AIX22027.pdf.
Full text
49
Elbahesh, Husni. "Functional Analysis of the Murine Oligoadenylate Synthetase 1b (Oas1b)." Digital Archive @ GSU, 2006. http://digitalarchive.gsu.edu/biology_theses/3.
Full textAbstract:
The flavivirus resistance gene, Flv, in mice has been identified as 2'-5' oligoadenylate synthetase 1b (Oas1b). Susceptible mice produce a protein that is truncated (Oas1btr) at the C-terminus due to a premature stop codon encoded by a C820T transition. Mice produce 8 Oas1 proteins, Oas1a-Oas1h. In the present study, Oas1a, Oas1b and Oas1btr were expressed as MBP-fusion proteins in bacteria and purified. 2-5A synthetase activity was demonstrated using MBP-Oas1a, while neither MBP-Oas1b nor MBP-Oas1btr were functionally active. The 2-5A synthetase activity of MBP-Oas1a was inhibited in a dose-dependent manner by the addition of MBP-Oas1b but not MBPOas1btr. Finally, three RNA probes were synthesized from the 3' end of the WNV Eg101 genome and used to test the ability of the expressed Oas1 proteins to bind to viral RNA. Results of the RNA binding activity assays suggest Oas1 proteins may specifically interact with regions of WNV RNA.
50
Crochu, Sandrine. "Caractérisation génétique de virus atypiques du lignage des Flavivirus et mise en évidence d'un nouveau mécanisme évolutif des cellules eucaryotes." Aix-Marseille 2, 2004. http://www.theses.fr/2004AIX20674.
Full text