Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-06-18T17:05:33Z No. of bitstreams: 1 Dissertacao_BCM_JorgeGomesGoulartFerreira.pdf: 1660507 bytes, checksum: f8f7845b0fe11483b4ccf916a903b743 (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-06-18T17:05:48Z (GMT) No. of bitstreams: 1 Dissertacao_BCM_JorgeGomesGoulartFerreira.pdf: 1660507 bytes, checksum: f8f7845b0fe11483b4ccf916a903b743 (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-06-18T17:05:57Z (GMT) No. of bitstreams: 1 Dissertacao_BCM_JorgeGomesGoulartFerreira.pdf: 1660507 bytes, checksum: f8f7845b0fe11483b4ccf916a903b743 (MD5) / Made available in DSpace on 2015-06-18T17:05:57Z (GMT). No. of bitstreams: 1 Dissertacao_BCM_JorgeGomesGoulartFerreira.pdf: 1660507 bytes, checksum: f8f7845b0fe11483b4ccf916a903b743 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou / A família Bunyaviridae consiste em uma das maiores e mais diversificadas famílias de vírus de RNA, contendo cerca de 350 vírus sorologicamente distintos. O Apeu virus (APEUV) é um vírus da família Bunyaviridae que se destaca por seu grande potencial emergente. Isolado pela primeira no Brasil, este vírus pode causar uma doença que apresenta sintomas semelhantes aos da gripe, como febre alta, dor de cabeça e mialgia, associadas geralmente a náuseas, vômitos, fraqueza e fotofobia. Entretanto, apesar de seu potencial patogênico, pouco se sabe sobre a sua interação com o sistema imunológico humano. Com o objetivo de estudar alguns aspectos da resposta imune, principalmente a reposta inata desencadeada pela infecção do APEUV, a expressão de 19 genes (TLR3, TLR7, TLR8, TLR9, MyD88, IRF3, IRF5, IRF7, IRF9, IRAK4, TRAF3, TRAF6, TICAM1, JUN, ROBO-3(RIG-1), IFIH1(MDA-5), IFNα, IFNβ, IFNy) foi analisada. Para tal, foram feitos ensaios de qPCR baseados na metodologia TaqMan®, utilizando cDNA obtido a partir de RNA total extraído de células mononucleares do sangue periférico (PBMC) e de células A549 (linhagem derivada de carcinoma pulmonar humano), infectadas ou não com APEUV por períodos de 4 ou 8 horas. Como controles, foram utilizadas células infectadas com o vírus da estomatite vesicular (VSV) como controle positivo de uma infecção por vírus de RNA fita simples, e células tratadas com o mock das amostras de vírus como controle negativo. Os dados obtidos foram analisados em software específico. Nossos resultados indicam que PBMC infectadas com APEUV por 4 horas (m.o.i.=1 e m.o.i.=3) induzem um aumento na expressão de TLR9 e IFNβ. Quando quantificada a expressão de genes em células A549 infectadas com APEUV (m.o.i.=1 por 4 horas) comparadas com o mock, verificou-se um aumento da expressão dos genes TLR9, IRF3 e IRF7 e no período de 8 horas, também comparando com o mock, verificou-se aumento de forma significativa na expressão de TLR 9, além de um aumento na expressão de TLR3, TLR7, TRAF3 , IRF7 e IFNβ. Verificamos ainda que, após escolher um gene housekeeping através de método estatístico, células A549 infectadas com APEUV em uma m.o.i.=1, tende a aumentar a expressão dos genes IFNβ eTICAM-I, fundamentais na indução de um estado celular antiviral. Estudos posteriores são necessários para a determinação dos mecanismos envolvidos na resposta imune inata humana contra o Apeu virus / The Bunyaviridae family consists of the largest and most diverse families of RNA viruses, containing about 350 serologically distinct viruses. The Apeu virus (APEUV) is a member of the Bunyaviridae family that stands out for its large emerging potential. First isolated in Brazil, this virus can cause a flu-like disease with symptoms such as fever, headache and myalgia, usually associated with nausea, vomiting, weakness and photophobia. However, despite their pathogenic potential, little is known about their interaction with the human immune system. In order to study some aspects of the immune response, especially the innate response triggered by APEUV infection, the expression of 19 genes (TLR3, TLR7, TLR8, TLR9, MyD88, IRF3, IRF5, IRF7, IRF9, IRAK4, TRAF3, TRAF6, TICAM1, JUN, robot-3 (RIG-1) IFIH1 (MDA-5), IFNα, IFNβ, IFNy) was analyzed. For that, qPCR assays were performed based on the TaqMan method using cDNA obtained from mRNA extracted from peripheral blood mononuclear cells (PBMC) and A549 cells (cell line derived from human lung carcinoma) infected or not by APEUV for 4 or 8 hours. Cells infected with vesicular stomatitis virus (VSV) were used as a positive control of infection with single stranded RNA viruses; also mock infected cells were used as controls. The data were analyzed using specific software. Our results suggested that APEUV infection on PBMC induce an increase of TLR9 and IFNβ expression on both situations with m.o.i=1 or m.o.i=3. On the A549 cell line, APEUV infection increased the expression of TLR9, TLR3, TLR7, TRAF3, IRF7 and IFNβ using m.o.i=1 and 4h infection condition, compared to mock control. After using a statistical methodology to choose a housekeeping gene, we verified that APEUV infection with m.o.i=1, increase the expression of IFNβ and IRF9 in A549 cells. A cell antiviral state is induced by the presence of IFNβ and IRF9. Further studies are needed to determine the mechanisms involved in human innate immune response against APEUV.

Bunyaviridae is one of the biggest known viral families, and includes many viruses of clinical and economic importance. The major virulence factor of most bunyaviruses is the non-structural protein (NSs). NSs is expressed early in infection and inhibits the innate immune response of the host by blocking several steps in the interferon induction and signalling pathways. Hence, NSs significantly contributes to the establishment of a successful viral infection and replication, persistent infection and the zoonotic capacity of bunyaviruses. Although functions and structures of many viral interferon antagonists are known, no structure of a bunyavirus NSs protein has been solved to date. This strongly limits our understanding of the role and the mechanism of interferon antagonism in this large virus family. In this work the first structure for a bunyavirus interferon antagonist, the core domain crystal structure of NSs from the Rift Valley fever virus (RVFV) is presented. RVFV is one of the most clinically significant members of the Bunyaviridae family, causing recurrent epidemics in Africa and Arabia, often featuring high-mortality haemorrhagic fevers. The structure shows a novel all-helical fold. The unique molecular packing of NSs in the crystal creates stable fibrillar networks, which could correspond to the characteristic fibrillation of NSs observed in vivo in the nuclei of RVFV infected cells. This first NSs structure might be a useful template for future structure-aided design of drugs that target the RVFV interferon antagonism. Attempts at characterising other bunyavirus NSs proteins of other genera were made, but were hampered by problems with obtaining sufficient amounts of soluble and folded protein. The approaches that proved unsuccessful for the solubilisation of these NSs proteins, however, should inform future experiments aimed at obtaining recombinant NSs for structural studies.

No description available.

Emerging arthropod-borne viruses, such as alphaviruses and bunyaviruses, represent a serious threat to human and animal health worldwide, and for most of them, vaccines and specific treatments are unavailable. Viral host cell entry can be divided into several entry checkpoints, and the most important checkpoints for low pH-dependent enveloped viruses, such as bunyaviruses and alphaviruses, include receptor binding at the cell surface and, followed by endocytosis, low pH dependent membrane fusion from within intracellular compartments. A more thorough understanding of the detailed mechanisms allowing the viruses to pass these checkpoints is a pre-requisite for the design of viral entry inhibitors. This thesis reports the in vitro analysis of native alphavirus-receptor interactions, with the help of electron cryo-microscopy and icosahedral reconstruction of virus-recaptor complexes, using the prototypic alphavirus Semliki Forest virus (SFV) and the C-type lectin DC-SIGN. Together with results from collaborative work on SFV glycosylation, this study provides progress in defining the binding sites of DC-SIGN at the surface of SFV. Second, an in vitro system for phlebovirus fusion was developed using standard fluorometry, and has been characterized with the help of electron cryo-microscopy. It was discovered that negatively charged phospholipids with a conical shape, including the late endosomal phospholipid BMP, allow efficient phlebovirus fusion in vitro, thereby providing a possible rationale for phlebovirus fusion in late endosomes. Furthermore, electron cryo-microscopy of phlebovirus-liposome complexes allowed the capture of early stage fusion intermediates and laid the basis for possible future higher resolution studies of these fusion intermediates.

Doctor of Philosophy / Department of Plant Pathology / Anna E. Whitfield / Tomato spotted wilt virus (TSWV) is an economically important plant virus dependent on insects (thrips) for transmission to plant hosts. Like many animal-infecting viruses, TSWV replicates in the cells of its insect vector. The virus is an emergent disease threatening food and fiber crops worldwide. The aim of this work was to develop novel control strategies against TSWV through a better understanding of the virus-vector interaction. Previously, the TSWV GN protein was shown to be the viral attachment protein, a molecule mediating attachment of virus particles to the midgut epithelial cells of vector thrips. The specific goals of my research were to further examine the utility of disrupting the virus-vector interaction for effective virus control by exploiting GN properties, and to track the route of TSWV in thrips using confocal microscopy. To achieve these goals, I expressed soluble and insoluble forms of GN fused to green fluorescent protein (GFP) transiently and transgenically and examined their cellular localization in planta. GN::GFP recombinant protein localized to Golgi stacks throughout the cells as indicated by a punctate pattern or co-localization to a Golgi marker. In contrast, the soluble form of GN, GN-S::GFP, localized to the ER and apparently also to the cytoplasm. Virus acquisition and transmission assays with GN-S::GFP transgenic tomato plants demonstrated that transmission of TSWV by F. occidentalis was reduced by 35 to 100%. These results indicated that transgenic expression of GN-S in tomato plants may have the potential to prevent secondary spread of the virus. Novel features of the morphology of principal (PSGs) and tubular salivary glands (TSGs) of the insect vector F. occidentalis and of their infection with TSWV were described. The virus colonized different cell types and regions within the PSGs with variable intensity and distribution; and accumulated at the lumen of individual cells. The TSGs of F. occidentalis are proposed as a route for TSWV infection into the PSGs. The transgenic plants and the new knowledge of the virus vector interaction are promising tools to control TSWV and a model approach for the control of other vector-borne viruses.

No description available.

[EN] Tomato spotted wilt virus is one of the most widespread and economically important viruses worldwide. It infects a large number of plant species, being the tomato and pepper the most affected. TSWV is transmitted from one plant to another by various species of thrips in a circulative and propagative manner, being Flankliniella occidentalis the main vector. The best strategy for disease control in tomato and pepper has been breeding resistant cultivars, but only tomato with the gene Sw-5 and pepper with gene Tsw have been effective against a wide spectrum of TSWV isolates, but resistance-breaking isolates often arise. To obtain a more effective and durable control is necessary: A) the genetic and biological characterization of conventional and resistance-breaking isolates of TSWV; B) the understanding the evolutionary and epidemiological factors involved in the emergence and dispersion of resistance-breaking isolates; C) the development of tools for viral detection and quantification; and D) evaluation of new sources of resistance (total or partial, estimated as the difficulty to viral infection or/and accumulation) or tolerance (total or partial, estimated as the difficulty to symptoms development and damage without affecting viral infection). In this work, TSWV isolates from Spanish tomato and pepper crops have been biologically and molecularly characterized. The complete genome of three TSWV isolates with different biotypes were sequenced: N (unable to infect Sw-5 resistant tomato and Tsw resistant pepper), T (tomato Sw-5 resistance-breaking), and P (pepper Tsw resistance-breaking). No correlation between genetic variation and the ability of overcoming resistance was found. These nucleotide sequences and others retrieved from the GenBank database were used to develop RT-qPCR, with high sensitivity and dynamic range. Primers and one TaqMan MGB were designed from conserved sequence stretches with the aim of detecting and quantifying any TSWV isolate. It was not possible to develop a molecular method to differentiate between conventional and resistance-breaking isolates since there is no correlation between genotype and biotype. Instead, two TaqMan MGB probes were designed to quantify the two main genotypes (according to the M segment) in mixed infections. RT-qPCR was used to evaluate TSWV accumulation in non-resistant tomato, non-resistant pepper and Datura stramonium (an important reservoir), as well as in the main vector F. occidentalis, which was considered to evaluate transmission efficiency. The results showed that resistance breakdown was not associated to a fitness cost (tradeoff) in the infectivity of susceptible hosts or transmissibility by thrips and that the resistance-breaking isolates have the same potential for dispersion in field as the conventional isolates. Finally, the information obtained from the previous studies and the RT-qPCR technique were used to evaluate the resistance and tolerance to TSWV of a new accession of Capsicum baccatum. In addition to considering the genetic and biological variation of TSWV, a new approach was used based on analysis of longitudinal data (those measured in the same individuals over time). The resistance level was estimated with two variables: A) absolute fitness, calculated from the variation in time of the viral titer, quantified by RT-qPCR; and B) infectition survival, the median time in which the virus was detected in a plant by ELISA. The tolerance level was estimated as symptom survival, the median time in which a plant developed severe symptoms. This analysis showed that this new accession is partially resistant and totally tolerant to TSWV conventional and resistance-breaking isolates and therefore is a good candidate for a pepper breeding program / [ES] Tomato spotted wilt virus (TSWV) es uno de los virus más extendidos y de mayor importancia económica del mundo. Infecta a un gran número de especies vegetales, siendo los cultivos de tomate y pimiento los más afectados. Este virus se transmite de una planta a otra por trips en una manera propagativa y circulativa, siendo Flankliniella occidentalis el más eficaz. El cultivo de variedades resistentes de tomate y pimiento permitió el control de la enfermedad, ya que estos genes inducen una respuesta hipersensible impidiendo la infección sistémica del virus. Sin embargo, con frecuencia aparecen aislados del virus capaces de superar estas resistencias. Para obtener un control de la enfermedad más eficaz y duradero es necesario: A) la caracterización genética y biológica de los aislados del TSWV que superan y no superan las resistencias; B) el estudio de los factores evolutivos y epidemiológicos implicados en la aparición y establecimiento de los aislados que superan las resistencias; C) el desarrollo de herramientas que permitan la detección y cuantificación de estos aislados virales; y D) la evaluación de nuevas fuentes de resistencia o tolerancia. En este trabajo se han caracterizado biológicamente y molecularmente diferentes aislados del TSWV procedentes de cultivos de tomate y pimiento de España. Se determinó la secuencia nucleotídica del genoma completo de tres aislados españoles correspondientes a tres biotipos: N (incapaz de infectar variedades resistentes de tomate o pimiento); T (supera la resistencia Sw-5 de tomate); y P (supera la resistencia Tsw de pimiento). No se encontró correlación entre la variación genética y la capacidad de superar la resistencia. Estas secuencias nucleotídicas y otras obtenidas de la base de datos Genbank se utilizaron para desarrollar una técnica basada en la RT-PCR cuantitativa (RT-qPCR) que permite detectar el virus con un alto grado de sensibilidad y cuantificar en un amplio rango dinámico. Se diseñaron los iniciadores y una sonda TaqMan MGB a partir de segmentos de secuencia conservados para que fueran válidos para todos los aislados del TSWV. También se desarrollaron dos sondas Taqman que permitían cuantificar diferencialmente variantes genéticas del virus en infecciones mixtas. La RT-qPCR se utilizó para evaluar la acumulación de varios aislados del TSWV en tomate sin Sw-5, pimiento sin Tsw y Datura stramonium, así como, en su principal vector F. occidentalis que se usó para evaluar la eficiencia de la transmisión. Se observó que la superación de la resistencia no suponía un coste en la eficacia biológica (fitness) tanto en la multiplicación del virus en estas plantas como en la transmisión por trips. Por tanto, los aislados que superan las resistencias tienen la misma capacidad de dispersión en campo que los aislados convencionales. Por último, se utilizó la RT-qPCR y la información obtenida para evaluar la capacidad de resistencia y tolerancia al TSWV de una accesión de Capsicum baccatum. El nivel de resistencia se estimó a partir de dos variables: A) la eficacia absoluta, calculada a partir de la variación en el tiempo del título viral, cuantificado por RT-qPCR; y B) la infectividad, como la mediana del tiempo que tarda el virus en ser detectado en una planta por ELISA. El nivel de tolerancia se estimó como la mediana del tiempo que tarda una planta en mostrar síntomas graves. Esta nueva accesión es parcialmente resistente y totalmente tolerante tanto para aislados del TSWV convencionales como para aquellos capaces de infectar e inducir síntomas severos en variedades de pimiento con el gen Tsw. Por tanto, esta nueva variedad de C. baccatum es un buen candidato para usarlo en programas de mejora genética de pimiento. / [CAT] Tomato spotted wilt virus (TSWV) és un dels virus més estesos i de major importància econòmica del món. Infecta a un gran nombre d'espècies vegetals i els cultius de tomaca i pebrot són alguns dels més afectats. Aquest virus es transmet d'una planta a una altra, per trips de manera propagativa i circulativa, i Flankliniella occidentalis és el més eficaç. El cultiu de varietats resistents de tomaca i pebrot (en els quals s'han introduït per millora genètica els gens Sw-5 i Tsw, respectivament) va permetre el control de la malaltia, ja que aquests gens indueixen una resposta hipersensible i impedeixen la infecció sistèmica del virus (resistència total). No obstant açò, amb freqüència apareixen aïllats del virus que són capaços de superar aquestes resistències. Per a obtenir un control de la malaltia més eficaç i durador és necessari: A) la caracterització genètica i biològica dels aïllats del TSWV que superen i no superen les resistències; B) l'estudi dels factors evolutius i epidemiològics implicats en l'aparició i establiment dels aïllats que superen les resistències; C) el desenvolupament d'eines que permeten la detecció i quantificació d'aquests aïllats virals; i D) l'avaluació de noves fonts de resistència (total o parcial, que dificulten la infecció i multiplicació viral) o tolerància (total o parcial, que dificulten l'aparició de símptomes i danys encara que no tinguen un efecte en la infecció viral). En aquest treball s'han caracteritzat biològicament i molecularment diferents aïllats del TSWV procedents de cultius de tomaca i pebrot d'Espanya. Es va determinar la seqüència nucleotídica del genoma complet de tres aïllats espanyols corresponents a tres biotips: N (incapaç d'infectar varietats resistents de tomaca o pebrot); T (supera la resistència Sw-5 de tomaca); i P (supera la resistència Tsw de pebrot). Els resultats van mostrar que no hi havia una correlació entre la variació genètica i la capacitat de superar la resistència. Aquestes seqüències nucleotídiques i altres obtingudes de la base de dades Genbank es van utilitzar per a desenvolupar una tècnica basada en la RT-qPCR que permet detectar el virus amb un alt grau de sensibilitat i quantificar en un ampli rang dinàmic. Es van dissenyar els iniciadors i una sonda TaqMan MGB a partir de segments de seqüència conservats perquè foren vàlids per a tots els aïllats del TSWV. Tambe es van desenvolupar dos sondes Taqman que permetien quantificar diferencialment variants genètiques del virus en infeccions mixtes. La RT-qPCR es va utilitzar per a avaluar l'acumulació de diversos aïllats del TSWV en tomaca sense Sw-5, pebrot sense Tsw i Datura stramonium així com, en el seu principal vector F. occidentalis que es va usar per a avaluar l'eficiència de la transmissió. Es va observar que la superació de la resistència no suposava un cost en l'eficàcia biològica (fitness) tant en la multiplicació del virus en aquestes plantes com en la transmissió per trips. Per tant, els aïllats que superen les resistències tenen la mateixa capacitat de dispersió en camp que els aïllats convencionals. Finalment, es va utilitzar la RT-qPCR i la informació obtinguda per a avaluar la capacitat de resistència i tolerància d'una accessió de Capsicum baccatum al TSWV. El nivell de resistència es va estimar a partir de dues variables: A) l'eficàcia absoluta, calculada a partir de la variació en el temps del títol viral, quantificat per RT-qPCR; i B) la infectivitat, com la mitjana del temps que es tarda a detectar el virus en una planta per mitjèr d'ELISA. El nivell de tolerància es va estimar com la mitjana del temps que tarda una planta a mostrar símptomes greus. Aquesta nova accessió és parcialment resistent i totalment tolerant tant per a aïllats del TSWV convencionals com per a aquells capaços d'infectar i induir símptomes severs en varietats amb el gen Tsw. Per tant, aquesta nova varietat d / Debreczeni, DE. (2015). Caracterización de aislados del virus del bronceado del tomate (TSWV) que superan las resistencias de los genes Sw-5 en tomate y Tsw en pimiento. Identificación de una fuente de tolerancia en pimiento [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/51460 / TESIS

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-11T12:04:23Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EpidemiologiaMolecularVirusOropouche.pdf: 8072606 bytes, checksum: 4583fd996c0fca9e376db310de118adb (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-08T15:49:26Z (GMT) No. of bitstreams: 2 Dissertacao_EpidemiologiaMolecularVirusOropouche.pdf: 8072606 bytes, checksum: 4583fd996c0fca9e376db310de118adb (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2014-04-08T15:49:26Z (GMT). No. of bitstreams: 2 Dissertacao_EpidemiologiaMolecularVirusOropouche.pdf: 8072606 bytes, checksum: 4583fd996c0fca9e376db310de118adb (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2009 / O Virus Oropouche (VORO; Bunyaviridae, Orthobunyavirus) é um dos mais importantes arbovírus que infecta humanos na Amazônia brasileira, e é causador da febre do Oropouche. Entre 1961 e 2009, um grande número de epidemias foi registrado em diferentes centros urbanos dos Estados Brasileiros do Acre, Amapá, Amazonas, Maranhão, Pará, Rondônia e Tocantins, e também no Panamá, Peru e Trinidad & Tobago. Este trabalho teve por objetivo desenvolver um estudo retrospectivo dos aspectos epidemiológicos e moleculares do VORO enfatizando sua distribuição, a dinâmica das epidemias ocorridas no período, bem como a dispersão de diferentes genótipos na América Latina e no Brasil como contribuição à epidemiologia molecular do VORO. Para tanto 66 isolamentos do VORO pertencentes ao acervo do Instituto Evandro Chagas foram propagados em camundongos e em cultura de células VERO, seguida da extração do RNA viral e obtenção do cDNA por RTPCR; os amplicons foram purificados e submetidos ao sequenciamento nucleotídico para análises moleculares e evolução, incluindo o rearranjo genético, estudo de relógio molecular e análise de dispersão viral. Foi demonstrada a presença de quatro linhagens distintas do VORO na Amazônia brasileira (genótipos I, II, III e IV), sendo os genótipos I e II, respectivamente os mais frequentemente encontrados em áreas da Amazônia ocidental e oriental. Esses e o genótipo III estão constantemente evoluindo, mediante o mecanismo “boom and boost” que resulta na emergência seguida de substituição das sublinhagens (subgenótipos) circulantes por outras mais recentes. O genótipo III do VORO, previamente encontrado somente no Panamá, foi descrito na Amazônia e Sudeste do Brasil. Os dados obtidos pela análise filogenética comparativa das topologias para os segmentos PRNA e MRNA sugerem que o VORO utiliza o rearranjo genético como mecanismo de geração de biodiversidade viral, sendo o genótipo I o mais estável e o II o mais instável e, portanto, mais sensível às pressões evolutivas; foi reconhecido um novo genótipo do VORO neste estudo em amostras isoladas em Manaus no ano de 1980, que foi denominado de genótipo IV. O estudo do relógio molecular mostrou que a emergência do VORO se deu no Estado do Pará provavelmente há 223 anos e daí ao longo dos anos se dispersou pela PanAmazônia bem como para o Caribe, sendo que o genótipo I foi o que originou os demais genótipos do VORO. / Oropouche Virus (OROV; Bunyaviridae, Orthobunyavirus) is one of most important arbovirus which infects humans in the Brazilian Amazon, and is also the causal agent of Oropouche fever. Between 1961 and 2009, dozens of epidemics were registered in several urban centers of the Brazilian states of Acre, Amapá, Amazonas, Maranhão, Pará, Rondônia and Tocantins, and also in Panama, Peru and Trinidad & Tobago. This work aimed to develop a retrospective epidemiologic and molecular study of OROV emphasizing its distribution, epidemic dynamics in the period, as well as the dispersion of the OROV genotypes in Brazil and other Latin American countries as a contribution to understanding of the molecular epidemiology of it. A total of 66 OROV isolates of the Instituto Evandro Chagas collection were growth into VERO cells and suckling mice; then, RNA was extracted and cDNA prepared by RT-PCR; the amplicons were purified and submitted to nucleotide sequencing to further molecular and evolution analyzes including genetic reassortment, molecular clock and viral dispersion. It was demonstrated the circulation of four different genetic lineages of OROV in the Brazilian Amazon (genotypes I, II, III, and IV); the genotypes I and II were respectively the most distributed OROV genotypes in Occidental and Oriental Amazon areas. These and the genotype III have been continuously under evolution pressure and changing by the mechanism “boom and boost” which result in an emergence of new OROV sub-genotypes that replace the older circulating sub-lineages in an area. The genotype III which was previousouly recognized in Panama it was identified in the Amazon and Southeast regions. The results obtained by the comparative phylogenetic analyses of the SRNA and MRNA topologies suggest that OROV uses the genetic reassortment as mechanism to further generate its viral biodiversity, and the genotype I is the most stable, while the genotype II is the most unstable, and therefore under higher evolutionary pressure; it was recognized a new OROV genotype in this study, the genotype IV. The molecular clock analysis showed that OROV emerged in Pará State approximately 223 years ago, and along of the years did its dispersal and evolution through the Pan- Amazon as well as to the Caribbean and Central America regions, and the genotype I was responsible by the emergence of all other OROV genotypes.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-11T16:15:35Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InfeccaoExperimentalHamsters.pdf: 2790075 bytes, checksum: 7188971dc75068f0e71978c12c97f993 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-14T14:16:05Z (GMT) No. of bitstreams: 2 Dissertacao_InfeccaoExperimentalHamsters.pdf: 2790075 bytes, checksum: 7188971dc75068f0e71978c12c97f993 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2014-04-14T14:16:05Z (GMT). No. of bitstreams: 2 Dissertacao_InfeccaoExperimentalHamsters.pdf: 2790075 bytes, checksum: 7188971dc75068f0e71978c12c97f993 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2007 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os flebovírus (família Bunyaviridae; gênero Phlebovirus), possuem importância considerável em saúde publica, pois podem causar uma variedade de síndromes clínicas. Na região amazônica brasileira até o momento já foram isolados 23 flebovírus sendo que quatro se destacam por terem sido isolados de humanos: Virus Alenquer, Virus Candiru, Virus Morumbi e Virus Serra Norte. Estes mais o Virus Itaituba, isolado de Didelphis marsupialis, fazem parte do Complexo Candiru (grupo Candiru) e foram utilizados para estudos de caracterização genética e de infecção experimental em hamsters dourados (Mesocricetus auratus). Os Hamsters mostraram-se suscetíveis a infecção por esses flebovírus, apesar de não terem apresentado sinais de doença. A análise histopatológica demonstrou aspectos lesionais no fígado, nos rins, no baço, nos pulmões e no sistema nervoso central, sendo as lesões mais intensas no fígado comprovadas pela presença dos antígenos virais empregando a técnica de imunohistoquímica. A análise das seqüências nucleotídicas parciais dos segmentos PRNA e MRNA obtidas para os cinco flebovírus estudados mostraram maior similaridade genética entre si do que com outros membros do gênero Phlebovirus. Sendo as mesmas geneticamente mais relacionadas ao Virus Punta Toro. Filogeneticamente, independentemente do segmento genômico analisado, os cinco flebovírus constituem um grupo monofilético, sendo que a análise pelo método de máxima verossimilhança demonstrou diferentes origens evolutivas para os segmentos de RNA o que sugere a ocorrência de rearranjo genético em natureza entre estes cinco flebovírus amazônicos. / The phleboviruses (family Bunyaviridae, genus Phlebovirus) are of considerable public health importance, due to their association with human diseases causing different clinical syndromes. In the Brazilian Amazon region, 23 phlebovirus were presently isolated, four of them have been isolated from humans: Alenquer virus, Candiru virus, Morumbi virus, and Serra Norte virus. These and Itaituba virus, isolated from Didelphis marsupialis belong to the Candiru Complex (Candiru group) and were used for studies on genetic characterization and experimental infection using gold hamsters (Mesocricetus auratus) as animal model. Hamsters showed to be susceptible to infection by these viruses; however signs of disease were not observed. The histophatological analysis demonstrated damages in the liver, kidneys, lung, and central nervous system. More intense lesions were observed in the liver which were characterized by the presence of viral antigens detected by immunohistochemistry assay. The analysis of the partial SRNA and MRNA nucleotide sequences obtained for the five studied phleboviruses showed more similarity among them than with other members of the genus Phlebovirus.More over, is was demonstrated that these phleboviruses were more genetically related to Punta Toro virus. Phylogenetically, regardless of the genome segment analyzed, the five phleboviruses constitute a monophyletic group. In addition the analysis by maximum likelihood method demonstrated different evolutionary origins for the RNA segments, suggesting the occurrence of natural genetic reassortment among the five Amazonian phleboviruses.