Columbiformes sinantrópicos podem ter um importante papel na epidemiologia de patógenos com potencial zoonótico ou de impacto econômico para a indústria avícola. Dentre eles destacam-se: Mycoplasma spp., Salmonella spp., paramixovírus aviário tipo 1 (APMV-1), inseridos no Programa Nacional de Sanidade Avícola (PNSA) e a Chlamydophila psittaci, agente de uma das principais zoonoses relacionada com aves silvestres. Dentro desse contexto, este trabalho objetivou pesquisar, sazonalmente, a ocorrência destes patógenos em pombos-domésticos (Columba livia) em dois entrepostos no Estado de São Paulo. Ao longo de um ano, mensalmente 10 pombos foram capturados em cada entreposto para a colheita de amostras de suabe cloacal e sangue. A técnica de soroaglutinação rápida em placa (SAR) foi utilizada para a detecção de anticorpos anti-M. synoviae, anti-M. gallisepticum e anti-S.Pullorum/Gallinarum; para a confirmação dos sororeagentes foram utilizadas a prova de inibição da hemaglutinação e soroaglutinação lenta, respectivamente. Para a detecção do DNA de C. psittaci e RNA de AMPV-1 foram utilizados métodos moleculares, PCR e RT-PCR. Para investigação de anticorpos anti-APMV-1 foi empregada a técnica de HI. Na SAR, 3,3% dos soros foram reagentes para M. synoviae; 2,5% para M. gallisepticum e 0,4% para S. Pullorum/Gallinarum. No entanto, essas amostras foram negativas nas técnicas confirmatórias. A ocorrência do APMV-1 não foi detectada. O DNA de C. psittaci foi detectado em 13,3% das amostras sendo 10,8% provenientes de aves capturadas na estação seca e 15,8% na estação chuvosa. Tais resultados são relevantes, pois demonstram que a C. psittaci ocorre em pombos presentes em áreas públicas frequentadas por um grande número de pessoas. Frente à escassez de pesquisas realizadas em Columbiformes no país, novos estudos são necessários para a determinação do real risco que pombos-domésticos podem representar quanto à transmissão de patógenos para aves comerciais e a influência da sazonalidade na disseminação desses microrganismos. / Columbiformes may play an important role in the epidemiology of pathogens with zoonotic potential or economic impact in the poultry industry. Among these pathogens there are Mycoplasma spp., Salmonella spp., Avian Paramyxovirus type 1 (APMV-1), included in the National Poultry Health Program (PNSA) and Chlamydophila psittaci, etiologic agent of an important zoonosis associated with wild birds. Thus, the aim of this study was to investigate, seasonally, the occurrence of the pathogens listed above in feral pigeons (Columba livia) in two warehouses in São Paulo State. During one year, 10 birds were captured monthly in each locality and cloacal swabs and blood samples were collected from each pigeon. The rapid seroagglutination test was performed for the detection of antibodies against M. synoviae, M. gallisepticum and Salmonella Pullorum/Gallinarum. Positive results were submitted to the hemagglutination inhibition and slow seroagglutination test, respectively. For the C. psittacis DNA and APMV-1s RNA diagnosis, molecular techniques PCR and RT-PCR were performed. Hemagglutination inhibition test was also performed in order to detect antibodies against APMV-1. From the serum samples analyzed by rapid seroagglutination test, 3.3% were positive for M. synoviae, 2.5% for M. gallisepticum and 0.4% for S. Pullorum/Gallinarum. However, none of these samples was positive on the confirmatory tests. APMV-1 was not detected in any of the laboratory tests used. C. psittacis DNA was detected in 13.3% of the samples being, 10.8% from pigeons captured during the dry season and 15.8% in the rainy season. These results are relevant since they indicate that C. psittaci occurs in birds living in public areas frequented by a large number of people. The occurrence of the other pathogens was not detected. Nevertheless, due to lack of information about the pigeons sanitary status in the country, additional researches are necessary to determine the risk that feral pigeons can pose in the transmission of pathogens for poultry and the influence of each season in the spread of these microorganisms.

Orientador: Lucilene Delazari Santos / Resumo: O veneno de serpente tem sido utilizado para diversas finalidades terapêuticas; portanto, desde o início do século XX, a criação de serpentes em cativeiro tornou-se uma atividade cada vez mais relevante. Os principais motivos da existência e permanência destes cativeiros se atribuem à produção dos soros antiofídicos, e o uso de seus venenos e seus componentes isolados com potenciais aplicações à saúde animal e humana. Porém, a vida em cativeiro pode resultar na síndrome da má adaptação, a qual o animal sob estresse desenvolve inapetência e, consequentemente há o acometimento da imunidade, podendo desenvolver infecções por micro-organismos como fungos, bactérias e vírus. Desta forma, o controle e prevenção de infecções em serpentes mantidas em cativeiro tornaram-se assuntos muito importantes. Dentre as doenças infecciosas virais, têm-se a infecção pelo Paramixovírus ofídico, atualmente, denominado Ferlavirus reptiliano. Este vírus é altamente devastador em serpentes, pois atua no sistema nervoso central e respiratório, podendo levar à morte e/ou morbidade na fase aguda da doença, sendo que na fase crônica, o animal pode atuar como reservatório do vírus, e disseminar a doença no plantel. Atualmente, não há tratamento específico nem vacinas disponíveis para esta virose. Porém, testes sorológicos estão emergindo para a detecção de anticorpos. Dentre estes testes, encontram-se o ensaio de Inibição de Hemaglutinação (HI) e o ensaio de ELISA de Bloqueio de Fase Líquida (BFL-ELISA), ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Snake venom has been used for several therapeutic purposes. Therefore, breeding snakes in captivity has become an increasingly relevant activity since early20th century. The main reasons for the existence and permanence of these captivities are the production of snake antivenom and the use of venom and its isolate compounds with potential applications in human and animal health. However, living in captivity may result in the maladaptation syndrome, in which the animal under stress develops inappetence and, consequently, a decrease in immunity, becoming more likely to develop infections caused by microorganisms as fungi, bacteria and viruses. Thus, the control and prevention of infections in snakes kept in captivity became very important subjects. The infection by the ophidian paramyxovirus named reptilian ferlavirus is among the viral infectious diseases. This virus is highly devastating in snakes because its action in central and respiratory nervous system and may lead to death and/or morbidity in the acute stage of the disease, consideringthat the animal may act as a reservoir of the virus in the chronic stage and spread the disease in the breeding stock. Nowadays, there isn’t a specific treatment or vaccines available for strike this viral infection, however, serological tests are emerging for antibodies detection. Hemagglutination Inhibition (HI) and Liquid Phase Blocking ELISA (LPB-ELISA) are among these tests, which differs from each other in sensitivity and analytical ... (Complete abstract click electronic access below) / Mestre

Columbiformes sinantrópicos podem ter um importante papel na epidemiologia de patógenos com potencial zoonótico ou de impacto econômico para a indústria avícola. Dentre eles destacam-se: Mycoplasma spp., Salmonella spp., paramixovírus aviário tipo 1 (APMV-1), inseridos no Programa Nacional de Sanidade Avícola (PNSA) e a Chlamydophila psittaci, agente de uma das principais zoonoses relacionada com aves silvestres. Dentro desse contexto, este trabalho objetivou pesquisar, sazonalmente, a ocorrência destes patógenos em pombos-domésticos (Columba livia) em dois entrepostos no Estado de São Paulo. Ao longo de um ano, mensalmente 10 pombos foram capturados em cada entreposto para a colheita de amostras de suabe cloacal e sangue. A técnica de soroaglutinação rápida em placa (SAR) foi utilizada para a detecção de anticorpos anti-M. synoviae, anti-M. gallisepticum e anti-S.Pullorum/Gallinarum; para a confirmação dos sororeagentes foram utilizadas a prova de inibição da hemaglutinação e soroaglutinação lenta, respectivamente. Para a detecção do DNA de C. psittaci e RNA de AMPV-1 foram utilizados métodos moleculares, PCR e RT-PCR. Para investigação de anticorpos anti-APMV-1 foi empregada a técnica de HI. Na SAR, 3,3% dos soros foram reagentes para M. synoviae; 2,5% para M. gallisepticum e 0,4% para S. Pullorum/Gallinarum. No entanto, essas amostras foram negativas nas técnicas confirmatórias. A ocorrência do APMV-1 não foi detectada. O DNA de C. psittaci foi detectado em 13,3% das amostras sendo 10,8% provenientes de aves capturadas na estação seca e 15,8% na estação chuvosa. Tais resultados são relevantes, pois demonstram que a C. psittaci ocorre em pombos presentes em áreas públicas frequentadas por um grande número de pessoas. Frente à escassez de pesquisas realizadas em Columbiformes no país, novos estudos são necessários para a determinação do real risco que pombos-domésticos podem representar quanto à transmissão de patógenos para aves comerciais e a influência da sazonalidade na disseminação desses microrganismos. / Columbiformes may play an important role in the epidemiology of pathogens with zoonotic potential or economic impact in the poultry industry. Among these pathogens there are Mycoplasma spp., Salmonella spp., Avian Paramyxovirus type 1 (APMV-1), included in the National Poultry Health Program (PNSA) and Chlamydophila psittaci, etiologic agent of an important zoonosis associated with wild birds. Thus, the aim of this study was to investigate, seasonally, the occurrence of the pathogens listed above in feral pigeons (Columba livia) in two warehouses in São Paulo State. During one year, 10 birds were captured monthly in each locality and cloacal swabs and blood samples were collected from each pigeon. The rapid seroagglutination test was performed for the detection of antibodies against M. synoviae, M. gallisepticum and Salmonella Pullorum/Gallinarum. Positive results were submitted to the hemagglutination inhibition and slow seroagglutination test, respectively. For the C. psittacis DNA and APMV-1s RNA diagnosis, molecular techniques PCR and RT-PCR were performed. Hemagglutination inhibition test was also performed in order to detect antibodies against APMV-1. From the serum samples analyzed by rapid seroagglutination test, 3.3% were positive for M. synoviae, 2.5% for M. gallisepticum and 0.4% for S. Pullorum/Gallinarum. However, none of these samples was positive on the confirmatory tests. APMV-1 was not detected in any of the laboratory tests used. C. psittacis DNA was detected in 13.3% of the samples being, 10.8% from pigeons captured during the dry season and 15.8% in the rainy season. These results are relevant since they indicate that C. psittaci occurs in birds living in public areas frequented by a large number of people. The occurrence of the other pathogens was not detected. Nevertheless, due to lack of information about the pigeons sanitary status in the country, additional researches are necessary to determine the risk that feral pigeons can pose in the transmission of pathogens for poultry and the influence of each season in the spread of these microorganisms.

Measles is one of the most contagious diseases known to mankind. Despite the availability of a safe and effective vaccine, approximately 164,000 measles-related deaths were recorded in 2008. The inherent restricted host tropism of MV means that the development of authentic rodent models will be a valuable research tool in testing new vaccines and antivirals. In addition to the receptor requirement, mouse innate immunity has been shown to inhibit MV growth. In this thesis, the contributions of several key components of the mouse innate immune system on the inhibition of MV replication were examined. The transcription factor interferon regulatory factor 3 (IRF3), which normally plays a key role in mediating innate immune signaling, contributed relatively little in inhibiting MV replication both in vitro and in vivo. In contrast, the JAK/STAT pathway and the double-stranded RNA inducible protein kinase, PKR, played more important roles in controlling virus replication. The resurgence of measles in areas where the virus was once thought to be eradicated makes the development of anti-MV treatments essential. Concurrent to the development of an animal model to better study its pathogenesis, we wanted to look at the effect of MV inhibitors on its replication. The MV fusion inhibitor, carbobenzoxy-D-phenylalanine-L-phenylalanine-glycine (ZfFG), was developed in the past to study fusion; however, its mechanism of action has not yet been elucidated. To examine this, spontaneous ZfFG-resistant mutants were generated and characterized. Mutations were found in the HRB region of the fusion (F) protein, and when these were modeled using published paramyxovirus F crystal structures, data suggested that ZfFG targeted a small pocket present between the head and stalk regions of its pre-fusion conformation. An authentic mouse model of measles developed from findings in this study may allow for in vivo efficacy testing of ZfFG in the future.

Measles is one of the most contagious diseases known to mankind. Despite the availability of a safe and effective vaccine, approximately 164,000 measles-related deaths were recorded in 2008. The inherent restricted host tropism of MV means that the development of authentic rodent models will be a valuable research tool in testing new vaccines and antivirals. In addition to the receptor requirement, mouse innate immunity has been shown to inhibit MV growth. In this thesis, the contributions of several key components of the mouse innate immune system on the inhibition of MV replication were examined. The transcription factor interferon regulatory factor 3 (IRF3), which normally plays a key role in mediating innate immune signaling, contributed relatively little in inhibiting MV replication both in vitro and in vivo. In contrast, the JAK/STAT pathway and the double-stranded RNA inducible protein kinase, PKR, played more important roles in controlling virus replication. The resurgence of measles in areas where the virus was once thought to be eradicated makes the development of anti-MV treatments essential. Concurrent to the development of an animal model to better study its pathogenesis, we wanted to look at the effect of MV inhibitors on its replication. The MV fusion inhibitor, carbobenzoxy-D-phenylalanine-L-phenylalanine-glycine (ZfFG), was developed in the past to study fusion; however, its mechanism of action has not yet been elucidated. To examine this, spontaneous ZfFG-resistant mutants were generated and characterized. Mutations were found in the HRB region of the fusion (F) protein, and when these were modeled using published paramyxovirus F crystal structures, data suggested that ZfFG targeted a small pocket present between the head and stalk regions of its pre-fusion conformation. An authentic mouse model of measles developed from findings in this study may allow for in vivo efficacy testing of ZfFG in the future.

No description available.

Les paramyxovirus respiratoires humains sont des virus responsables d'infections chez les jeunes enfants, les personnes âgées et les patients immuno déprimés. Ces virus possèdent deux glycoprotéines à la surface de leur enveloppe, jouant un rôle dans l'entrée du virus dans la cellule cible. La glycoprotéine d’attachement (HN, G ou H) permet l’attachement du virus à son récepteur cellulaire et, dans le cas de HN, celle-ci est suspectée d’activer la seconde glycoprotéine, la protéinede fusion (F). Cette dernière réalise la fusion entre l'enveloppe du virus et la membrane cellulaire.Le mécanisme par lequel la protéine HN "active" la protéine F reste mal caractérisé, malgré la détermination récente de leurs structures en cristallographie. Plusieurs modèles sont actuellement proposés. Ce travail de thèse s’est focalisé principalement sur les glycoprotéines d’enveloppe des virus parainfluenza humain de type 2 (hPIV-2) et parainfluenza de type 5 (PIV-5), ainsi que sur la glycoprotéine de fusion du métapneumovirus humain (hMPV). La première partie de ce projet a consisté à caractériser une mutation retrouvée sur la protéine F de souches circulantes hPIV-2. Cette étude a notamment souligné l’importance de la sous-unité F2 dans la régulation de la fusion membranaire. Puis, dans un second temps, l’une des étapes du mécanisme d’entrée du métapneumovirus a été étudiée : la fusion membranaire induite par la glycoprotéine F. Il a été démontré qu’il était possible dans une certaine mesure, et par une approche de mutagenèse combinatoire, de dissocier les caractéristiques de F hMPV et ainsi de pouvoir mieux les étudier. Ce travail d’ingénierie de la glycoprotéine F hMPV s’est également inscrit dans un objectif de recherche appliquée afin de contribuer au développement de nouveaux outils prophylactiques et thérapeutiques. Cette perspective thérapeutique de F PIV-5 a été évaluée dans le cadre d’un vecteur oncolytique basé sur l’adénovirus de type 5 (AdV-5). L’expression de cette glycoprotéine hyperfusogène a ainsi contribué à un effet cytotoxique amplifié des vecteurs armés in vitro ainsiqu’en modèle animal immunocompétent. / Human respiratory paramyxoviruses are responsible for infectious diseases and hospitalisations among infants, children, elderly and the immunocompromised. These viruses harbour two glycoproteins implicated in virus entry into the cell. The attachment glycoprotein (HN,G or H) is implicated the virus attachment on a target cell receptor, and HN is also suspected to activate the second glycoprotein, the fusion protein (F). This latter glycoprotein can perform the fusion between the cellular membrane and the viral envelope. The mechanism of activation of the F protein, is not well-defined, even with the structural characterisation for some viruses studied inthis thesis. This thesis work is focussed on the viral glycoprotein of parainfluenza virus type 2 (hPIV-2), parainfluenza virus type 5 (PIV-5), and the fusion glycoprotein of human Metapneumovirus (hMPV).The first part of this project was the characterization of a mutation observed in the F protein natural variants of hPIV-2. This work highlights the importance of the F2 subunit of F in the fusion regulation. A second part of the project consisted of the study of the mechanism of F hMPV entry into the cell, induced by F glycoprotein. This work showed that it was possible to dissociate the characteristics of the F glycoprotein, in order to allow a better understanding of these characteristics. This engineering work on the F protein was used to understand the basic science but could also be used in the development of therapeutic tools.The therapeutic use of F PIV-5 was also evaluated in an oncolytic vector based on adenovirus type 5 (AdV-5). Its expression in tumours showed a highly cytotoxic activity for the target cells in vivo, but also in vitro on immunocompetent rodents.

Die für die Vermehrung der Paramyxoviren notwendige Freisetzung des Virusgenoms in die Wirtszelle findet nach einer Verschmelzung der Virushülle mit der Zellmembran statt. Die Membranfusion wird durch eine Konformationsänderung des membranständigen Fusionsproteins (F-Protein) der Paramyxoviren vermittelt. Der Auslöser der Strukturumwandlung des F-Proteins ist bislang unbekannt. Man nimmt an, daß eine Wechselwirkung mit dem zweiten membranständigen Protein der Hämagglutinin-Neuraminidase (HN-Protein) die Strukturumwandlung des F-Proteins induziert. Das F-Protein kann jedoch auch in Abwesenheit des HN-Proteins eine Membranfusion vermitteln. Für das Verständnis des Mechanismus der F-proteinvermittelten Fusion ist die Kenntnis der dreidimensionalen Struktur des F-Proteins notwendig. In der vorliegenden Arbeit wurden die F-Proteine der Paramyxoviren, Sendaivirus und Simianvirus 5, in fusionskompetenter Form isoliert und in kleine Lipidvesikel rekonstituiert, um deren Struktur mittels Kryoelektronenmikroskopie und Einzelpartikelanalyse aufzuklären. Die 3D-Struktur des Sendaivirus-F-Proteins konnte mit einer Auflösung von 16 Angström aufgeklärt werden. Es ist die erste 3D-Struktur des F-Proteins eines Paramyxovirus in der fusionskompetenten Form. Um geeignete Bedingungen herauszufinden, die das Auslösen der Konformationsänderung der F-Proteine bzw. das "Einfangen" von Strukturintermediaten während der Fusion ermöglichen, wurde das Fusionsverhalten von Sendaivirus und Simianvirus 5 bei unterschiedlichen Temperatur- und pH-Werten sowie in Anwesenheit von Lysolipiden mittels Fluoreszenzdequenchingassays untersucht. Ein signifikanter Anstieg der Fusionsaktivität der untersuchten Viren konnte durch eine Erhöhung der Temperatur erreicht werden. Mittels ESR-Spektroskopie unter Einsatz von spinmarkierten Lysolipiden konnte gezeigt werden, daß Lysolipide die proteinvermittelte Fusion von Hüllviren in einem späten lipidabhängigen Schritt hemmen. Diese Untersuchungen bilden damit eine Grundlage zur Aufklärung der 3D-Struktur des F-Proteins im fusionsaktiven Zustand. Desweiteren wurde die Rolle der transmembranalen und zytoplasmatischen Domäne des F-Proteins bei der Membranfusion und der Wechselwirkung mit dem HN-Protein mittels Fluoreszenzmikroskopie untersucht. Die Befunde der 3D-Strukturaufklärung und der fluoreszenzmikroskopischen Studien wurden unter anderem in Hinblick auf die Bedeutung der Wechselwirkung zwischen den F- und HN-Proteinen für die Fusion diskutiert. / Paramyxoviruses infect their host cells by fusion of the viral envelope with the cell membrane. The membrane fusion is mediated by a confomational change of a viral envelope glycoprotein called the fusion (F) protein. The trigger of the F protein conformational change is still unknown. It is suggested, that an interaction of the F protein with the second envelope glycoprotein hemagglutinin-neuraminase (HN) induces its conformational change. However the F protein can mediate membrane fusion in absense of HN. The knowledge of the three dimensional structure of the F protein is reqiured to understand the F mediated membrane fusion. In the present work the fusion competent form of the fusion proteins of the paramyxoviruses Sendai virus and Simian virus 5 were isolated and incorporated each of them into small lipid vesicles. The 3D-structure of the entire ectodomain of the Sendai virus F protein has been determined in fusion potential conformation by cryo electron microscopy of single molecules and 3D-reconstruction at a resolution of 16 Angström. To detect usefull conditions for triggering the conformational change of F, the fusion of Sendai virus and Simian virus 5 have been studied at different temperature and pH, respectively, using a fluorescence dequenching assay. A significant increase of virus fusion activity has been found due to temperature enhancement. Using ESR-spectroscopy and spin-labeled lysolipids it has been shown that lysolipids inhibit the protein mediated fusion of enveloped viruses at a late lipid-dependent intermediate. Thus lysolipids are capable to freeze a conformational intermediate of the F protein during fusion. Furthermore the role of the transmembrane and the cytoplasmic domain of the Sendai virus F protein for membrane fusion was investigated using fluorescence microscopy. The results of the fluorescence microscopy study and the detection of the 3D-structure have been discussed in view of the relevance of F-HN-interaction for membrane fusion.

No description available.