Orientador : Clarice Weis Arns / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-02T11:17:26Z (GMT). No. of bitstreams: 1 Camargo_LourdesMariaAraujoQuaresmade_D.pdf: 4406461 bytes, checksum: 508e9bc44cd8f6790c59eeedab1bff1d (MD5) Previous issue date: 2002 / Doutorado

Orientador: Clarice Weis Arns / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T11:34:54Z (GMT). No. of bitstreams: 1 D'Arce_ReginaCeliaFreitas_D.pdf: 5035070 bytes, checksum: 70450102e2aa6208d35d5a038918c516 (MD5) Previous issue date: 2004 / Resumo: O presente estudo teve como objetivo o desenvolvimento de técnicas moleculares diferenciais para o diagnóstico do Vírus da Bronquite Infecciosa (VBI), responsável por infecções respiratória e urogenital agudas, altamente contagiosas em frangos e galinhas, e do Pneumovírus Aviário (PVA), agente etiológico da Rinotraqueíte dos Perus e associado à Síndrome da Cabeça Inchada (SCI) em galinhas. Sorotipos do VBI e subtipos do PV A foram cultivados em ovos embrionados e em células CER (Chicken Embryo Related), respectivamente. Foram adaptadas as técnicas de extração do RNA viral, RT -PCR, nested-PCR subtipo-especmco. para o PVA e RT -PCR-RFLP para o VBI a partir dos vírus cultivados, sendo testadas posteriormente em material coletado de aves infectadas experimentalmente. Tanto o PVA como o VBI foram detectados por RT-PCR e/ou nested-PCR nos grupos infectados com os agentes isoladamente ou em co-infecção, principalmente nos dias 3 e 6 pós-inoculação. As técnicas diagnósticas desenvolvidas foram então utilizadas na detecção viral de materiais clínicos, obtidos de plantéis avícolas com histórico de problemas respiratórios e sorologia positiva para os agentes estudados. Para o PVA obteve-se a detecção de oito vírus de campo, todos pertencentes ao subtipo A, donde foi possível o isolamento de quatro amostras. No caso do VBI foram detectadas três amostras de campo, todos diferenciados da amostra vacinal por RFLP, dos quais isolou-se duas amostras em ovos embrionados. Amostras positivas tiveram seus fragmentos de PCR seqüenciados, confirmando a classificação obtida através da nested-PCR para o PVA e do RFLP para o VBI. A análise das seqüências do VBI mostrou que os isolados de campo diferem de todos os sorotipos descritos até o momento. As técnicas molecu1ares otimizadas nesse estudo confinaram sua capacidade em diferenciar os subtipos A e B do PV A, bem como as variantes detectadas de VBI de campo e o sorotipo vacinal, demonstrando ser adequadas alternativas diagnósticas e valiosas ferramentas para estudos de epidemiologia molecular / Abstract: The aim of this study was the development of molecu1ar techniques able to differentiate Infectious Bronchitis Virus (IBV), responsible for high 'contagious acute respiratory and renal infections in chickens, and Avian Pneumovirus (APV), causal agent of Turkey Rhinotracheitis and associated to Swollen head Syndrome (SHS) in chickens. APV and IBV strains, representatives of subtypes A and B and serotype Massachussets, were multiplied in Chicken Embryo Related (CER) cell line and embrionated eggs, respectively. The techniques of RNA extraction, RT -PCR, APV subtype-specific nested-PCR and RT -PCR-RFLP for IBV diagnosis were adapted, and then tested in experimentally infected chickens. Both viruses were detected by RT -PCR and/or nested-PCR in the groups infected with the agents separately or in co-infection, mainly at 3rd end 6th day post infection. Moreover, these techniques were utilised in clinical field samples from chicken and turkey flocks with respiratory problems and positive APV and IBV serology. Eight APV were detected, all of them classified as subtype A, where four samples were isolated. In the case of IBV, three field samples were detected, originating two viral isolation, all different from vaccine serotype by RFLP. The PCR products were sequenced, confirming the APV nested-PCR and IBV RFLP classification. IBV sequence analysis showed that the Brazilian field samples detected in this study are distinct from all serotype described 50 far. These optimised techniques could be useful to direct differentiation of APV subtypes A and B, and IBV field and vaccine strains, proving to be a valuable molecular epidemiological tool / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 5 uppsatser.

Le métapneumovirus humain (hMPV) est un virus responsable d'infections aiguës des voies respiratoires telles que des bronchiolites, des bronchites ou des pneumonies, principalement chez les populations à risques que sont les jeunes enfants de moins de 5 ans, ainsi que les personnes âgées ou immunodéprimées. Découvert en 2001, ce virus et sa pathogénèse ne restent encore aujourd'hui que partiellement caractérisés. De ce fait et malgré les besoins, il n'y a aucun vaccin ou traitement thérapeutique spécifique et efficace contre le HMPV disponible sur le marché. Dans ce contexte, mon projet de thèse s'est articulé autour de deux axes principaux : (i) L'étude de la protéine de fusion F du virus hMPV, protéine majeure antigénique de surface et responsable de l'entrée du virus dans la cellule cible. Elle a pour particularité d'induire de manière autonome la fusion membranaire in vitro et d'être associée à des effets cytopathiques variable selon les souches virales. De par son rôle clé pour le virus hMPV, la protéine F a déjà fait l'objet de plusieurs études structurales et fonctionnelles mais les déterminants de cette activité fusogénique ne sont pas encore entièrement caractérisés. Nous nous sommes donc intéressés à l'identification de déterminants du phénotype viral hyperfusogénique, localisés dans les domaines heptad repeats de la protéine F du hMPV. (ii) L'atténuation de deux souches virales cliniques (CAN98-75 et C-85473) par délétion de gènes accessoires dans le but de développer des candidats vaccinaux adaptés aux enfants en bas âge. Différents virus ont été générés par génétique inverse et les délétions des gènes accessoires SH et G dans les deux fonds génétiques viraux ont été étudiées pour leur impact sur l'infectivité, la réplication et la pathogénèse virale in vitro et in vivo ainsi que leur contribution pour le développement de virus atténués candidats vaccinaux / Human metapneumovirus (hMPV) is a major pathogen responsible of acute respiratory tract infections, such as bronchiolitis or pneumonia, affecting especially infants, under five years old, elderly individuals and immunocompromised adults. Identified since 2001, this virus and its pathogenesis still remain largely unknown and no licensed vaccines or specific antivirals against hMPV are currently available. In this context, my research project was built over two main subjects: (i) The study of the fusion F glycoprotein which is the major antigenic protein of hMPV and is responsible of viral entry into host cell. By its crucial role for the virus, the F protein has already been characterized in several structural and/or functional studies. Thus, it has been described that the hMPV F protein induces membrane fusion autonomously, resulting in variable cytopathic effects in vitro, in a strain-dependent manner. However, as the determinants of the hMPV fusogenic activity are not well characterized yet, we focused on identification of some of these, located in heptad repeats domains of the protein. (ii) The evaluation of hMPV SH and G gene deletion for viral attenuation. Liveattenuated hMPV vaccine candidates for infants’ immunization has been constructed thank to this deletion approach at the beginning of hMPV vaccine development efforts. Despite encouraging results, these candidates have not been further characterized and the importance of the viral background has not been evaluated

Orientador: Helio Jose Montassier / Resumo: As infecções do trato respiratório de aves têm-se constituído em problemas crescentes e com marcantes consequências negativas sobre a produção avícola em várias partes do mundo, notadamente onde a avicultura é mais desenvolvida como no Brasil. Dentre essas enfermidades, destacam-se as micoplasmoses aviárias e a pneumovirose aviária, que, apesar de suas relevâncias em sanidade avícola, não têm sido investigadas de forma sistematizada no Brasil, em especial no que concerne à interação entre esses agentes ou a ocorrência de co-infecção em frangos de corte. O presente estudo investigou a ocorrência de infecção por Mycoplasma gallisepticum (MG) e pelos subtipos A ou B de Metapneumovírus aviário (AMPV) em frangos de corte de plantéis avícolas comerciais mantidos em granjas mais tecnificadas localizadas nas regiões Sudeste e Centro-Oeste do Brasil. Para tanto, as técnicas de PCR e de RT-Nested-PCR foram usadas na detecção e/ou identificação, respectivamente de MG e AMPV em amostras de suabes nasais e traqueais colhidos de 87 lotes de frangos de corte com problemas respiratórios e oriundos de 15 granjas de produção comercial de frangos de corte. Dos lotes amostrados, dois deles em um total de 87 (2,3%) e de uma única granja da região Sudeste, mostraram-se positivos para MG, enquanto que nenhum dos lotes investigados revelou-se positivo para AMPV. A baixa ou nenhuma incidência desses agentes pode ser explicada pela utilização de medidas cada vez mais efetivas para o controle sanitário... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Respiratory infections of poultry may be an increasing and negative problem with poultry production in several parts of the world, especially when poultry farming is more widely used than in Brazil. These diseases include avian mycoplasmosis and avian pneumovirosis, which, despite their relevance in poultry health, have not been systematically investigated in Brazil, especially with regard to the interaction between these agents or the occurrence of co-infection in broilers. The present study investigated the occurrence of infection by Mycoplasma gallisepticum (MG) and A or B subtypes of avian Metapneumovirus (AMPV) in broiler commercial poultry farms located in the Southeast and Center-West regions of Brazil. For this, PCR and RT-Nested-PCR techniques were used in the detection and / or identification, respectively of MG and AMPV in nasal and tracheal swab samples collected from 87 lots of broilers with respiratory problems and from 15 commercial production of broilers. Of the sampled lots, two of them in 87 (2.3%) were positive for MG, whereas none of the lots were positive for AMPV. The low or no incidence of these agents can be explained by the use of increasingly effective measures for the sanitary control of these agents in the commercial farms of sampled broilers. Our findings also suggest, however, that other bacterial and viral infectious agents not investigated in this study may be involved in the etiology of the respiratory problems of these birds, since those that... (Complete abstract click electronic access below) / Mestre

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