Orientador: Maria Rita Donalisio Cordeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T22:14:31Z (GMT). No. of bitstreams: 1 Ramalheira_RaquelMariaFerreira_M.pdf: 783396 bytes, checksum: 4c4dede3c21fce6bd0892ad98627ec66 (MD5) Previous issue date: 2006 / Resumo: Surtos de infecções respiratórias, particularmente pelo vírus influenza são comuns em instituições asilares, sendo a vacina a maneira mais eficaz de prevenção. Os objetivos desse trabalho foram: estudar a incidência de síndrome gripal no período de 15 meses e analisar a ocorrência de eventos adversos após vacinação contra influenza nos idosos de instituição asilar de Campinas, SP. Foram visitados semanalmente os indivíduos institucionalizados, SUS dependentes (N=154), moradores do Lar dos Velhinhos de Campinas para detecção de casos de síndrome gripal (SG) de junho de 2004 a agosto de 2005. Foram incluídos na pesquisa os indivíduos com condições de compreender e responder as perguntas e que consentiram em participar. Após a campanha vacinal contra influenza em abril de 2004, os idosos vacinados somente contra a influenza (cobertura de 95%) foram investigados no 5o dia após a aplicação do imunobiológico para detecção de sintomas sugestivos de eventos adversos, com nexo temporal com a vacina. Foram investigadas em modelo logístico múltiplo, co-variáveis possivelmente associadas à ocorrência de SG e de eventos adversos (variáveis dependentes). Dentre os 154 identificados acompanhados, 50 (32,5 por 100 moradores) apresentaram sintomas gripais. Foram encontrados 76 episódios de SG cuja distribuição temporal concentrou-se nos meses de junho de 2004 e 2005. A incidência de pelo menos um evento adverso foi de 14,3 por 100 vacinados. Entre as queixas pós-vacinais referidas, 12 (57,1%) foram de sintomas locais, sendo a dor no local da aplicação o evento de maior freqüência tanto nas mulheres (53,3%) quanto nos homens (50,0%). A ocorrência de eventos adversos bem como de quadro de SG não se associaram a nenhuma co-variável testada nos modelos logísticos. Embora com altas coberturas, houve indícios de circulação de vírus de tropismo respiratório na instituição. Ressalta-se a importância de manter altas as coberturas vacinais contra influenza nos moradores e funcionários, além de incrementar medidas de prevenção de doenças de transmissão por secreções. A baixa reatogenicidade da vacina pode facilitar a adesão às campanhas. A vigilância etiológica da SG poderá dar mais subsídios para a compreensão da dinâmica de circulação de patógenos em populações de risco / Abstract: Respiratory infection, particularly influenza outbreaks are common in institutionalized individuals. Vaccination is the most efficient prevent measure. The aim of this work were: to study respiratory infection incidence in institutionalized elderly subjects in 15 months period and to analyze the occurrence of adverse effects after influenza vaccination in a nursing home in Campinas, São Paulo State. SUS dependent elderly people (N= 154) living in Lar dos Velhinhos de Campinas were visited weekly to detect flu like syndrome from June 2004 to August 2005. It was included individuals with ability to understand and to answer research questions and those who accept to participated. After influenza vaccination campaign of April 2004 (coverage 95%) individuals were investigated in the 5-day to discover vaccine side events. A multiple logistic models were applied to investigate association between co variables and flu like syndrome and adverse events (dependent variables). Among the 154 subjects, it was identified 50 (32.5%) individuals witch present flu like symptoms during study period. Temporal trend of the 76 episodes showed concentration in June 2004 and 2005. Incidence of at least one side effect was 14.3 per 100 vaccinated residents. Local symptoms were referred by 12 (57.1%) individuals, whereas the most frequent complain was local pain in women (53.3%) and men (50%). Neither side effects nor flu like syndrome were associated with co variables in the logistic models. Despite high influenza coverage levels it was detected respiratory virus circulation in the nursing home. It is reinforced the importance of influenza vaccination of residents and staff, and also improvement of routine measures to prevent secretion transmission disease. The safety and low reatogenity of influenza vaccine may help campaign adhesion. Etiologic surveillance of flu like syndrome could improve understanding of the pathogens circulation among risk population / Mestrado / Epidemiologia / Mestre em Saude Coletiva

Over 80% of the newly emerging infectious diseases are caused by RNA viruses. Major global problems associated with the development of vaccines against the RNA virus are their high genetic and antigenic diversity. Hence, effective control of epidemics with newly emerging RNA viruses require improved vaccines which are either specific to the new strain or broadly effective even when new viral strains emerge. The main focus of this dissertation is to develop epidemic vaccines using these two approaches. Using a newly emerged swine enteric virus called porcine epidemic diarrhea virus (PEDV) as a model, our first goal was to develop a quick and easy method for rapid response vaccines with potential applicability to a range of RNA viruses. We hypothesized that the methods which can disrupt genomic RNA without impacting the structural integrity of the virus would result in attenuated vaccine with minimum replication in the host while inducing immune responses. As hypothesized, developed rapid response PEDV vaccine induced complete protection against the virulent challenge virus, while vaccine viral shedding was not detected in vaccinated pigs. To address the second problem of rapid viral evolution leading to vaccines becoming obsolete, we used swine influenza virus (SIV) as a model to develop and test a universal vaccine composed of peptides encoding conserved antigenic epitopes which are present in most influenza A viruses. Importantly, a novel amphiphilic invertible polymer (AIP) was used to address the well-recognized problem of poor antigenicity of peptides. We hypothesized that peptides encoding conserved epitopes when conjugated with an AIP will induce strong immune responses and protect against challenge virus. While the conserved epitopes were previously tested by others in mice, we were the first to test a combination of these epitopes in pigs. Pigs vaccinated with the peptide polymer vaccine mounted strong antibody responses against the epitopes indicating that the delivery system was effective. However, protection against replication of the challenge virus was delayed. In summary, the methods developed and tested in this body of work significantly contribute to the area of emergency response management in infectious disease outbreaks. / United States Department of Agriculture, National Institute of Food and Agriculture (USDA-NIFA) / North Dakota State Agricultural Products Utilization Committee (ND APUC) / North Dakota State Board of Agricultural Research (ND SABRE)

Influenza viruses cause between 3 and 5 million cases of respiratory infection each year and are responsible for between 250 and 500 thousand deaths. There are principally two avenues for the treatment and prevention of influenza. They are vaccination and antiviral regimens. Prevention of infection is largely accomplished through vaccination. While vaccines remain the preferred method for controlling the spread of influenza, treatment with antiviral drugs is important for treatment of severe infections that are caused by viruses that are different from the vaccination strains. The two major classes of antiviral drugs for influenza treatment are the adamantanes and the neuraminidase inhibitors. While most viruses have become resistant to the adamantanes, the neuraminidase inhibitors remain the primary choice for treatment of infections. Oseltamivir is the most important of the neuraminidase inhibitors. Data from an experiment run at Utah State University displayed a characteristic that is reflected in other published data. Oseltamivir, which has been shown to be effective against influenza virus strains in vitro, is unable to sufficiently protect mice from lethal infections. The focus of the present research was to identify viral differences that might explain for this discrepancy. Four viral strains were chosen that display differing susceptibilities to oseltamivir in mice. The viruses used were Influenza A/Duck/MN/1525/81 (H5N1), A/Victoria/3/75 (H3N2), A/NWS/33 (H1N1) and A/California/04/2009 (Pandemic H1N1). Oseltamivir was unable to protect mice that were infected with the A/Duck/MN/1525/81 and A/Victoria/3/75 viruses. These two viruses, along with the A/NWS/33 and the A/California/04/2009 viruses, were compared in vitro using virus replication kinetics, neuraminidase inhibition assays, and antiviral assays in cell culture. The viruses were studied in vivo by comparing survival, weight loss, lung scores and weights, lung virus titers, complete blood counts, cytokine assays, and histopathology. A second in vivo experiment was run to determine the effects of oseltamivir on survival, weight loss, lung scores and weights, lung virus titers, and histopathology. The two in vivo experiments summarized in this study confirmed previous data since oseltamivir was unable to protect mice infected with the influenza A/Duck and A/Victoria viruses. Overall, the virus infections behaved remarkably similar. The most interesting difference was that the A/Duck/MN/1525/81 and A/Victoria/3/75 viruses were able to induce more severe histopathological damage in mouse lungs earlier in the infection. The ability to cause severe disease more quickly might explain why the A/Duck/MN/1525/81 and A/Victoria/3/75 viruses remain lethal, despite oseltamivir treatment.

La grippe est une maladie infectieuse qui se traduit par des épidémies saisonnières et pandémies occasionnelles et qui a des conséquences importantes sur la santé publique. Elle est causée par le virus influenza, un virus à ARN négatif segmenté. Chaque segment du génome est transcrit et repliqué dans le noyau de la cellule hôte par la polymérase virale, une ARN polyméraseARN-dépendente. Afin de pouvoir produire un ARN messager (ARNm) viral qui peut être reconnu par la machinerie de traduction de la cellule, la polymérase de la grippe se sert d’un mécanisme appelé ‘cap-snatching’ (mécanisme de ‘vol de coiffe’). La polymérase virale intéragit avec un ARNm coiffé, produit par l’ARN polymérase II de la cellulle hôte, coupe cet ARN à proximité de la coiffe et l’utilise comme amorce pour l’initiation de la transcription de son propre matériel génétique. Par conséquence, la transcription virale est fonctionnellement liée à la transcription cellulaire. L’objet des recherches présentées dans cette thèse portait sur l’intéraction entre la polymérase de la grippe et la machinerie de transcription de la cellule hôte et plus précisément, la caractérisation structurale et fonctionelle de l’association entre la polymérase de la grippe et le domain C-terminal de l’ARN polymérase II (CTD), qui sert de support pour la coordination des processus de synthèse et les modifications post-transcriptionelles de l’ARNm. Les structures cristallographiques obtenues de la polymérase d’influenza A et B, associées aux peptides dérivés du domain CTD de l’ARN polymérase II, ont permis d’élucider comment la polymérase virale reconnait spécifiquement la polymérase cellulaire. La perturbation de cette intéraction mène à l’inhibition de l’amplification du virus, ce qui confirme que cette association est essentielle. Dans la seconde partie de la thèse, un complexe actif de ‘cap-snatching’ a été assemblé in vitro et caractérisé fonctionellement, constitué du complexe d’élongation contenant l’ARN polymérase II avec un ARNm coiffé et son domain CTD phosphorylé, lié à la polymérase virale elle-même transcriptionnellement active. De plus, l’intéraction de la polymérase virale avec deux facteurs impliqués dans la régulation de l’élongation par l’ARN polymérase II, DRB sensitivity-inducing factor et Tat stimulatory factor 1, a été caractérisée. En conclusion, le travail de thèse présenté ici a contribué à élucider le mécanisme de recrutement de la polymérase d’influenza à la machinerie de transcription cellulaire et a montré que l’association entre les polymérases virale et cellulaire est essentielle pour la réplication du virus. La perturbation de cette intéraction est une piste prometteuse pour la conception de nouveaux médicaments contre le virus influenza. / Influenza is an infectious disease causing seasonal epidemics and occasional pandemics, which are a significant health burden for the global human population. The causative agent, influenza virus, is a negative-strand segmented RNA virus. Each segment of the viral genome is transcribed and replicated by a virally-encoded RNA-dependent RNA polymerase in the nucleus of the infected cell. In order to produce a functional viral messenger RNA (mRNA) that can be processed by the cellular translation machinery, influenza polymerase employs a mechanism called‘cap-snatching’. The viral polymerase binds to a nascent, capped transcript, produced by the cellular RNA polymerase II, cleaves it shortly after the cap and uses it as a primer for the transcription of its own genomic segments. Viral transcription is therefore functionally dependent on cellular transcription. The studies described in this thesis aimed to investigate the interaction between influenza polymerase and the cellular transcription machinery. The main focus was to characterize structurally and functionally the association of influenza polymerase with the C-terminal domain (CTD) of RNA polymerase II, which serves as a scaffold for coordinating co-transcriptional events during mRNA synthesis and processing. Crystal structures of influenza A and B polymerase bound to phosphorylated peptides mimicking the CTD of RNA polymerase II gave new insight on how the viral polymerase directly recognizes the transcribing cellular polymerase. Moreover, disrupting this interaction was found to be severely detrimental to viral replication, confirming the essentiality of this association. In the second part of this work, an active cap-snatching complex was assembled in vitro and characterized functionally. This comprised a reconstituted RNA polymerase II elongation complex with emerging capped transcript and phosphorylated CTD with bound and transcriptionally active influenza polymerase. Additionally, the interaction of the viral polymerase with two factors involved in the regulation of transcription elongation by RNA polymerase II, DRB sensitivity-inducing factor and Tat stimulatory factor 1, was analyzed biochemically. Overall, the work presented here gives insights into the mechanism of recruitment of the influenza polymerase to the cellular transcription machinery and shows that the association of the viral and cellular polymerase is essential for the viral replication. Targeted disruption of this interaction is therefore a promising avenue for the design of novel anti-influenza drugs.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20782号 / 生博第388号 / 新制||生||51(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 朝長 啓造, 教授 永尾 雅哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Periodic introductions of influenza A viruses (IAVs) from wild birds contribute to emergence of novel strains that infect domestic poultry, lower mammals, and humans, but the mechanisms of emergence are unclear. The objectives of this dissertation research are to infer the genesis of two emerging IAVs, low pathogenic avian influenza (LPAI) H10N8 and highly pathogenic avian influenza (HPAI) H7N8 viruses, and to characterize the antigenic diversity and genetic evolution of contemporary H7 avian influenza viruses (AIVs) from North America. First, AIVs that are genetically close to the human H10N8 isolate were recovered at the live poultry market (LPM) visited by the first H10N8 patient. High seroprevalence of H10 virus was observed in ducks and chickens from five LPMs in the region. These findings suggested that LPM was the most probable source of human infection with the H10N8 virus, and this virus appeared to be present throughout the LPM system in the city. Second, the novel H7N8 virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkey, in which it evolved from LPAI into HPAI. H4N8 IAVs from diving ducks possess a gene constellation comprising five H7N8–like gene segments. These findings suggest that viral gene constellations circulating among diving ducks could contribute towards the emergence of IAVs that can affect poultry. Diving ducks may serve as a unique reservoir, contributing to the maintenance, diversification, and transmission of IAVs in wild birds. Third, antigenic and genetic characterization of 93 H7 AIVs from North America showed limited antigenic diversity. Gradual accumulation of nucleotide and amino acid substitutions in the H7 gene of AIVs from wild and domestic birds caused a wide genetic diversity. These findings suggested that continuous genetic evolution has not led to significant antigenic diversity for contemporary H7 AIVs isolated from wild and domestic birds in North America. In summary, these findings not only improve our understanding of the ecology and evolution of IAVs but also provide information for formulation of effective disease prevention and control strategies.

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