O Zika vírus (ZIKV) é um flavivírus transmitido pelo mosquito Aedes aegypti e que se espalhou rapidamente pelas Américas, causando uma epidemia no Brasil em 2015 . Um número crescente nos casos de infecções veio acompanhado de um aumento no número de fetos e bebês nascidos com microcefalia, levando a um chamado de emergência mundial de saúde. Históricamente, o ZIKV não havia causado infecções de destaque em humanos e a reermegência dessa ameça viral associada à defeitos do nascimento foi logo relacionada à evolução e consequente distinção entre os genótipos virais, o original Zika africano e seu descendente Zika asiático, que chegou ao Brasil. A hipótese da cepa brasileira do ZIKV ser a causadora de microcefalia e de outros defeitos do nascimento ganhou mais respaldo após a identificação do vírus em amostras de tecido cerebral e líquido amniótico de fetos. Posteriormente, a associação direta entre microcefalia e a síndrome congênita com o ZIKV foi confirmada por meio da aplicação de modelos biológicos experimentais que se revelaram susceptíveis à infecção viral, como células do sistema nervoso central em sistemas 2D e 3D in vitro e camundongos prenhês. Esse trabalho teve como objetivo investigar a infecção da cepa brasileira do ZIKV (ZIKVBR) em diferentes células humanas neurais in vitro diferenciadas a partir de células-tronco pluripotentes induzidas, além de criar uma plataforma para teste de fármacos in vitro contra o vírus. Nossos resultados comprovaram a susceptibilidade e permissividade celular à infecção do ZIKVBR em células neuronais e, em especial, progenitoras neurais, causando morte celular por apoptose. Além disto, quando células progenitoras neurais foram cultivadas em suspensão, formando neuroesferas, o ZIKVBR foi capaz de causar uma redução na população de células, gerando uma anormalidade morfológica semelhante à microcefalia. Além do mais, quando células progenitoras neurais infectadas com ZIKVBR foram diferenciadas em neurônios maduros, a análise da sinaptogênese revelou que esses neurônios apresentavam uma menor densidade de puncta sináptica, indicando um comprometimento no funcionamento das sinapses que pode estar contribuindo para os problemas associados com a síndrome congênita do ZIKV. Por fim, o tratamento dessas células com a droga Sofosbuvir, um inibidor de RNA polimerase dependente de RNA aprovado para uso clínico, foi capaz de resgatar NPCs e neurônios apoptóticos. Em suma, nossos dados indicam que o ZIKVBR infecta preferencialmente células progenitoras neurais, replicando-se eficientemente e causando morte por apoptose nessas células e neurônios maduros diferenciados de células progenitoras neurais infectadas apresentam uma menor desidade de puncta sináptica. Finalmente, a reutilização de compostos farmacêuticos já aprovados para uso clínico pode acelerar o tratamento para indivíduos infectados pelo ZIKV onde a prevenção já não é mais opção, como no caso de mulheres grávidas. / Zika virus (ZIKV) is a mosquito-borne flavivirus transmitted by Aedes aegypti that has rapidly spread through the Americas, causing a widespread epidemic in Brazil in 2015. A increasing number of infection cases was followed by a rise in the number of fetuses and babies born with microcephaly, leading to a global health emergency call. Up to then, ZIKV had not caused meaningful infections in humans and the reemergency of this viral threat associated with birth defects was soon related to viral genotype mutations and its consequent distinction from the original african Zika strain to its descendent asian Zika strain, which reached Brazil. The hypotesis of the brazilian ZIKV strain being responsible for microcephaly and other birth defects gained support after the isolation and identification of the virus in samples of cerebral tissue and amniotic fluid of fetuses. Subsequently, the direct association between microcephaly and congenital syndrome with ZIKV was confirmed through the application of biologic experimental models which proved susceptible to viral infection, as for cells from the central nervous system cultured in 2D and 3D models as well as pregnant mice. The aim of this study was to investigate the brazilian ZIKV strain (ZIKVBR) infection in different human neural cells in vitro differentiated from induced pluripotent stem cells, as well as creating a platform for in vitro drug testing with antiviral capabilities. Our results showed cellular infection susceptibility and permissiveness to ZIKVBR in neurons and, specially, neural progenitor cells, displaying cell death by apoptosis. Futhermore, when neuronal progenitor cells cultured in suspension, forming neurospheres, were infected with ZIKVBR, it caused a reduction in cell population, displayed by evident morphological abnormalities resembling to microcephaly. Additionally, when neural progenitor cells infected with ZIKVBR were diferentiated further into mature neurons, synaptogenesis analysis revealed these neurons displayed fewer synaptic puncta density, indicating a compromise in synapse functioning that may be contributing to problems associated with ZIKV congenital syndrome. Moreover, cell treatment with Sofosbuvir, a RNA polymerase RNAdependent inhibitor approved for clinical use, was able to rescue apoptotic NPCs and neurons. In summary, our results reveal that ZIKVBR preferentially infects neural progenitor cells, efficiently replicating itself and causing death by apoptosis in these cells and mature neurons differentiated from infected neural progenitor cells display reduced synaptic puncta density. Lastly, the repurpose of FDA approved compounds may aid in accelerating treatment for infected individuals whose prevention is no longer an option, as it is for pregnant women.

Após setenta anos de sua descoberta, o vírus da zika surgiu no Brasil, espalhou-se rapidamente pelas Américas e trouxe complicações incomuns em doenças causadas por Flavivirus, como a microcefalia. A Organização Mundial da Saúde classifica a zika como a doença viral mais preocupante da atualidade e considera urgente desenvolver novos métodos de diagnóstico para ela e doenças correlatas como a dengue. Embora existam exames para identificar infecções pelos vírus dessas duas doenças, ainda não há um método rápido, específico e de baixo custo para o diagnóstico precoce. Visando preencher essa lacuna, este trabalho teve como objetivo construir dois tipos de biossensores eletroquímicos de DNA para detecção label-free desses dois vírus. Foram fabricados eletrodos descartáveis em substrato de politereftalato de etileno metalizado com filme fino de ouro nas configurações com um e três contatos. As sequências genéticas de iniciadores e sondas de captura foram desenhadas especialmente para este trabalho com base na análise dos genomas dos vírus. O primeiro biossensor utilizou o eletrodo em uma célula eletroquímica e foi capaz de identificar sequências de DNA da zika ou da dengue. As análises por espectroscopia de impedância eletroquímica mostraram que o biossensor é seletivo à sequência alvo com limite de detecção de (9,86 ± 0,89) nmol L-1. O segundo biossensor utilizou um eletrodo de três contatos para identificação de sequências de DNA em uma gota da amostra. No contato central, usado como eletrodo de trabalho, foi imobilizada a sequência de captura e os contatos laterais funcionaram como eletrodos de referência e auxiliar. Nesse sistema as medidas de impedância indicaram limite de detecção de (25,0 ± 1,7) nmol L-1. Os biossensores desenvolvidos mostraram seletividade para identificar o material genético dos vírus da zika e da dengue nos ensaios com DNA sintético e, portanto, são promissores para a análise de amostras reais, principalmente de produtos da polimerase da cadeia reversa. / After seventy years of its discovery, zika virus has emerged in Brazil, spread rapidly throughout the Americas, bringing unusual complications in diseases caused by flaviviruses, such as microcephaly. The World Health Organization classifies zika as the most harmful viral disease today and considers urgent the development of new diagnostic methods for zika and related diseases, such as dengue. Although there are tests to identify both infections, no current diagnostic method is rapid, specific and cost-efective. This thesis describes two types of electrochemical DNA biosensors for label-free detection of these zika and dengue. Disposable electrodes were fabricated on polyethylene terephthalate substrates covered with a nanometric gold layer by thermal evaporation, manufactured in one- and three-contact configurations. Genetic sequences of primers and complementary capture probes were designed based on the analysis of the virus genomes. The first biosensor we developed used the new electrode in an electrochemical cell and was able to identify zika or dengue DNA sequences. Analyses by electrochemical impedance spectroscopy showed that these biosensors are selective for zika or dengue with a detection limit of (9.86 ± 0.89) nmol L-1. A second type of biosensor used a three-contact electrode to identify DNA sequences in a drop of sample. In the central contact, used as a working electrode, the capture sequence was immobilized and the lateral contacts acted as reference and auxiliary electrodes. In this system the impedance measurements indicated a limit of detection of (25.0 ± 1.7) nmol L-1. The developed biosensors showed selectivity for zika and dengue in the synthetic DNA assays, and therefore are promising for the analysis of real samples, especially the polymerase chain reaction amplicon.

Brain development is an exquisitely coordinated process of progenitor cell proliferation followed by the migration of progeny to their final location in the developing brain. There are a myriad of points at which this process can be disturbed, and the examination of these perturbations help us further understand basic science, as well as epidemics sweeping through the world around us. Microcephaly, which is defined as a head circumference greater than 2 standard deviations below the mean, can occur through genetic, infectious, vascular, or metabolic etiologies, and the studies herein examine two forms by which microcephaly occurs. First, we investigate the role of the dynein regulatory protein Nde1 in the development of the neocortex, which is the outer region of the forebrain. NDE1 mutations are associated with severe microcephaly, and we find that unlike most microcephaly genes whose products have one role in the cell cycle, Nde1 is required at three discrete points in neuronal progenitors, termed radial glia progenitors (RGPs). We initially find that Nde1 is required to recruit dynein to the nuclear envelope to allow for interkinetic nuclear migration (INM) during G2. Additionally, Nde1 helps to initiate primary cilia resorption at the G1-to-S transition. Finally, there is a necessity for Nde1 at the G2-to-M transition after the completion of INM and prior to nuclear envelope breakdown. These three distinct roles for Nde1 illustrate the breadth of functions that the protein has during RGP proliferation, and help to explain why patients with NDE1 mutations have such severe microcephaly. As this work was ongoing there was a global outbreak of a new pathogen that had previously been dormant throughout Africa and Asia, only to emerge at epidemic proportions in the Western Hemisphere. This pathogen, the Zika Virus (ZIKV), is particularly alarming because of its subclinical course in adults but devastating consequences for fetal development, with the hallmark symptom being microcephaly. Using our organotypic brain slice model system, we demonstrate the ability of a variety of ZIKV isolates to infect and replicate in embryonic brain tissue. All ZIKV isolates that infect the organotypic slices lead to increases in apoptosis, though these increases are particularly pronounced in isolates from the Asian/American lineages. Notably, one isolate from a patient in Nigeria (termed 30656) does not replicate in mouse neuronal tissue, but electroporation of the 30656 ZIKV genome allows for a single cycle replication, suggesting that this isolate is unable to enter RGPs. All infectious isolates are pathogenic in early- and mid- gestation embryonic tissue, but only one isolate infects and replicates in late- gestation embryonic tissue. This was the most recently isolated sample tested, and it demonstrates a predilection for neurons, suggesting that ZIKV may be mutating as it spreads. These results provide foundational insight into the pathogenesis of ZIKV- associated microcephaly, and illustrate how studies of genetic forms of microcephaly can enhance and facilitate our understanding of infectious causes of the disease.

Submitted by admin tede (tede@pucgoias.edu.br) on 2018-04-17T17:46:02Z No. of bitstreams: 1 ALYNE APARECIDA FERREIRA FREITAS.pdf: 750774 bytes, checksum: f4eb9631ba2e7c7ff4dcdee8b5719580 (MD5) / Made available in DSpace on 2018-04-17T17:46:03Z (GMT). No. of bitstreams: 1 ALYNE APARECIDA FERREIRA FREITAS.pdf: 750774 bytes, checksum: f4eb9631ba2e7c7ff4dcdee8b5719580 (MD5) Previous issue date: 2018-03-05 / The objective of this study was to evaluate the family impact in parents of children diagnosed with microcephaly by Zika virus. This is a cross-sectional analytical study with a quantitative approach, using a sociodemographic questionnaire and family impact scale. A total of 76 parents with children undergoing rehabilitation and rehabilitation treatment were surveyed at a reference center in Goiânia. After the application of the instruments, a database was made using the IBM SPSS Statistics 18 software. Descriptive analyzes were performed using frequency, mean and standard deviation. The tests used to evaluate the existence or not of a statistically significant difference (p≤0.05) between independent and multiple variables were the Student's T-Test and the ANOVA Scheffé test, respectively. The mother is the main caregiver, the majority of whom are young mothers, divorced or divorced, with a corresponding monthly family income of 1 to 3 minimum wages, belonging to the low income class. It was observed that the predominant period of the diagnosis of the microcephalic child was in prenatal care. The greater the tendency to engage in activities with friends, parties and to go to bars, the individuals were more likely to perform physical and leisure activities. It was evidenced a difficulty on the part of the parents to find reliable persons to take care of the child, as well as, lack of understanding of other people for the burden that is to take care of the deficient son and expressed the desire in not having more children. It is concluded that after the initial shock of receiving the diagnosis of the child, the parents go through the reorganization phase, adapting to the challenges, changes in routine and family structure. The involvement of health professionals is essential, providing support and guidance to these families. Coping strategies emphasized social support in the institutional network, optimism, resilience and spirituality. / O objetivo deste estudo foi avaliar o impacto familiar em pais de crianças diagnosticadas com microcefalia pelo Zika vírus. Trata-se de um estudo transversal analítico com abordagem quantitativa, utilizando-se de um questionário sociodemográfico e a escala de impacto familiar (EIF). Foram pesquisados 76 pais com filhos em tratamento de reabilitação e readaptação em um centro de referência de Goiânia/Go. Após a aplicação dos instrumentos, foi confeccionado um banco de dados utilizando o software IBM SPSS Statistics 18. Por meio deste, foram realizadas análises descritivas utilizando-se frequência relativa e absoluta, média e desvio padrão. Os testes utilizados para avaliar a existência ou não de diferença estatisticamente significativa (p≤0,05) entre amostras independentes e múltiplas variáveis, foi utilizado o teste de análise de variância (ANOVA) Scheffé. A mãe é a principal cuidadora, sendo na sua maioria mães jovens, divorciadas ou desquitadas, apresentando renda mensal familiar correspondente de 1 a 3 salários mínimos, pertencentes à classe renda baixa. Observou-se ser no pré-natal o período predominante do recebimento do diagnóstico do filho microcefálico. Quanto maior a tendência de empreender atividades com amigos, festas e a frequentar bares, os indivíduos se mosraram mais propensos a realizarem atividades físicas e de lazer. Foi evidenciado uma dificuldade por parte dos pais em encontrar pessoas de confiança para cuidar do filho, bem como, falta de compreensão de outras pessoas pelo fardo que é cuidar do filho deficiente e expresso o desejo em não ter mais filhos. Conclui-se que após o choque inicial do recebimento do diagnóstico do filho, os genitores passam pela fase de reorganização, adaptando aos desafios, alterações na rotina e estrutura familiar. É fundamental o envolvimento dos profissionais de saúde, fornecendo suporte e orientação a essas famílias. Destacaram-se como estratégias de enfrentamento o apoio social na rede institucional, otimismo, resiliência e espiritualidade.

Após setenta anos de sua descoberta, o vírus da zika surgiu no Brasil, espalhou-se rapidamente pelas Américas e trouxe complicações incomuns em doenças causadas por Flavivirus, como a microcefalia. A Organização Mundial da Saúde classifica a zika como a doença viral mais preocupante da atualidade e considera urgente desenvolver novos métodos de diagnóstico para ela e doenças correlatas como a dengue. Embora existam exames para identificar infecções pelos vírus dessas duas doenças, ainda não há um método rápido, específico e de baixo custo para o diagnóstico precoce. Visando preencher essa lacuna, este trabalho teve como objetivo construir dois tipos de biossensores eletroquímicos de DNA para detecção label-free desses dois vírus. Foram fabricados eletrodos descartáveis em substrato de politereftalato de etileno metalizado com filme fino de ouro nas configurações com um e três contatos. As sequências genéticas de iniciadores e sondas de captura foram desenhadas especialmente para este trabalho com base na análise dos genomas dos vírus. O primeiro biossensor utilizou o eletrodo em uma célula eletroquímica e foi capaz de identificar sequências de DNA da zika ou da dengue. As análises por espectroscopia de impedância eletroquímica mostraram que o biossensor é seletivo à sequência alvo com limite de detecção de (9,86 ± 0,89) nmol L-1. O segundo biossensor utilizou um eletrodo de três contatos para identificação de sequências de DNA em uma gota da amostra. No contato central, usado como eletrodo de trabalho, foi imobilizada a sequência de captura e os contatos laterais funcionaram como eletrodos de referência e auxiliar. Nesse sistema as medidas de impedância indicaram limite de detecção de (25,0 ± 1,7) nmol L-1. Os biossensores desenvolvidos mostraram seletividade para identificar o material genético dos vírus da zika e da dengue nos ensaios com DNA sintético e, portanto, são promissores para a análise de amostras reais, principalmente de produtos da polimerase da cadeia reversa. / After seventy years of its discovery, zika virus has emerged in Brazil, spread rapidly throughout the Americas, bringing unusual complications in diseases caused by flaviviruses, such as microcephaly. The World Health Organization classifies zika as the most harmful viral disease today and considers urgent the development of new diagnostic methods for zika and related diseases, such as dengue. Although there are tests to identify both infections, no current diagnostic method is rapid, specific and cost-efective. This thesis describes two types of electrochemical DNA biosensors for label-free detection of these zika and dengue. Disposable electrodes were fabricated on polyethylene terephthalate substrates covered with a nanometric gold layer by thermal evaporation, manufactured in one- and three-contact configurations. Genetic sequences of primers and complementary capture probes were designed based on the analysis of the virus genomes. The first biosensor we developed used the new electrode in an electrochemical cell and was able to identify zika or dengue DNA sequences. Analyses by electrochemical impedance spectroscopy showed that these biosensors are selective for zika or dengue with a detection limit of (9.86 ± 0.89) nmol L-1. A second type of biosensor used a three-contact electrode to identify DNA sequences in a drop of sample. In the central contact, used as a working electrode, the capture sequence was immobilized and the lateral contacts acted as reference and auxiliary electrodes. In this system the impedance measurements indicated a limit of detection of (25.0 ± 1.7) nmol L-1. The developed biosensors showed selectivity for zika and dengue in the synthetic DNA assays, and therefore are promising for the analysis of real samples, especially the polymerase chain reaction amplicon.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2018-06-05T17:44:09Z No. of bitstreams: 2 Dissertação - Thiago Henrique Moreira da Fé - 2018.pdf: 1985149 bytes, checksum: 20d3ea58da14d653dfbb18d338dbb1b8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-06-06T11:04:33Z (GMT) No. of bitstreams: 2 Dissertação - Thiago Henrique Moreira da Fé - 2018.pdf: 1985149 bytes, checksum: 20d3ea58da14d653dfbb18d338dbb1b8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-06T11:04:33Z (GMT). No. of bitstreams: 2 Dissertação - Thiago Henrique Moreira da Fé - 2018.pdf: 1985149 bytes, checksum: 20d3ea58da14d653dfbb18d338dbb1b8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Dengue and zika are viral infectious diseases occurring in countries with a tropical and subtropical climate in which around 3.6 billion people live. It is estimated that in 50 to 100 million new cases of dengue occur annually, generating economic, social and public health impacts. Dengue and zika have usually been diagnosed by serological methods which are generally of low confidence, as they can generate false-positive results, which are related to the presence of antibodies produced against previous infections of the virus. The molecular methods are more accurate, however the molecular method most commonly used (PCR) requires a long time of reaction and requires sophisticated instrumentation and high cost, making point of care applications difficult,, especially in developing countries. This work presents the development of a molecular diagnostic methodology in a paper-based platform that allowed the detection of the virus through the reverse transcription -loop mediated isothermal amplification (RT-LAMP). The reactions were carried out on 6 mm diameter FTA paper discs, confined in a multi-layered polyester-toner device, incubated at 65 ° C for 45 minutes in a dry bath and then performed visual detection using the SYBR Green intercalator. Positive reactions were identified by the green fluorescence emitted after the addition of the intercalator. The results were recorded through the capture of the images by a photodocumentator and/or by smartphone and later analyzed by the software ImageJ, allowing the comparison between negative and positive reactions. The methodology developed for the detection of the virus by RT-LAMP in paper substrate was sensitive, being able to detect the virus in initial concentrations of 0.1 pg μL -1 of RNA in the master mixture. In addition, it was possible to detect the virus directly in complex samples (serum of infected patients) without the need of previous viral RNA extraction step. Elimination of the RNA extraction step together with the visual detection on the paper produce the final result in 46 minutes. The results demonstrated that the detection of the virus by RT-LAMP in paper substrates is a valuable tool for the molecular diagnosis of infectious diseases, presenting great potential for point-of-care applications for both diagnostics and epidemiological studies, especially in developing countries. / A dengue e a zika são doenças infecciosas virais de ocorrência nos países de clima tropical e subtropical no qual vivem cerca de 3,6 bilhões de pessoas, estimando-se, no caso da dengue, que 50 a 100 milhões de novos casos da doença ocorram anualmente, gerando impactos econômicos, sociais e de saúde pública. A dengue e a zika têm sido usualmente diagnosticadas por métodos sorológicos que são em geral de baixa confiança, pois podem gerar resultados falso-positivos, que estão relacionados à presença de anticorpos produzidos contra infecções anteriores do vírus. Os métodos moleculares são mais precisos, porém o método molecular mais utilizado atualmente (PCR) requer longo tempo de realização e necessita de instrumentação sofisticada e de alto custo, dificultando sua aplicação no ponto de atendimento, especialmente em países em desenvolvimento. Este trabalho apresenta o desenvolvimento de uma metodologia de diagnóstico molecular em uma plataforma a base de papel que permitiu a detecção do vírus por meio da reação de transcrição reversa seguida pela amplificação isotérmica mediada por loop (RTLAMP). As reações foram realizadas em discos de papel FTA com 6 mm de diâmetro, confinado em dispositivo multicamadas de poliéster-toner, incubados à 65 °C por 45 minutos em banho seco e posteriormente realizado a detecção visual onchip, através da utilização do intercalador SYBR Green. As reações positivas foram identificadas pela fluorescência verde emitida após a adição do intercalador. Os resultados foram registrados por meio da captura das imagens por uma fotodocumentadora e/ou por câmera de celular e posteriormente analisados pelo software ImageJ, permitindo a comparação entre reações negativas e positivas. A metodologia desenvolvida para a detecção do vírus por RT-LAMP em substrato de papel apresentou-se sensível, sendo capaz de detectar o vírus em concentrações iniciais de 0,1 pg µL-1 de RNA na mistura reacional. Além disso, foi possível detectar o vírus diretamente em amostras complexas (soro de pacientes infectados) sem a necessidade da etapa prévia de extração do RNA viral. A eliminação da etapa de extração do RNA juntamente com a realização da detecção visual no próprio papel proporcionou a obtenção do resultado final em 46 minutos. Os resultados demonstraram que a detecção do vírus por RT-LAMP em substrato de papel é uma importante ferramenta para o diagnóstico molecular de doenças infecciosas, apresentando grande potencial para aplicações no ponto de atendimento tanto para diagnósticos quanto para estudos epidemiológicos, especialmente em países em desenvolvimento.

No Brasil a confirmação do primeiro caso de febre zika, resultante da infecção pelo vírus zika (ZIKV), foi no primeiro semestre de 2015, na região nordeste. Os achados característicos eram de uma doença que apresentava sintomas semelhantes aos sintomas causados pelo vírus dengue (DENV), porém mais amenos, sendo inicialmente denominada de \"Síndrome Dengue-Like\". Além desta semelhança no quadro clínico, ZIKV e os DENV são arboviroses endêmicas no Brasil e pertencem à mesma família viral, sendo muito próximos filogeneticamente, resultando em uma forte reação cruzada entre os anticorpos induzidos por estas infecções. O diagnóstico específico para o ZIKV requer cuidados uma vez que o DENV circula há muito mais tempo no Brasil e muitas pessoas já são imunes para, no mínimo, um sorotipo da doença. Desta forma, anticorpos contra os DENV nos testes sorológicos podem reagir inespecificamente contra o ZIKV, originando um resultado falso-positivo. As maneiras para diferenciar as duas doenças são o isolamento e a detecção do RNA viral durante a fase aguda da doença e dos anticorpos neutralizantes durante a fase de convalescença. Atualmente com a escassez de testes registrados pela ANVISA e a dificuldade em diferenciar as duas viroses usando testes sorológicos, amostras bem caracterizadas e confirmadas quanto à infecção pelo ZIKV foram utilizadas para o teste de comparação com vários kits comerciais aprovados ou não pela ANVISA, para detecção de anticorpos. Estas amostras também foram testadas por um ensaio soro-molecular padronizado e desenvolvido no Laboratório de Virologia Molecular. / In Brazil, the first case of zika fever, resulting from zika virus (ZIKV) infection, was confirmed in the first half of 2015, in the northeast region. The characteristic findings were of a disease that presented symptoms similar to the symptoms caused by dengue viruses (DENV), but milder, being initially called \"Dengue-Like Syndrome\". Although this similarity in the clinical presentation, ZIKV and (DENV) are arboviruses endemic in Brazil and belonging to the same viral family, being very close phylogenetically, and resulting in a strong cross-reaction between the antibodies induced by these infections. The specific diagnosis for ZIKV requires careful evaluation since DENV has been circulating much longer in Brazil and many people are already immune to at least one serotype of the disease. Thus, the antibodies against DENV may react non-specifically against ZIKV in serological tests, resulting in a false-positive result. The ways to differentiate the two diseases are the isolation and detection of viral RNA during the acute phase of the disease and the neutralizing antibodies during the convalescent phase. Actually, with the scarcity of tests registered by ANVISA and the difficulty in differentiating the two viruses using serological tests, well-characterized and confirmed samples for ZIKV infection were used for the comparison of several commercial kits for detection of antibodies approved or not by ANVISA. These samples were tested by a standardized serummolecular assay developed at the Molecular Virology Laboratory.

Le virus Zika (ZIKV), virus découvert pour la première fois à la fin des années quarante, est un arbovirus émergent récemment arrivé sous le feu des projecteurs à l’occasion d’une pandémie rapide à l’échelle mondiale. Appartenant à la famille des Flaviviridae, ce flavivirus est transmis par les moustiques du genre Aedes. Alors qu’on le croyait relativement peu pathogène, ce virus se révèle être la cause probable d’une vague de complications neurologiques, incluant l’apparition de microcéphalies et de syndromes de Guillain-Barré. De plus, il n’existe à l’heure actuelle ni vaccins ni traitements spécifiques, la lutte contre le virus se résumant largement à la mise en place de mesures de prévention contre la piqûre de moustiques et la lutte anti-vectorielle.Une meilleure connaissance de l’ensemble de la biologie du virus, depuis les modalités d’entrée dans l’organisme, en particulier au niveau cutanée, jusqu’aux mécanismes moléculaires intimes de la réplication du virus s’avère nécessaire. Par des approches moléculaires et cellulaires, nous avons mis en évidence le tropisme du virus, identifié ses récepteurs et déterminé les réponses cellulaires induites par ce dernier. Nos travaux ont également identifié un potentiel mécanisme d’évasion mise en place par le ZIKV. Nous avons également entrepris un travail original sur un mécanisme moléculaire favorisant la pathogénicité des flavivirus. Une meilleure connaissance de ce mécanisme pourrait déboucher sur l’identification de potentiels cibles thérapeutiques. Enfin, le tropisme neuronal avéré du ZIKV nous a amené à travailler sur la réponse immune des astrocytes humain. En effet, les astrocytes forment une population cellulaire très importante dans le système nerveux central qui est fortement impliquée dans les mécanismes de neurogénèse dans le cerveau des fœtus. / The Zika virus (ZIKV) was first isolated from non-human primates the late 1940s. This emerging arbovirus has recently been under the spotlight due to a rapid world pandemic. Belonging to the Flaviviridae family, this flavivirus is transmitted by Aedes’ genus mosquitoes. Historically low pathogenic, a new major concern is the possible association of ZIKV with diverse of neurological complications, including the development of microcephaly and Guillain-Barré syndrome, particularly in newborns of infected mothers. In addition, there is currently no vaccine or specific treatment to cure the disease, so the main preventive measures to fight the spreading of the virus are to prevent mosquitoes’ bites and to plan an effective vector control. A better understanding of the biology of the virus, from the entry in the body, especially at the skin level, to the molecular mechanisms of viral replication, is therefore necessary.Using different molecular and cellular strategies, we investigated the tropism of the virus, identified cell surface receptors and determined the cell’s responses to the infection. Our work also permitted to identify a potential mechanism by which ZIKV evades the host immune system to facilitated his own replication. We also have undertaken original work on a molecular mechanism increasing the pathogenicity of flavivirus. A better knowledge of this mechanism may lead to the identification of potential therapeutic targets. Finally, considering the neuronal tropism of the ZIKV, we studied the immune response of human astrocytes, a very important cell population in the central nervous system, playing a major role in the mechanisms of neurogenesis during the fetus’ brain development.

The Zika virus is associated with the devastating birth defect microcephaly, and while a vaccine was not yet available in early-2017, several were under development. It is imperative to identify effective communication strategies to promote uptake of a new vaccine, particularly among women of reproductive age. Moreover, though the Zika outbreak has received much social media attention, little is known about these conversations on Instagram. The purpose of this dissertation, therefore, was to understand current Zika-focused communication on Instagram and to inform effective communication strategies to promote future Zika vaccine uptake intent. The study aims were: (1) explore Zika conversations on Instagram; (2) determine effective message characteristics to increase Zika vaccine uptake intent; and (3) explore salient demographic, healthcare, and psychosocial factors related to Zika vaccine uptake intent. A content analysis of 1,000 Zika-focused Instagram posts, found that these messages primarily focus on perceived threat constructs, yet they elicited little engagement. In addition, 10% of all Instagram posts mentioned conspiracy theories, and these messages elicited high engagement. A 2x2 online experiment tested the effect of message framing and visual type on Zika vaccine uptake intent. The 339 participants – all women of reproductive age – each were exposed to one of four messages (gain vs. loss-framed, and infographic vs. photo). There was no interaction effect of framing and visual type (p=.116), nor main effect of either framing (p=.185) or visual type (p=.724) on vaccine uptake intent. When testing the effect of these variables on those known to be predictors of behavioral intent, gain-framed messages were associated with higher subjective norms, perceived benefits, and self-efficacy. Data from the same online survey was used to examine whether demographics, healthcare-related variables, and psychosocial variables predict Zika vaccine uptake intent. Attitude (p<.001), subjective norms (p=.002), perceived benefits (p=.001), self-efficacy (p=.031), perceived susceptibility (p=.030), and cues to action (p=.020) were predictive of higher Zika vaccine uptake intent, as was being African-American (p=.042). In summary, messages promoting the Zika vaccine should be designed to complement the high perceived threat of Zika while activating positive social norms and perceived benefits in order to allow the public to respond efficaciously.

Depuis 2007, le Gabon est régulièrement confronté à des infections par les virus Chikungunya (CHIKV) et Dengue (DENV). Au total, près de 4300 prélèvements provenant de patients se présentant avec un syndrome fébrile algique, en phase clinique aiguë, ont pu être collectés et analysés pour la période de 2007 à 2010. En effet, deux importantes épidémies concomitantes de CHIKV et de DENV ont sévi au Gabon (i.e. Libreville en 2007 et Franceville en 2010). Entre ces deux flambées épidémiques, de nombreux cas sporadiques d'infection à CHIKV ou à DENV ont continué à être enregistrés à travers le pays. Des cas de co-infection à CHIKV/DENV ont également été diagnostiqués lors des deux flambées épidémiques. Ces deux arbovirus se sont ainsi propagés en quelques années dans un mouvement de nord-ouest à sud-est à travers le pays. L’étude plus avancée des cas de co-infection à CHIKV/DENV a pu démontrer que ce phénomène pouvait survenir soit de manière simultanée soit séquentielle au cours du repas sanguin d'Aedes albopictus, principal vecteur du CHIKV et du DENV au Gabon.(…)En conclusion, ce travail de thèse décrit précisément la survenue brutale d’épidémies imputables à plusieurs arbovirus circulant simultanément au Gabon et responsables de nombreux cas cliniques se présentant sous la forme d'un syndrome fébrile algique. La co-circulation de ces virus suggère l’apparition d’une dynamique de type épidémique/endémique et implique un problème de santé publique latent dans cette région d’Afrique, voire dans l’ensemble de la sous-région d’Afrique Centrale. / Following to the first simultaneous Chikungunya (CHIKV) and Dengue (DENV) viruses outbreak in 2007, an active surveillance of febrile syndromes was set up in Gabon, a central African country. During a three-year period, we observed a rapid spread of CHIKV and DENV in a southward movement from north-west to south-east of the country. Indeed, CHIKV and DENV have disseminated within a non-immune population, widely favored by the extraordinary capacity of Aedes albopictus vector to colonize diverse environments and to replace local mosquito’s species. In 2010, a second outbreak occurred in Gabon with further CHIKV/DENV co-infections in both human and mosquito. This is the first documented evidence of co-infection in a wild-caught Aedes albopictus. Additionally, an underlying Zika (ZIKV) virus epidemic transmission by the same invasive vector was retrospectively recorded during the outbreak in 2007. These data reveal an unusual ZIKV natural life cycle, occurring in an urban environment and potentially representing a new arboviral emerging threat from Aedes albopictus.(…)In conclusion, these data highlighted the recent introduction and rapid dissemination of CHIKV and DENV in Gabon. The Aedes albopictus vector has shown its extraordinary capacity to sustain epidemic transmissions, leading to arboviral co-circulations (i.e. CHIKV, DENV-2, DENV-1, DENV-3, ZIKV) and notably to some CHIKV/DENV-2 co-infection cases. This multiple arboviral circulation suggests an epidemic/endemic dynamic in Gabon, involving a latent public health problem in this region of Africa.