A febre Zika é uma enfermidade que afeta pessoas de países tropicais e subtropicais. O agente etiológico da doença é o vírus Zika (ZIKV), um flavivirus de genoma RNA de fita simples, transmitido principalmente por mosquitos do gênero Aedes. Devido a associação do ZIKV à microcefalia e síndrome de Guillain-Barré é necessário desenvolver estratégias para a prevenção da doença. Entre eles, a melhor medida profilática é a vacinação. A proteína NS3 tem sido relatada como um potencial ativador da imunidade celular contra o ZIKV e outros flavivírus, cuja atuação tem sido utilizada como um alvo contra infecções virais. Neste contexto, o objetivo deste projeto foi testar a eficiência da NS3 como ativadora da resposta imunológica e, em combinação com o vírus inativado, produzir uma vacina capaz de desenvolver proteção total contra a infecção viral. Uma sequência da NS3 foi clonada no vetor pET-26b, expresso em E. coli Rosetta e purificado por cromatografia de afinidade em coluna de níquel. A completa inativação do ZIKV foi realizada por meio de adição de formaldeído 0,05% e incubação por 3 dias. Combinações vacinais de NS3 e vírus inativado foram inoculados em vários grupos de camundongos 129 Sv/Ev e A129 nos dias 0 e 14. No dia 21, os camundongos A129 vacinados foram desafiados com o ZIKV selvagem e analisados segundo a perda de peso e sobrevivência por 21 dias. Os camundongos vacinados com NS3 e vírus inativado, na mesma formulação vacinal, tiveram 100% de proteção contra o ZIKV infeccioso. O sangue total e o baço foram coletados dos camundongos 129 Sv/Ev e A129 após 21 dias de uma nova imunização. Foi avaliado a produção de anticorpos específicos e neutralizantes contra ZIKV por ELISA e PRNT, mostrando que animais imunizados com a formulação vacinal \"vírus inativado com NS3\" produzem mais anticorpos contra o vírus, porém não foi detectado neutralização do vírus em nenhum grupo imunizado. O perfil de citocinas expressas por linfócitos do baço, analisado por FACS, sugere que a NS3 participa na modulação para uma resposta Th1. Esta abordagem possibilitou a avaliação de uma combinação vacinal que se mostrou capaz de prevenir a infecção com o ZIKV em camundongos susceptíveis / Zika Fever is a disease that affects many people in tropical and subtropical countries. The etiologic agent is Zika Virus (ZIKV), a single-stranded RNA flavivirus, transmitted mostly by Aedes mosquitoes, but sexual, congenital and blood transfusion transmission has also been reported. Due to the possible association of ZIKV to microcephaly and Guillain-Barré Syndrome, it is necessary to develop strategies for disease prevention. Among them, the best prophylactic measure is vaccination. The non-structural protein NS3 has been shown to stimulate cellular immunity response against others flaviviruses, and such activity has been used as a target against other flaviviruses infection. In this context, the aim of this project is to test the efficiency of NS3, in combination with the inactivated virus, to produce a vaccine capable of developing full protection against viral infection. The NS3 sequence was cloned into the pET-26b vector, expressed in E. coli Rosetta and purified by nickel column affinity chromatography. The complete inactivation of ZIKV was performed by addition of 0.05% formaldehyde and incubation for 3 days. Vaccine combinations of NS3 and inactivated virus were inoculated into 129 Sv/Ev and A129 mice on days 0 and 14. On day 21, vaccinated A129 mice were challenged with wild ZIKV and analyzed for weight loss and survival by 21 days. Mice vaccinated with NS3 and inactivated virus, in the same vaccine formulation, had 100% protection against infectious ZIKV. Whole blood and spleen cells were collected from 129 Sv/Ev and A129 mice after 21 days of new immunization. The production of specific and neutralizing antibodies against ZIKV was evaluated by Imunnofluorescence, ELISA and PRNT, showing that animals immunized with the vaccine formulation \"virus inactivated with NS3\" increased the antibodies\' level against the virus, but no virus neutralization was detected in any immunized group. The cytokine profile expressed by spleen lymphocytes, analyzed by FACS, suggests that NS3 participates in modulation for a Th1 response. This approach gave us the opportunity to evaluate this vaccinal combination that was shown to be able to prevent infection with ZIKV in susceptible mice

Introdução: Em 2015, a infecção pelo zika começou a chamar a atenção de profissionais de saúde da região Nordeste. Apesar dos sintomas semelhantes aos da dengue, tratava-se de uma enfermidade que até então não estivera presente no território brasileiro. No segundo semestre de 2015, houve um aumento nos casos de microcefalia em recém-nascidos e com isso, pesquisadores descobriram que essa condição neurológica estava associada à infecção pelo referido vírus em gestantes. Desta forma, a gravidez em tempos de zika tornou-se uma preocupação para mulheres. Vivenciar uma gestação nestas condições poderia resultar em sofrimento mental para as mulheres, principalmente ao considerar que as áreas mais afetadas pela epidemia são marcadas por vulnerabilidades econômicas e sociais. Grupos e instituições que defendem o direito ao aborto nos casos de infecção pelo zika colocam seus argumentos em circulação na esfera pública, ao mesmo tempo em que atores com posicionamento contrário procuram defender seus discursos. Objetivos: Buscou-se mapear a controvérsia em torno do zika e a interrupção da gravidez para gestantes infectadas pelo vírus em dois jornais brasileiros, Folha de S. Paulo e O Estado de S. Paulo. Metodologia: Trata-se de uma pesquisa qualitativa, envolvendo análise documental de notícias. Foram selecionados textos jornalísticos do período de novembro de 2015 a dezembro de 2017. Por meio da Teoria Ator-Rede de Latour, buscou-se mapear a controvérsia sobre o direito ao aborto para gestantes infectadas pelo zika, identificando os atores e os argumentos que a compõem, por meio da elaboração de categorias de análise. Resultados e discussão: Observou-se que os atores que se posicionaram diante do problema foram médicos, pesquisadores, representantes da esfera jurídica, representantes governamentais, movimentos sociais, organismos internacionais e grupos religiosos. Os argumentos favoráveis recorrentes foram a gravidade da microcefalia, negligência do Estado, sofrimento das mulheres atingidas pela epidemia, defesa da descriminalização do aborto. Enquanto que os argumentos contrários se concentraram na incerteza sobre a relação causal entre zika e microcefalia e o valor da vida humana. Além disso, a presença de vozes das mulheres afetadas pela epidemia foi menor em relação aos outros atores. Considerações finais: O debate sobre a relação entre o vírus e o direito ao aborto foi perdendo força após o ano de 2016, porém reacendeu o debate do aborto. O zika pode ser um objeto adequado para refletir como a comunidade médica e de pesquisadores constroem controvérsias nos veículos midiáticos, bem como outros atores envolvidos no tema, como movimentos sociais, grupos religiosos, representantes governamentais, jurídicos, organismos internacionais. No entanto, nos veículos de comunicação, há uma hegemonia do discurso científico, enquanto que as vozes das mulheres não ecoam neste meio. / Introduction: In 2015, the zika infection began to catch the attention of health professionals in the Northeast region. Despite the symptoms similar to those of dengue, it was a disease that had not been present in Brazil until then. In the second half of 2015, there was an increase in the cases of microcephaly in newborns and with that, researchers found that this neurological condition was associated with the infection by said virus in pregnant women. In this way, pregnancy in times of zika has become a concern for women. Experiencing a pregnancy in these conditions could result in mental suffering for women, especially considering that the areas most affected by the epidemic are marked by economic and social vulnerabilities. Groups and institutions that defend the right to abortion in cases of zika infection place their arguments in the public sphere, while opposing actors try to defend their speeches. Objectives: We aimed to map the controversy around zika and the interruption of pregnancy to pregnant women infected by the virus in two Brazilian newspapers, Folha de S. Paulo and O Estado de S. Paulo. Methodology: This is a qualitative research, involving documentary analysis of news. Journalistic texts were selected from November 2015 to December 2017. Through the Latour Actor-Network Theory, we tried to map the controversy over the right to abortion for pregnant women infected with zika, identifying the actors and the arguments that the compose, through the elaboration of categories of analysis. Results and discussion: It was observed that the actors who posed the problem were doctors, researchers, legal representatives, government representatives, social movements, international organizations and religious groups. Favorable arguments were the seriousness of microcephaly, neglect of the State, the suffering of women affected by the epidemic, and the defense of the decriminalization of abortion. While the opposing arguments focused on uncertainty about the causal relationship between zika and microcephaly and the value of human life. In addition, the voices of the women affected by the epidemic were lower than the other actors. Final Considerations: The debate over the relationship between the virus and the right to abortion was losing strength after 2016, but it rekindled the abortion debate. The zika can be an appropriate object to reflect how the medical community and researchers construct controversies in the media vehicles, as well as other actors involved in the theme, such as social movements, religious groups, government representatives, jurists, international organizations. However, in the media, there is a hegemony of scientific discourse, while the voices of women do not echo in media vehicles.

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.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-11-22T11:48:56Z No. of bitstreams: 1 CarolinaRodriguesChanesDissertacao2018.pdf: 2882728 bytes, checksum: 9c589bc58e82d7391f2534b1161bc575 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-11-22T11:49:14Z (GMT) No. of bitstreams: 1 CarolinaRodriguesChanesDissertacao2018.pdf: 2882728 bytes, checksum: 9c589bc58e82d7391f2534b1161bc575 (MD5) / Made available in DSpace on 2018-11-22T11:49:14Z (GMT). No. of bitstreams: 1 CarolinaRodriguesChanesDissertacao2018.pdf: 2882728 bytes, checksum: 9c589bc58e82d7391f2534b1161bc575 (MD5) Previous issue date: 2018-08-24 / Zika virus is an emergent flavivirus transmitted by mosquitos of Aedes genus, was first isolated in 1947 from Zika forest in Africa of the sentinel?s monkeys. The virus spread to other continents and was related with congenital and neurological syndromes in South America and became a public health issue. The genomic sequencing arouses as a crucial tool to obtain answers in real time about infections diseases outbreaks and has helped to understand the origin, phylogeny and trace Zika virus propagation. The main goals of this work are to obtain the complete Zika virus genome sequences that circulated in three different states of Brazil, DF, MT and TO, using the amplification technic multiplex PCR, the MinION portable platform of Oxford Nanopore. Besides that, we had the goal to generate the phylogenetic tree by maximum likelihood to better understand the origin, propagation and dissemination of the virus in these states, and consequentially, in Brazil. The sequences obtain in this present work shown high coverage, 94% to 100% was the coverage of the completeness of the genomes, that presented four distinct lineages, two different clades and related with Northeast samples of Brazil. Finally, our study provides new complete genome sequences of Zika virus in the state of Mato Grosso, demonstrate the relationship of the genome sequences of MT with sequences of Northeast of Brazil and suggests two possible entrances of Zika virus in the state of MT. / Zika virus ? um flaviv?rus emergente transmitido por mosquitos do g?nero Aedes, primeiramente isolado em 1947 de macacos sentinelas na floresta de Zika na ?frica. O v?rus se espalhou para outros continentes at? ser relacionado com s?ndromes neurol?gicas e cong?nitas na Am?rica do Sul sendo taxado como um problema de sa?de p?blica. O sequenciamento gen?mico emergiu como uma ferramenta crucial de respostas em tempo real a surtos de doen?as infecciosas e tem ajudado a entender a filogenia, origem e delinear a propaga??o do Zika virus. Este trabalho tem como objetivos obter a sequ?ncia completa do Zika virus circulante em tr?s diferentes estados do Brasil, DF, MT e TO, utilizando a t?cnica de amplifica??o por PCR multiplex, a plataforma port?til MinION da Oxford Nanopore. Al?m disto, tivemos como objetivo realizar a ?rvore filogen?tica por m?xima verossimilhan?a a fim de compreender melhor a origem, propaga??o e dissemina??o do v?rus nestes estados e, consequentemente, no Brasil. As sequ?ncias obtidas no presente trabalho apresentaram alta cobertura, foi poss?vel cobrir entre 94% e 100% dos genomas que se apresentaram em quatro linhagens distintas e se associaram em dois clados diferentes relacionados as amostras do Nordeste do Brasil. Finalmente, nosso estudo fornece novas sequ?ncias completas do Zika virus no estado do Mato Grosso, demonstra a rela??o das sequencias do MT com as sequ?ncias do Nordeste do Brasil e sugere duas poss?veis entradas do Zika virus no estado do MT.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2018-04-03T14:49:52Z No. of bitstreams: 1 HaryelleNarymaConfessorFerreira_DISSERT.pdf: 903183 bytes, checksum: 512cec0622808aed442e5d8b7f4b59a6 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2018-04-10T20:46:08Z (GMT) No. of bitstreams: 1 HaryelleNarymaConfessorFerreira_DISSERT.pdf: 903183 bytes, checksum: 512cec0622808aed442e5d8b7f4b59a6 (MD5) / Made available in DSpace on 2018-04-10T20:46:08Z (GMT). No. of bitstreams: 1 HaryelleNarymaConfessorFerreira_DISSERT.pdf: 903183 bytes, checksum: 512cec0622808aed442e5d8b7f4b59a6 (MD5) Previous issue date: 2018-02-26 / Introduction: The increase in the number of cases of microcephaly in Brazil and its association with the Zika virus (ZIKV) is a global public health problem. The International Classification of Functioning Disability and Health (ICF) model is a powerful tool and extremely relevant in managing disability. Objective: Describe the functioning profile of children with microcephaly associated with ZIKV in two states of northeastern Brazil. Methods: This is a descriptive cross-sectional study. The sociodemographic characteristics, head circumference and other clinical data were collected from medical charts, physical examinations, measuring instruments and interviews with the children and their parents. The Brazilian Portuguese version of the ICF core set for cerebral palsy (CP) was used. Each ICF category was assigned a qualifier, which ranged from 0 to 4 (no disability, mild disability, moderate disability, severe disability and complete disability). For environmental factors, 0 represents no barrier and 4 total barrier; +0, no facilitator +4, total facilitator. Results: A total of 34 children with microcephaly caused by ZIKV were recruited (18 girls and 16 boys) at four rehabilitation facilities in Rio Grande do Norte and Para?ba states, Brazil. The average age of the participants was 21 months and head circumference z-scores ranged from 0.92 to -5.51. The functioning profile revealed complete disability in most of the body function categories (b). The activity and participation areas (d) were highly impacted, particularly in mobility-related categories. With respect to environmental factors (e), most of the sample reported a total facilitator for the nuclear family, friends and health services, systems and policies, as well as a total barrier to social attitudes. Conclusion: This is the first study that describes the functioning profile of children with microcephaly associated with ZIKV, using a tool based on the ICF in Brazil. Our findings reinforce the need to maximize health care and access to information ? based on the ICF ? for multiprofessional teams, administrators, family members and children.

Submitted by Taiwany Oliveira (taiwanyrodriguess@gmail.com) on 2018-10-22T14:48:18Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE DE DOUTORADO LIGIA FERNANDES ABDALLA.pdf: 4835970 bytes, checksum: 41de8abe90eea5b202c2800675761f12 (MD5) / Approved for entry into archive by Marcos Roberto Gomes (mrobertosg@gmail.com) on 2018-10-22T15:27:12Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE DE DOUTORADO LIGIA FERNANDES ABDALLA.pdf: 4835970 bytes, checksum: 41de8abe90eea5b202c2800675761f12 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-10-23T14:07:04Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE DE DOUTORADO LIGIA FERNANDES ABDALLA.pdf: 4835970 bytes, checksum: 41de8abe90eea5b202c2800675761f12 (MD5) / Made available in DSpace on 2018-10-23T14:07:04Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE DE DOUTORADO LIGIA FERNANDES ABDALLA.pdf: 4835970 bytes, checksum: 41de8abe90eea5b202c2800675761f12 (MD5) Previous issue date: 2018-09-26 / Zika virus (ZIKV) is an emergent arbovirus of the family Flaviviridae and the genus Flavivirus, which until 2007 was restricted to some cases of mild disease in Africa and Asia. In Brazil, it`s suspected that the entry of the virus occurred during the 2013 Confederations Cup and in the first half of 2015, there were already confirmed cases in states in all regions of the country. The Brazilian epidemic revealed that the usually mild and self-limiting infection could be related to neurological disorders. The objectives of this study were to clarify numerous questions regarding ZIKV infection, aiming to contribute to a better understanding of the mechanisms involved in the immunopathogenesis and course of the disease. As a result, the first report of atrial fibrillation in patients with Zika was described and could be considered an atypical manifestation during the virus infection; elevation of proinflammatory cytokines during ZIKV infection was observed; CXCL10 chemokine was identified as a potential biomarker for infection; saliva was characterized as the fluid of choice for the detection of Zika virus in the acute phase of the disease and a logistic regression model for the classification of cases of zika in relation to dengue was set up based on the clinical evaluation. / O Zika virus (ZIKV) é um arbovírus emergente da família Flaviviridae e do gênero Flavivirus, que até 2007 estava restrito a alguns casos de doença leve na África e na Ásia. No Brasil, suspeita-se que a entrada do vírus tenha se dado durante a Copa das Confederações de 2013 e no primeiro semestre de 2015, já havia casos confirmados em estados de todas as regiões do país. A epidemia brasileira revelou que a infecção geralmente leve e autolimitada poderia estar relacionada à distúrbios neurológicos. Os objetivos deste trabalho era esclarecer inúmeros questionamentos existentes em relação à infecção pelo ZIKV, visando contribuir com uma melhor compreensão dos mecanismos envolvidos na imunopatogênese e curso da doença. Como resultados, descreveu-se o primeiro relato de fibrilação atrial em pacientes com Zika, podendo ser considerado uma manifestação atípica durante a infecção pelo vírus; observou-se elevação de citocinas pró-inflamatórias durante a infecção ZIKV; identificou-se a quimiocina CXCL10 como um biomarcador potencial de infecção; caracterizou-se a saliva como fluído de escolha para o detecção do vírus Zika na fase aguda da doença e, montouse um modelo de regressão logística para a classificação de casos de zika em relação à dengue, com base na avaliação clínica.

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.

Background Arboviral diseases are one of the most common causes of acute febrile illness (AFI) and a significant health problem in South America. In Peru, laboratory etiologic identification of these infections occurs in less than 50% of cases, leading to underdiagnoses of important emerging arboviruses. Aim To assess the prevalence of the Dengue (DENV), Oropouche (OROV), Chikungunya (CHIKV), Mayaro (MAYV) and Zika (ZIKV) viruses in patients with acute febrile illness from Puerto Maldonado (Peru). Methodology Serum samples were obtained from patients with AFI during January 2016 to March 2016. A total of 139 specimens were analyzed for the presence of DENV, OROV, CHIKV, MAYV, and ZIKV using polymerase chain reaction (PCR). Results CHIKV in 9.4% and OROV in 8.6% were the most prevalent arboviruses, followed by DENV and ZIKV, with a prevalence of 6.5% and 5%, respectively. Among all patients, the most common symptoms accompanying fever were headaches 79.9%, muscle pain 65.5% and joint pain 63.3%. Conclusions During this short 3-month period, 4 arboviruses were detected by PCR, CHIKV and OROV being the most common arboviruses in Puerto Maldonado (Peru). Thus, it is crucial to include OROV detection in the national health surveillance. Furthermore, the etiologic clinical diagnosis of arboviral infections is not possible due to the low specificity of symptoms; therefore an increase of cases confirmed by molecular diagnostic methods will enhance arboviral surveillance in Peru.

<p>A successful flaviviral life cycle involves several coordinated events between viral proteins and host factors. The polyprotein processing at the surface of the ER membrane results in the formation of several replication proteins that bring about changes in the ER membrane making it permissive for viral genome amplification. Non-structural proteins 4A (NS4A) and non-structural protein 4B (NS4B) are two of the most important integral membrane proteins of DENV that are essential part of the viral replicase complex. The cleavage at NS4A-2K-NS4B is temporally and spatially regulated. The cleavage at the N-terminal of 2K is carried out by viral NS2B/3 protease while host signalase cleaves on the C-terminal side at the ER lumen to give rise to a mature NS4B protein. This thesis primarily focuses on demonstrating the function of 2K as an independent peptide rather than simply a signal sequence, and the role 2K plays, when present as 2K-NS4B vs NS4B. Moreover, this thesis has attempted to explore the function of transmembrane domains (TMDs) in replication separating them from their membrane anchor function. This thesis will also describe the development of a ZIKV replicon and its use in screening small molecule inhibitors in the last chapter.</p><p>In Chapter 2 of the thesis, we established 2K as an independent, information carrying peptide rather than just a signal peptide. A strategy involving chimeric virus generation and mutational analysis supported the notion that 2K is rather unique and important for viral replication and infectious particle production. Using an interserotypic 2K chimeric virus, it was established that the 2Ks of DENV are serotype specific, however, they are interchangeable with a huge fitness cost in infectious particle production. We further showed that individual amino acid residues towards then end of h-region and C-terminus of the 2K peptide affect viral replication and infectious particle production. Moreover, it was shown that the 2K peptide consists of a highly conserved ‘DNQL’ region at its N-terminal that plays an important role in viral replication.</p><p>Chapter 3 details the mechanistic aspect of the effects observed in interserotypic 2K chimeric viruses. The interserotypic chimeric viruses were comparable to wild type in replication, however, they were deficient in infectious particle production early in the life cycle. The major change to be noted in the chimeric viruses was the absence of signalase cleavage at the 2K-NS4B junction. We demonstrated that in a virus infected system, 2K-NS4B and NS4B populations are always present which led us to look for any specific functions of the cleaved vs uncleaved 2K-NS4B protein. Using a transcomplementation system where NS4B was presented in the absence of 2K, we showed that particle production can be rescued in the interserotypic 2K chimeric viruses. It was further concluded using NS4B truncations that the property of NS4B to rescue particle production was concentrated in the ER luminal loop. Further, alanine scanning mutagenesis of the conserved residues of ER loop resulted in pinpointing T198 and its involvement in the early stages of viral packaging.</p><p>Chapter 4 examined the role of TMDs of NS4A and NS4B and attempted to define their roles separately from their membrane anchoring functions. Several interserotypic TMD chimeric viruses were generated to address the function of these domains. We concluded that TMD1 and TMD3 of NS4A could be replaced with partial success across the DENV serotypes, whereas, TMD2 was serotype specific. The specificity of TMD2 of NS4A is not contributed by a single amino acid and should be a function of the secondary structure formed by TMD2 as it sits on the inner leaflet of the ER membrane. We demonstrated the variable roles different TMDs of NS4B play in viral replication using a similar strategy of reverse genetics of chimeric viruses. TMD1 of NS4B was replaceable with no to minimal effect, whereas, the remaining four showed variable effect upon substitution. More importantly, we demonstrated how the reorientation of TMD5 of NS4B post NS2B/3 cleavage might vary in different serotypes of DENV using revertant virus obtained from the TMD5 interserotypic chimera. Analysis of interserotypic cytosolic and ER luminal loop chimeras of NS4B pointed to functional conservation of the cytosolic loop between DENV-2 and DENV-3, whereas, the remaining cytosolic loops and the ER loops showed variable level of defects upon substitution, suggesting their functions in serotype-dependent manner.</p><p>Chapter 5 describes the construction and characterization of a ZIKV replicon system and use of it to screen several small molecule inhibitors of the flaviviruses MTase. Several small molecule inhibitors of flavivirus N-7-MTase were designed/synthesized in Dr. Arun K Ghosh’s lab which would target the extra pocket unique to the flavivirus SAM-binding site. We analyzed the docking of a set of these compounds into MTase domain of NS5 of ZIKV, DENV and YFV and screened them for their ability to inhibit replication of ZIKV, DENV and YFV. A huge variation in the activity profile of these compounds were observed against different flaviviruses even though these compounds were targeted against the highly conserved MTase domain of flavivirus NS5. GRL-002- and GRL-004-16-MT specifically inhibited ZIKV replication with low micromolar IC₅₀ value, while these compounds showed little to no effect on DENV and YFV.<b> </b>On the other hand, compounds GRL-007-, GRL-0012- and GRL-0015-16-MT demonstrated a dual inhibitory effect against DENV and YFV albeit the CC₅₀ values of the GRL-012 and GRL-015 were concerning. Compounds GRL-007-16-MT showed broad spectrum activity against ZIKV, DENV and YFV even though it was slightly cytotoxic to Vero cells. Moreover, GRL-002-16 was inhibitory to YFV while ineffective against DENV, whereas, GRL-016-16 had the opposite effect. Our results reveal the differential efficacies of the small molecule inhibitors targeting N-7-MTase. The experimental data suggests these compounds have different cytotoxicities in different cell lines and the compounds act in a virus-specific way. Nonetheless, we were able to shortlist some potent compounds for future modifications.</p>