Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 719 pages. Includes vita. Includes bibliographical references.

SACRAMENTO, R. H. M. Dengue e o Aedes aegypti na população indígena dos Tremembé da aldeia Tapera no Ceará: prevalência, conhecimentos e práticas. 2017. 84 f. Dissertação (Mestrado em Patologia) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-05-09T13:22:46Z No. of bitstreams: 1 2017_dis_rhmsacramento.pdf: 15673570 bytes, checksum: 580afc157dc04fd616e5fc54e94b158a (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-05-10T15:13:07Z (GMT) No. of bitstreams: 1 2017_dis_rhmsacramento.pdf: 15673570 bytes, checksum: 580afc157dc04fd616e5fc54e94b158a (MD5) / Made available in DSpace on 2017-05-10T15:13:07Z (GMT). No. of bitstreams: 1 2017_dis_rhmsacramento.pdf: 15673570 bytes, checksum: 580afc157dc04fd616e5fc54e94b158a (MD5) Previous issue date: 2017-03-23 / INTRODUCTION: Dengue remains an important public health problem. The indigenous peoples of the Northeast were the first to be contacted in the Portuguese colonial invasion, and have since suffered the social and health consequences of this historical moment. Despite recent efforts to address this vulnerable population, there are few records of dengue among Brazilian Indians. PURPOSE: to determine the risk of dengue transmission and the seroprevalence of the disease among TREMEMBÉ natives (Tapera village in the city of Itarema, Ceará), as well as aspects related to infestation by mosquitoes of the genus Aedes. MATERIAL AND METHODS: a cross-sectional study, a seroepidemiological survey (part I) and a prospective environmental study of monthly traps for mosquito eggs collection (part II) were carried out. The sub-study I involved 350 Tremembé Indians living in the village of Tapera, municipality of Itarema, Ceará. The association between the results of serological screening (IgG) and the epidemiological variables investigated were analyzed using contingency tables, using the Prevalence Ratio and tested by the Fisher's exact test, with a 95% confidence interval and with defined statistical significance As p <0.05. For the sub-study II, 42 houses were randomly randomized. In each residence a trap (ovitrampa) was installed inside and outside the house, composing the 84 traps monitored. These traps were inspected for 12 consecutive months. The reeds were removed weekly, put to dry and then the eggs were counted. The data were entered and analyzed using Epiinfo software in version 3.5.1. The project was approved by all the ethical instances required for projects involving special populations. RESULTS: The Tremembé da Tapera are mostly in the economically active age (65.1%) and 62% consider themselves illiterate or with low schooling. More than half (62.5%) have a family income below a salary and only 35.5% of them move more frequently to the city. Fever was the most cited symptom among the natives (82.7%), followed by pain in the muscles (66.2%), eyes (63.4%), joints (57.6%) and vomiting %). The most cited measure of control was to eliminate standing water (76.4%) and to place sand in the plant pots (76.4%). Most houses are masonry (95.7%), garbage is burned by 44.4% of families and 77% has regular water supply. Seroprevalence was 22.1% (62/280), of which 39 were women (63%) and 23 were men (37%). Positivity increased with age, being 4.2%; 26.8% and 42.3% for the groups of <15 years, 15 to 59 and> 59 years; Respectively (p <0.001). He was also associated with the belief that he had dengue, a higher frequency of dislocation to the city and presence of basic diseases. Eggs were collected in all months of the year and in all households. The highest collection occurred in March (3,145 eggs) and the lowest in September (379 eggs). More eggs were collected in traps located outside the home (57%). In six houses there were trapping eggs in 11 of the 12 months of monitoring. CONCLUSION: the prevalence of dengue among the Tremembé was high compared to other rural regions, even though they have a reasonable knowledge about symptoms and prevention measures. The mosquito infestation of the genus Aedes was very high in the village, with persistence throughout the year. / INTRODUÇÃO: dengue permanece como importante problema de saúde pública. Os povos indígenas do Nordeste foram os primeiros contatados na invasão colonial portuguesa, e vem sofrendo, desde então, as consequências sociais e sanitárias deste momento histórico. Apesar do esforço recente em atender essa população vulnerável, há poucos registros de dengue entre indígenas brasileiros. OBJETIVO: determinar o risco de transmissão de dengue e a soroprevalência da doença entre indígenas da etnia TREMEMBÉ (aldeia Tapera no município de Itarema, Ceará) além dos aspectos relacionados à infestação por mosquitos do gênero Aedes. MATERIAL E MÉTODOS: foi realizado estudo transversal analítico, tipo inquérito soroepidemiológico (parte I) e um estudo ambiental prospectivo de acompanhamento mensal de armadilhas para coleta de ovos de mosquitos (parte II). O sub-estudo I envolveu 350 indígenas da etnia Tremembé, residentes na aldeia da Tapera, município de Itarema, Ceará. A associação entre os resultados de triagem sorológica (IgG) e as variáveis epidemiológicas investigadas foram analisadas utilizando tabelas de contingência, utilizando-se a Razão de Prevalência e testadas pelo teste exato de Fisher, com intervalo de confiança de 95% e com significância estatística definida como p<0,05. Para o sub-estudo II foram sorteadas aleatoriamente 42 casas. Em cada residência foi instalada uma armadilha (ovitrampa) dentro e outra fora de casa, compondo as 84 armadilhas monitoradas. Essas armadilhas foram inspecionadas por 12 meses consecutivos. As palhetas eram retiradas semanalmente, postas para secar e posteriormente os ovos eram contados. Os dados foram digitados e analisados utilizando o software Epiinfo na versão 3.5.1. O projeto foi aprovado por todas as instâncias éticas necessárias aos projetos que envolvem populações especiais. RESULTADOS: os Tremembé da Tapera estão em sua maioria na idade economicamente ativa (65,1%) e 62% se consideram analfabetos ou com baixa escolaridade. Mais da metade (62,5%) tem renda familiar inferior a um salário e apenas 35,5% deles se deslocam com maior frequência para cidade. A febre foi o sintoma mais citado pelos indígenas (82,7%), seguido de dor nos músculos (66,2%), nos olhos (63,4%), articulações (57,6%) e vômitos (57,0%). A medida de controle mais citada foi eliminar água parada (76,4%) e colocar areia nos vasos de planta (76,4%). A maior parte das casas é de alvenaria (95,7%), o lixo é queimado por 44,4% das famílias e 77% tem abastecimento de água regular. A soroprevalência foi de 22,1% (62/280), sendo 39 mulheres (63%) e 23 homens (37%). A positividade aumentou com a idade, sendo de 4,2%; 26,8% e 42,3% para os grupos de <15 anos, 15 a 59 e >59 anos; respectivamente (p<0,001). Foi associada também a acreditar que teve dengue, maior frequência de deslocamento para cidade e presença de doenças de base. Foram coletados ovos em todos os meses do ano e em todas as casas. A maior coleta ocorreu no mês de março (3.145 ovos) e a menor em setembro (379 ovos). Foram coletados mais ovos em armadilhas localizadas fora de casa (57%). Em seis casas houve detecção de ovos nas armadilhas em 11, dos 12 meses de monitoramento. CONCLUSÃO: a prevalência de dengue entre os Tremembé foi alta se comparada a outras regiões rurais, mesmo que eles apresentem um razoável conhecimento sobre sintomas e medidas de prevenção. A infestação por mosquitos do Gênero Aedes foi muito alta na aldeia, com persistência durante todo o ano.

Orientadora: Profa. Dra. Maria Aparecida Sperança / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2016. / O arbovírus Mayaro (MAYV), pertence ao gênero Alphavirus, família Togaviridae e grupo sorológico A. Como parte do projeto de pesquisa sobre "Arboviroses Emergentes na Amazônia Ocidental", foi isolada uma estirpe de um alphavirus Mayaro a partir de um indivíduo febril de Acrelândia, Acre. O MAYV ocorre no ambiente silvestre ou rural em áreas próximas a florestas e seu ciclo de transmissão envolve a participação de primatas não humanos, aves e mosquito Haemagogus ssp. As características patogênicas do vírus MAY em humanos são pouco compreendidas e o potencial de dispersão desses vírus e a possibilidade de participação de espécies de mosquitos do gênero Aedes em seu ciclo de transmissão fazem desse vírus um importante organismo para estudos biológicos e epidemiológicos. Devido à natureza não específica dos sinais e sintomas de doenças febris agudas causadas por MAYV, o diagnóstico clínico é difícil e pode ser confundido com outras doenças febris, como: a febre da dengue, febre do chikungunya, febre amarela e a malária. Já o diagnóstico laboratorial por isolamento viral é laborioso e depende de infraestrutura e pessoal especializado, disponível em poucos laboratórios brasileiros. O diagnóstico imunoenzimático possui limitações quanto à utilização de partículas virais infecciosas e também por produzirem reações cruzadas com vírus da mesma família. Sendo assim, com o intuito de obter antígeno recombinante específico para detecção de MAYV, este projeto teve por objetivo expressar a glicoproteína E2 do vírus em células de insetos utilizando baculovírus recombinante como vetor. A glicoproteína E2 dos alphavirus tem a função de se ligar a receptores presentes na membrana plasmática da célula alvo para a entrada do vírus através de uma vesícula endossomal. Como resultado, foi obtido o baculovírus recombinante para a glicoproteína E2 de MAYV, e a mesma foi expressa pela primeira vez em células de inseto High-FiveTM. Apesar de utilizar vetor com sinal de exportação da proteína recombinante para o meio extracelular, a glicoproteína recombinante E2 de MAYV não foi exportada, permanecendo na fração celular da cultura. Análises da estrutura tridimensional da E2 homóloga de alphavirus indicaram que esta glicoproteína apresenta um domínio transmembrana, o que justifica sua permanência na bicamada fosfolípidica da célula hospedeira. / The arbovirus Mayaro (MAYV), belongs to the genus Alphavirus, family Togaviridae and serological group A. As part of the research project on "Emerging Arboviroses in the Western Amazon", a strain of Mayaro alphavirus was isolated from a febrile individual of Acrelândia, Acre. MAYV occurs in the wild or rural environment, in areas near to forests and its life cycle includes the participation of non-human primates, birds and mosquito Haemagogus ssp. The pathogenic characteristics of MAYV in humans are poorly understood and the dispersal potential of these viruses and the possibility of participation of species of Aedes mosquitos in its transmission cycle make this virus an important organism for biological and epidemiological studies. Because of the nonspecific nature of the signs and symptoms of acute febrile illness caused by MAYV, clinical diagnosis is difficult and can be confused with other infectious diseases, such as dengue fever, chikungunya fever, yellow fever and malaria. Moreover laboratory diagnosis by viral isolation is laborious and depends on infrastructure and trained personnel available in a few Brazilian laboratories. The enzyme immunoassay diagnosis has limitations on the use of infectious viral particles and also for producing cross-reactions with other virus from the same group. Thus, in order to obtain recombinant antigen for specific detection of MAYV, this project aimed to express the MAYV E2 glycoprotein in insect cells using recombinant baculovirus as vector. The E2 glycoprotein of the alphaviruses has the function of binding to receptors in the plasma membrane of the target cell for viral entry through an endosomal vesicle. The recombinant baculovirus was obtained for the MAYV E2 glycoprotein, and this protein was expressed for the first time in High- FiveTM insect cells. Despite using a vector with signal to exportation of the recombinant protein to the extracellular medium, the MAYV E2 glycoprotein was not exported, remaining in the cell fraction of the culture. Structural analyses of alphavirus E2 homologous protein indicated that the glycoprotein has a transmembrane domain, which justifies its permanence in the host cell phospholipid bilayer.

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-04Bitstream added on 2014-06-13T20:16:32Z : No. of bitstreams: 1 lavezzo_lc_me_sjrp.pdf: 1608901 bytes, checksum: fe41b66f249b2bbe571fc89130581c47 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O Brasil é um país de dimensões continentais, com 8.512.000 km 2 , tem a maioria de seu território situado em área tropical, que associado a grande diversidade de flora e fauna, o que torna apropriada à ocorrência de zoonoses transmitidas por artropodes. Estudos sobre arbovírus na região, pelo Instituto Evandro Chagas, em 44 anos (1954-1998) identificaram um grande número de vírus, e pelo menos 187 diferentes arboviroses foram isoladas. A prevalência mundial de arboviroses cresceu dramaticamente nas últimas décadas, e nas áreas onde estas doenças são endêmicas, a transmissão por transfusão dificilmente é investigada. A preocupação com a transmissão de arboviroses, principalmente do Vírus Dengue (DENV), por transfusão sanguínea aumentou após a documentação da transmissão de West Nile Vírus (WNV) por transfusão nos EUA. DENV e WNV são semelhantes em alguns aspectos, o que suporta a hipótese de que dengue possa ser transmitida por transfusão sanguínea: são eficientemente transmitidos ao homem através da picada de mosquitos infectados; grande proporção das infecções é assintomática; níveis de viremia durante a fase de incubação pode exceder 106 virions por mL, e a transmissão de ambos foi documentada após transplante de órgão e em acidentes com profissionais da saúde. Desta forma, o objetivo deste trabalho foi investigar a presença de arboviroses em 205 amostras clínicas de doadores de sangue da Região Amazônica e em amostras obtidas durante uma epidemia de Dengue, em 2006, na cidade de Tupã/SP. O RNA viral foi extraído, Multiplex-Nested-PCR foi realizada com o uso de primers gênero e espécie-específico para Flavivirus, Alphavirus, e, para a detecção e identificação do vírus Oropouche, realizou-se RT- Nested-PCR usando primers que se ligam no segmento S do genoma viral. Apesar da complexidade e número, a transmissão... / O Brasil é um país de dimensões continentais, com 8.512.000 km 2 , tem a maioria de seu território situado em área tropical, que associado a grande diversidade de flora e fauna, o que torna apropriada à ocorrência de zoonoses transmitidas por artropodes. Estudos sobre arbovírus na região, pelo Instituto Evandro Chagas, em 44 anos (1954-1998) identificaram um grande número de vírus, e pelo menos 187 diferentes arboviroses foram isoladas. A prevalência mundial de arboviroses cresceu dramaticamente nas últimas décadas, e nas áreas onde estas doenças são endêmicas, a transmissão por transfusão dificilmente é investigada. A preocupação com a transmissão de arboviroses, principalmente do Vírus Dengue (DENV), por transfusão sanguínea aumentou após a documentação da transmissão de West Nile Vírus (WNV) por transfusão nos EUA. DENV e WNV são semelhantes em alguns aspectos, o que suporta a hipótese de que dengue possa ser transmitida por transfusão sanguínea: são eficientemente transmitidos ao homem através da picada de mosquitos infectados; grande proporção das infecções é assintomática; níveis de viremia durante a fase de incubação pode exceder 106 virions por mL, e a transmissão de ambos foi documentada após transplante de órgão e em acidentes com profissionais da saúde. Desta forma, o objetivo deste trabalho foi investigar a presença de arboviroses em 205 amostras clínicas de doadores de sangue da Região Amazônica e em amostras obtidas durante uma epidemia de Dengue, em 2006, na cidade de Tupã/SP. O RNA viral foi extraído, Multiplex-Nested-PCR foi realizada com o uso de primers gênero e espécie-específico para Flavivirus, Alphavirus, e, para a detecção e identificação do vírus Oropouche, realizou-se RT- Nested-PCR usando primers que se ligam no segmento S do genoma viral. Apesar da complexidade e número... (Complete abstract click electronic access below)

Orientador: Maurício Lacerda Nogueira / Banca: Paula Rahal / Banca: Carlos Eugênio Cavasani / Resumo: A região Amazônica apresenta extensas áreas de floresta e ecossistemas naturais, provendo condições favoráveis para a existência de muitos arbovírus. Mais de 200 arbovírus foram isolados na região Amazônica, e aproximadamente 40 estão associados a doenças humanas. Quatro dos 40 são considerados ser de importância para a saúde pública no Brasil: vírus da Dengue (sorotipos 1 a 4), Oropouche, Mayaro e Febre Amarela. Juntamente com os arbovírus, a malária é uma doença endêmica, e aproximadamente 98% dos casos estão restritos á região da Amazônia Legal. Este trabalho teve como objetivo analisar 111 amostras clínicas de soro de pacientes que residiam em Novo Repartimento (Pará), Porto Velho (Rondônia), Plácido de Castro (Acre) e Oiapoque (Amapá) previamente confirmados para malária. Para tal finalidade, foi utilizada a técnica de Multiplex-Nested-PCR e RT-Nested-PCR para a detecção e identificação dos principais arbovírus brasileiros, pertencentes aos gêneros Flavivirus, Alphavirus e Orthobunyavirus. Duas amostras de pacientes de Novo Repartimento foram positivas para Dengue sorotipo 2, e também, ambos tinham infecção por Plasmodium vivax . Apesar de dados escassos, infecções duplas por dengue e malária deveriam ser comuns em áreas onde ambas as doenças são co-endêmicas em muitas partes do mundo. Na região Amazônica Brasileira, esta situação provavelmente ocorre mais frequentemente que a detectada. Apesar de ambas as doenças causarem sintomas similares, e infecções simultâneas com dois agentes etiológicos podem resultar em uma doença com sintomas sobrepostos, e possivelmente, tanto o espectro clínico da doença e/ou o tratamento pode ser afetado. Neste contexto uma coinfecção por malaria e dengue não poderia ser descartada, e o diagnóstico deveria ser realizado concomitantemente para dengue e malária... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Amazon region has extensive forested areas and natural ecosystems, providing suitable conditions to the existence of several aboviruses. Over 200 arboviruses were isolated in the Amazon region, and about 40 are associated to human diseases. Four out of 40 are considered to be of public health importance in Brazil: dengue virus (serotypes 1-4), Oropouche, Mayaro and Yellow Fever. Along with aboviruses, malaria is an endemic disease caused by protozoans Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae in Brazil, and about 98% of the cases are restricted to the Legal Amazon region. The aim of this study was to test 99 serum clinical samples from patients living in Novo Repartimento (Pará), Porto Velho (Rondônia), Plácido de Castro (Acre) and 12 plasma samples from patients living in Oiapoque (Amapá), previously confirmed for malaria. For this purpose, we used Multiplex-Nested-PCR and RT-Nested-PCR assays for detection and identification of the major Brazilian arboviruses, belonging to the genus Flavivirus, Alphavirus and Orthobunyavirus. We detected Dengue virus serotype 2 in two serum samples from patients living in Novo Repartimento, and both had active Plasmodium vivax infections. Despite scant data, dual dengue and malaria dual infections should be common in areas where both diseases are co-endemic in many places of the world. In the Amazon region, this situation is likely to occur more frequently than detected. In spite of having similar clinical findings, and simultaneous infections with two etiological agents can result in a disease with overlapped symptoms, possibly either clinical spectrum of the disease or treatment can be affected. In this context, concurrent dengue and malaria could not be ruled out, and diagnosis should be made concomitantly in febrile patients living or returning from areas where both diseases are co-endemic... (Complete abstract click electronic access below) / Mestre

O vírus Oropouche pertence à família Bunyaviridae, gênero Orthobunyavirus, sorogrupo Simbu, e é segunda causa mais freqüente de arbovirose febril no Brasil. Estima-se que mais de meio milhão de casos de febre do Oropouche tenham ocorrido no Brasil nos últimos 30 anos, havendo também ocorrências no Panamá, Peru, Suriname e Trinidad. Epidemias de febre do Oropouche têm sido registradas quase que exclusivamente na Amazônia. Porém, com o aquecimento global do planeta, desmatamentos e conseqüente redistribuição de insetos vetores e animais reservatórios, há risco de disseminação de vírus Oropouche para outras regiões do Brasil e da América do Sul. A patogenia da infecção por Oropouche não é bem entendida. Modelo em roedores, usando inoculação intracerebral, tem sido descrito, mas não com uso da via subcutânea, que mais se assemelha à rota natural da infecção. O objetivo deste trabalho foi estabelecer e caracterizar um modelo experimental de infecção com Oropouche, usando inoculação subcutânea em hamster, a fim de contribuir para o entendimento da patogenia do mesmo. Oropouche da linhagem BeAn19991, passado em cérebro de camundongo recém-nascido, foi inoculado (106,25 TCID50/100?L) por via subcutânea na coxa de hamsters sírios com 2-3 semanas de idade. Em torno de 3 dias alguns animais desenvolveram doença com sinais clínicos brandos caracterizados por: eriçamento dos pêlos e agressividade, outros com características mais graves: perda de peso, tremores sugestivos de calafrios, letargia e paralisia. Os animais foram sacrificados 1, 3, 5, 8 e 11 dias pós-inoculação ou quando eram encontrados em estado agônico. Baço, cérebro, coração, fígado, músculo e sangue foram colhidos para titulação viral, histologia e imunohistoquímica. Infiltrado inflamatório estava presente no cérebro, principalmente em torno dos vasos, meninges e discretamente no coração. O vírus Oropouche foi detectado no tecido cerebral (107,23 TCID50/g), no fígado (106,67TCID50/g) e no sangue (106 TCID50/g). Antígeno de Oropouche foi encontrado, difusamente no fígado, associado com hepatócitos e em neurônios de diversos locais do cérebro. Este modelo reproduz uma infecção sistêmica por Oropouche e pode tornar-se útil no estudo da patogênese, bem como para testar drogas antivirais e possíveis candidatos à vacina. / Oropouche virus belongs to the family Bunyaviridae, genus Orthobunyavirus, serogroup Simbu and is the second most frequent arboviral febrile illness in Brazil. There are estimates of more than half million cases of Oropouche fever in Brazil in the past 30 years, with cases registered in Panama, Peru, Suriname and Trinidad. Oropouche fever has been registered almost exclusively in the Amazon, but with the global warming, deforestation and the consequent redistribution of vectors and reservoir animals, the risk of Oropouche virus dissemination to others areas of Brazil and South America increases. However, the pathogenesis of Oropouche infection is not well understood. Rodent models using intracerebral inoculation have been described, but no attempts to use a route that more closely resembles the natural route of infection have been published. This study was conducted to establish and characterize an experimental model of infection with Oropouche, using subcutaneous inoculation of hamsters, in order to contribute to the understanding of Oropouche pathogenesis. We have established an experimental model through subcutaneous inoculation of hamsters in order to study pathogenesis. Suckling mouse brain passaged Oropouche strain BeAn19991 was inoculated (106,25 TCID50/100?L) subcutaneously in the thigh of syrian hamsters 2-3 weeks old. Around day 3 animals developed disease characterized by lethargy, paralysis, chill-like shaking and ruffled fur. Animals were sacrificed on days 1, 3, 5, 8 and 11 post-inoculation or whenever found to be agonic. Brain, heart, liver, spleen, muscle and blood were harvested for virus titration, histology and Oropouche immunohistochemistry. Inflammatory infiltrate was present in the brain, mostly as perivascular cuffs, meninges and some in the heart. Oropouche titers were 107,23 TCID50/g of tissue in brain 106,67 TCID50/g in liver, and 106 TCID50/g in blood. Oropouche antigen was detected diffusely distributed in the liver in association with hepatocytes, and in neurons in several regions of the brain. This model reproduces systemic Oropouche infection and may become useful in pathogenesis studies, as well as to test antiviral drugs and possible vaccine candidates.

The state of Florida is the third most populous state in the United States of America, with six (6) of its metropolitan areas dubbed as the fastest growing in the entire country. A mosquito bite may mean the transmission of a virus or disease which might be fatal. Hence, there is a need for the state to control mosquitoes through the various Departments of Mosquito Control in each of its sixty-seven (67) counties. Six locally acquired mosquito-borne viruses which affect humans and animals in the state of Florida were considered. This thesis used statistical methods to examine data for rainfall, population estimate, as well as, the data on six (6) arboviruses, over the course of thirteen (13) years, namely 2002 to 2014. The first hypothesis that was tested, was that greater precipitation increased the likelihood of a greater number of arbovirus cases. It was important to also examine the relationship that this growing human population had with mosquito-borne diseases, and so the second hypothesis that was tested, was that, an increase in the human population would increase the likelihood of a greater number of arbovirus cases. Subsequently, an analysis was done for eleven (11) of Florida*s 67 counties with the greatest cumulative occurrence of human and animal arbovirus cases combined. Of the eleven counties, seven exhibited a weak associated between the size of the human population and the spread of animal and human arbovirus cases; three exhibited a somewhat moderate association; and one – Osceola County – had a strong negative association. This indicated that, as the size of the human population increased in Osceola County, the combined number of human and animal arbovirus cases decreased, which refuted the second hypothesis of this thesis. A linear regression model for the data for Osceola County was derived and that model was used to simulate what will occur in future years with the use of population projection data. In each simulated year, the number of combined human and arbovirus cases was negative. This prediction meant that, as the projected population increased from year to year, then the number of cases should be zero in each year. The reliability of these predictions are questionable, since Osceola County does not exist in a vacuum and it cannot be isolated from the surrounding counties which may be experiencing an outbreak of arboviruses.

Mosquito-borne viruses, including dengue virus (DENV), Usutu virus (USUV), West Nile virus (WNV), and Japanese encephalitis virus (JEV), are rapidly emerging, global pathogens. Though the number of people impacted by each virus varies, there have been thousands to millions of people infected. The focus of this thesis work centers around USUV and WNV; both have RNA genomes and belong to the Flaviviridae virus family. Both WNV and USUV were initially isolated in Africa and have since spread to Europe; interestingly, WNV has also spread globally and is considered endemic in the Americas. Similar to other flaviviruses, USUV and WNV are maintained in a mosquito vector-avian host transmission cycle, with spillover infection into humans. Human infections of WNV and USUV are usually asymptomatic, but in severe cases can cause neuroinvasive disease. WNV and USUV belong to the JEV serocomplex group, which indicates that antibodies produced against these viruses share a common antigen; the common antigen is hypothesized to be the envelope (E) protein on the outside of the virion. Neutralizing antibodies against both WNV and USUV have been found in birds and humans across Europe. In vitro cross-neutralization of WNV and USUV has been modeled experimentally and been observed in clinical settings. The neutralizing antibody response generated against WNV has been studied extensively in mouse models; however, there are few studies which examine the neutralizing antibody response generated against USUV. Whether prior WNV exposure protects against USUV disease is also unknown. The main goal of this thesis was to characterize how a primary flavivirus exposure would influence a secondary flavivirus exposure; specifically, we wanted to observe if WNV exposure would protect against USUV disease in vivo and generate a cross-neutralizing antibody response in vitro. For the WNV exposure, we used an attenuated vaccine strain of WNV that contains the WNV E gene (D2/WN-V3) developed by our collaborators. We hypothesized that treatment with D2/WN-V3 would protect against USUV infection. Two in vivo models were used: CD-1 mice and interferon alpha-beta receptor 1 deficient (Ifnar1-/-) mice. We discovered that sera from mice vaccinated with D2/WN-V3 neutralized both WNV and USUV in vitro. In the Ifnar1-/- model, we observed that vaccinated mice had higher survival rates and lower USUV viremia levels after USUV challenge. This work helps characterize the consequences of flavivirus antibody cross-neutralization in vitro and cross-protection in vivo. As the flavivirus field moves toward the goal of creating a pan-flavivirus vaccine, both cross-reactive antibodies and cross-protection need to be considered. / Master of Science / West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses that were originally isolated in Africa during the 20th century. Both viruses are maintained through a transmission cycle between mosquito vectors and avian hosts. Mosquitos transfer the infectious agent (WNV or USUV) through feeding on a bird (usually a passerine species); once in the bird, the virus can replicate to high levels. Human infections of WNV and USUV from mosquitos can also occur, with symptoms ranging from mild febrile illness to severe encephalitis or meningitis. Over the past few decades, WNV and USUV have spread to Europe, most likely through infected migratory birds. Interestingly, mosquito surveillance studies in mainland Europe have found mosquitos that tested positive for both USUV and WNV. In Europe, antibodies for both viruses have been found in humans and birds, indicating a previous exposure to WNV, USUV, or both. The neutralizing antibody response is a critical immune defense against viral infections. Neutralizing antibodies bind strongly to the outside of the virion (virus particle), preventing the virion from interacting with and infecting the host cell. For WNV and USUV, one of the targets that neutralizing antibodies bind to is the outer envelope (E) protein of the virion. In clinical settings and experimental studies, cross-neutralization of WNV and USUV has been documented. During cross-neutralization, a serum sample containing neutralizing antibodies against WNV can also neutralize USUV, and vice versa. Although the neutralizing response against WNV has been characterized in humans and lab animal models such as mice, there is little research regarding the neutralizing response against USUV. Importantly, whether prior WNV exposure provides protection against USUV infection is currently unknown. The main goal of this thesis was to characterize the disease outcome and neutralizing response against USUV after a WNV exposure. For the WNV exposure, we used a vaccine strain of WNV that contains the E gene (D2/WN-V3) developed by our collaborators. We predicted that vaccinated mice would avoid USUV clinical signs of disease and generate neutralizing responses to WNV and USUV. To do this work, we used two laboratory mouse models: mice with an intact immune response system (CD-1) and mice with a stunted immune response (Ifnar1-/-). We discovered that serum from vaccinated mice did cross-neutralize WNV and USUV. In the Ifnar1-/- model, vaccinated mice had higher survival rates and lower levels of virus in blood after USUV infection compared to unvaccinated mice. Ultimately, this work highlights the importance of characterizing the immune response against similar viruses and will inform the development of human vaccines for both viruses.

Les alphavirus sont un groupe de virus enveloppés à ARN simple brin positif retrouvés sur la totalité du globe et responsables de nombreuses maladies humaines et animales. Durant la dernière décennie, une réémergence du virus du chikungunya (CHIKV) a été observée causant de nombreuses épidémies sur tous les continents. Malgré les nombreuses études, les mécanismes moléculaires de réplication du CHIKV et les interactions hôte-virus restent peu caractérisées. L’objectif de mon travail était de mieux comprendre et caractériser l’entrée du virus du chikungunya et les facteurs de l’hôte impliqués dans la réplication chez les mammifères. Plusieurs approches distinctes ont été utilisées dans ce projet. Dans un premier temps, nous avons mis en avant une diminution de l’infection du CHIKV après un traitement avec du fer sous forme de citrate d’ammonium ferrique et nous avons étudié le rôle potentiel dans l’entrée virale de NRAMP2 et TFRC, deux protéines impliquées dans le transport cellulaire du fer et connus comme récepteurs d’entrée de plusieurs virus. D’autre part, nous nous sommes intéressés à deux autres protéines, CD46 et TM9SF2, identifiés à travers un criblage par ARNi réalisé en collaboration, dans le but de déterminer si elles sont utilisées comme facteurs d’entrée par le virus du chikungunya. Dans un dernier axe, nous avons mis en place et réaliser un criblage perte de fonction sur le génome entier en utilisant la technologie CRISPR/Cas9 afin d’identifier des facteurs de l’hôte importants pour l’entrée du CHIKV, sa réplication ou la mort viro-induite. Bien qu’il soit apparu que l’approche utilisée pour le criblage devrait être optimisée, nous avons pu identifier des candidats potentiellement nécessaires pour l’infection par le CHIKV. Ces candidats sont testés individuellement afin de confirmer leur implication dans la biologie du virus / Alphaviruses are a group of enveloped, positive-sense RNA viruses which are distributed almost worldwide and are responsible for a considerable number of human and animal diseases. Among these viruses, the Chikungunya virus (CHIKV) has recently re-emerged and caused several outbreaks on all continents in the past decade. Despite many studies, molecular mechanisms of chikungunya virus replication and virus-host interactions remain poorly understood. The aim of my project was to better understand and characterize the CHIKV entry and the host factors involved during replication steps in mammals. Several different approaches have been used in this work. As a first step, we have demonstrated a decrease of CHIKV infection after iron treatment in form of ferric ammonium citrate and we have studied the potential role in viral entry of NRAMP2 and TFRC, two proteins involved in iron transport and known receptors for other viruses. On the other hand, we have also focused on two proteins, CD46 and TM9SF2, identified through an RNAi screen in collaboration, in order to determine if they are required as entry factors for chikungunya virus. In a last axis, we have set up and carried out a genome-wide loss of function screen with the CRISPR/Cas9 technology in order to identify host factors important for chikungunya virus entry, replication or virus-induced cell death. Although it appears that screen conditions should be optimized, we have identified potential candidates required for CHIKV infection and we are currently testing them

L'épidémie de West-Nile en Amérique du Nord en 2002, qui a touché plus de quarante états aux Etats-Unis, a conduit les Agences de santé à s'interroger sur le risque d'émergence, à l'extérieur de la zone intertropicale, de zoonoses vectorielles. Cette épidémie associée au changement climatique, a bien mis en évidence le rôle central de l'avifaune migratrice dans la diffusion du virus. La biologie des oiseaux, tout particulièrement le phénomène migratoire, permet un transport des virus sur de longues distances et entre espèces très diversifiées. Le Moyen-Orient, qui est situé au carrefour de différents continents, est extrêmement propice à la propagation des virus émergents dans les pays du Nord. La circulation du virus West Nile a été rapportée dans différents pays de la région, tels que l'Egypte, Israël, Liban, Irak, Emirats Arabes Unis et Iran. Saidi et al. (1970) ont montré la présence d'anticorps anti-virus du Nil occidental au sein de la population de la côte caspienne (Nord de l'Iran), des provinces du Khorassan (Nord-Est) et du Khuzestan (Sud-Ouest). Notre étude, conduite dans le cadre d'un programme associant TIMC-IMAG UMR 5525 UJF CNRS VetAgroSup, le Réseau International des Instituts Pasteurs et le Centre National d'Etudes Spatiales, vise: (i) à caractériser la circulation du virus de West-Nile au sein des populations équines d'Iran ; et (ii) et à modéliser l'impact sanitaire de l'environnement et du climat sur la transmission. Les résultats acquis permettent d'apprécier le risque associé à la dissémination spatio-temporelle du virus par les oiseaux migrateurs. Une attention toute particulière est portée à l'étude des déterminants environnementaux et climatiques susceptibles d'accroitre le potentiel de transmission du virus. / The outbreak of West Nile in North America in 2002, which affected more than forty states in the United States, has led Public Health Agencies to adress the risk of emergence of vectorial zoonosis, outside of the area tropical. This epidemic events associated with climate change, has highlighted the central role of migratory birds in spreading the virus. The biology of birds, especially the migratory phenomenon, ensures a transport of viruses over long distances and across very diverse species. The Middle East, located at the crossroad between different continents, is extremely prone to the spread of zoonotic diseases, like West-Nile, in the Northern countries. The circulation of WN virus has reported from different countries in the region; such as Egypt, Israel, Lebanon, Iraq, United Arab Emirates and Iran. Saidi et al. (1970) have established the presence of antibodies to West Nile among the population of the Caspian coast (northern Iran), the provinces of Khorasan (Northeast) and Khuzestan (Southwest). Our study, in the framework of a collaborative programme associating TIMC-IMAG UMR CNRS UJF VetAgroSup, International Network of Institut Pasteur and Centre National d'Etudes Spatiales, aim to: (i) characterizing the circulation of the virus from West Nile in the equine population of Iran, and (ii) and modelling the health impact of the environment and climate on the transmission. The results obtained allow us to assess the risk associated with spatial and temporal spread of the virus by migratory birds. Particular attention is given to the study of environmental and climatic determinants that may increase the potential for transmission.