Introdução - No litoral sul do Estado de São Paulo, no período de 1975 a 1978, ocorreu uma epidemia, a Encefalite pelo arbovírus Rocio. A região foi objeto de estudo de diversos investigadores. Altas taxas de morbidade e mortalidade devido ao processo epidêmico foram observadas e causaram impacto socioeconômico. A maioria dos indivíduos infectados, no início da epidemia, era do sexo masculino e estava em idade produtiva, sendo trabalhadores rurais da região. Diante das limitações hospitalares e inespecificidade do tratamento na época, houve desde uma lenta convalescença, seqüelas até a ocorrência de óbitos, afetando a economia da região, que repercutiu principalmente na queda no turismo. Mediante este fato, justifica-se a importância deste estudo histórico-documental da encefalite por arbovírus Rocio. Objetivo - Objetivou-se relatar acontecimentos sociais e naturais, medidas clínicas, impactos midiáticos e avanços científicos relacionados à doença, verificando, a trajetória do Rocio. Método - Foi feita uma revisão de literatura de trabalhos publicados desde o início da epidemia até os dias atuais. As fontes consultadas foram teses, dissertações, livros, periódicos, bancos de dados de jornais e revistas, bases de dados cooperativas, relatórios de instituições públicas, contato com especialistas e comunicações em eventos. Resultados - Foi possível analisar como a mídia impressa relatou os acontecimentos sociais relacionados à epidemia e como foi a reação popular às notícias veiculadas, além de discutir a possibilidade de o homem voltar a ser acometido pelo Rocio, diante das atuais mudanças climáticas, acelerada urbanização e pressão sobre a cobertura vegetal no litoral sul do Estado, o que altera a ecologia das populações dessa região. Conclusões - Houve o desencontro entre informações veiculadas pela mídia e dados científicos fornecidos por pesquisadores e autoridades sanitárias, o que dificultava a aceitação da epidemia pela população e viabilizava a distorção de informações e criação de barreiras aos métodos de combate ao possível vetor. Ainda não se sabe como o vírus Rocio tornou-se emergente no litoral sul do Estado em 1975 e o porquê do seu silenciamento, entretanto é conhecido que esse arbovírus ainda mantém atividade, possibilitando o retorno da epidemia no país. / Introduction - On the south coast of São Paulo State, Brazil, there was an encephalitis epidemic due to the arbovirus Rocio from 1975 to 1978. Thus, the region has been the object of study by researchers. High rates of morbidity and mortality caused an enormous social impact. The most infected individuals at the beginning of the epidemic was male and was in the productive age. Facing the limitations of the hospital and nonspecific treatment at the time, there was since a slow convalescence, sequels and even the occurrence of deaths, affecting the regional economy, which mainly reflected the drop in local tourism. In view of this fact, it is the importance of this historical and documentary study of encephalitis caused by arbovirus Rocio during the epidemic period. Aim - The aim of this study was to report social and natural events, clinical measurements, media impacts and scientific advances related to the disease, checking the trajectory of virus Rocio. Method It was made a literature review of studies published since the beginning of the epidemic until the present day. The sources which were consulted: theses, dissertations, books, periodicals, databases of newspapers and magazines, electronic databases, reports from public institutions, contact with specialists in communications and events. Results - In this study, it was possible to analize how the written press communicated the social reactions related to the epidemic and how the population reacted the news communicated, in addition was possible to discuss the possibility of the human being be affected by Rocio again due to the current climate change, accelerated urbanization and pressure on the vegetation on the coast south of the State, thus changing the ecology of the population of that region. Conclusions - There was a contradiction between the information carried by the media and scientific data provided by researchers and health authorities, which hindered the acceptance of the epidemic by the population and facilitated the distortion of information. This fact created barriers to methods of combating the possible vector. It is not known how the virus Rocio has become emerging on the south coast of the State in 1975 and why it became silent, however it is know that the arbovirus still has activity, what allows the return of the epidemic in the country.

Background: Mosquito-borne diseases are a growing concern for temperate regions including the northeastern US. There the two primary mosquito vectors, Cx. pipiens and Ae. albopictus are widespread, endemic circulation of West Nile virus causes sporadic outbreaks, and imported arboviruses such as dengue, chikungunya, and Zika are on the rise. With temperate mosquito-borne disease outbreaks likely to increase in frequency, it is critical to reduce mosquito populations in the northeastern US. Community-based source reduction is heralded as the most sustainable component of integrated mosquito management. Yet mosquitoes develop rapidly, requiring weekly maintenance of mosquito habitat. This is onerous and community commitment flags. The development of predictive models to inform focused vector-control efforts is therefore of great utility. Objectives and Methods: The overarching objective of this research is to make robust predictive modeling frameworks based on empirically derived relationships of the ecology and epidemiology of mosquito-borne disease systems in the northeastern US. We aim to quantify the relationships between local environmental and meteorological conditions and mosquito vectors. In Chapters 2 and 4 we use lengthy surveillance records to develop models and use model ensembles to generate predictions based on out-of-sample data. For chapter 3 we use more spatially refined data to investigate the influence of intra-urban heterogeneities and how climatic conditions influence mosquito populations across these defined differences. Results: In Chapter 2, we model and forecast WNV infection rates among mosquito vectors using meteorological and hydrological conditions. We show that real-time climate information can predict WNV Culex infection rates prior to when human risk is greatest. In Chapter 3, we link infrastructure degradation and vegetation patterns with Ae. albopictus infestation levels as well as the interactive effect of precipitation across these environmental conditions. In Chapter 4, we identify key land use characteristics and meteorological conditions associated with annual Ae. albopictus abundance. Further we use imported chikungunya cases to delineate areas of high arboviral importation and, in combination with areas of high Ae. albopictus abundance, areas at heightened risk for arboviral transmission. Conclusions: While temperate outbreaks are often self-limiting they may be increasing in frequency and severity. Due to the multitude of invasive vectors and arboviruses, vector control techniques that work for multiple mosquito species are likely more effective and sustainable. Here we build build empirical models that accurately predict mosquito dynamics before populations peak which is critical for vector control. We recommend integrating predictive modeling into mosquito management guidelines as this could focus valuable resources to when and where mosquito-borne transmission risk is greatest. Further we find social and ecological determinants of mosquito dynamics, supporting further study that combine socio-ecological processes into model frameworks.

Eastern equine encephalitis virus (EEEV) is a highly pathogenic arbovirus that causes severe disease, with a mortality rate of approximately 30-35% in humans and 80-90% in horses. Studies dating back to the 1930's have identified many of the epidemiological and ecological aspects of the virus. However, there are several aspects of EEEV's transmission cycle that remain unclear. In the northeastern states, transmission is seasonal, peaking in the late summer months, while in Florida there is year-round transmission of EEEV. Recent phylogenetic studies have also suggested that Florida may serve as a reservoir for EEEV; the virus may periodically be introduced from Florida to the northeastern US where it locally amplifies, overwinters, and can remain stable for several years. How EEEV is able to migrate from Florida and how it persists during the winter in North America is not yet known, however several theories exist and are examined further by this research. The first part of this study investigates the hypothesis that snakes may serve as overwintering reservoir hosts for EEEV. Rates of exposure and infection of wild snakes were examined by testing serum samples from wild snakes at a focus of EEEV in Alabama. Two species of vipers, the cottonmouth and the copperhead, were found to be positive for EEEV RNA. The second part of the study attempts to identify the hosts and vectors of enzootic winter transmission of EEEV in Florida, with a focus on avian host preference. EEEV was detected in two mosquito species, Culiseta melanura and Anopheles quadrimaculatus, and were both from the month of February. In addition, the results also suggest that EEEV vectors preferentially feed upon wading birds during the winter months.

Within the state of Florida, there are three arboviruses of public health importance that can cause neuroinvasive disease in humans: West Nile Virus, Saint Louis Encephalitis Virus, and Eastern Equine Encephalitis Virus. Mosquitoes (Diptera: Culicidae) within the genus Culex are known and suspected vectors of these diseases. The vectors of these diseases can be present in urban wetland habitats that allow for exposure to residential communities. Vector ecology must be investigated in order to understand the dynamics of disease transmission. In Hillsborough County, Florida the spatial and temporal distribution of these vectors are not well established. An ecological study was conducted in the University of South Florida's Ecopreserve using trapping methodologies to sample the adult and gravid females as well as collect the egg population. Collections were made at three spatial points for the duration of July through December 2013 and compared to meteorological variables. Culex erraticus, a proposed bridge vector of Eastern Equine Encephalitis, was the most abundant adult species and gravid female captured. Culex nigripalpus, primary Floridian vector of Saint Louis Encephalitis and bridge vector of West Nile Virus, was the second most abundant adult species caught as well as the majority of eggs collected. Based on the results collected, the presence of Culex erraticus and Culex nigripalpus was confirmed. The majority of Culex erraticus adults were collected in September and October and Culex nigripalpus adults were the highest in July and August. The results of the gravid and egg collection generated crucial insight regarding methodology for studying vector ecology within this urban wetland habitat. However, modeling at spatial points based on meteorological variables yielded inconsistent results that illicit further investigation regarding these arboviral vectors of disease.

ABSTRACT Eastern Equine Encephalitis virus (EEEV) is an alphavirus with high pathogenicity in both humans and horses. Florida continues to have the highest occurrence of human cases in the USA, with four fatalities recorded in 2010. Unlike other states, Florida supports year-round EEEV transmission. This research uses Geographic Information Science (GIS) to examine spatial patterns of documented sentinel seroconversions and horse cases in order to understand the relationships between habitat and transmission intensity of EEEV in Florida. Sentinel sites were categorized as enzootic, periodically enzootic, and negative based on the amount of chicken seroconversions to EEEV. Sentinel sites were analyzed based on land classification data d using the Kruskal-Wallis test to determine which habitats were associated with disease transmission. Cluster analyses were performed for the horse cases using density-based spatial clustering of applications with noise (DBSCAN). Ecological associations of EEEV were examined using compositional analysis and Euclidean distance analysis to determine if the proportion or proximity of certain habitats played a role in transmission. The research in these studies provides evidence of ecological associations for EEEV transmission in Florida that hasn't been previously analyzed. Furthermore, these studies provide the groundwork for better understanding of why there is a disproportionate number of horse and human cases of EEEV in Florida than in any other state.

Thesis (M.Sc. (Microbiology))--Mahidol University, 1984.

Wolbachia are widely spread, maternally transmitted insect endosymbiotic intracellular bacteria. They have been implicated in the control of several insect transmitted diseases, including dengue, yellow fever, Zika and malaria. Effective pathogen suppression in the insect host is shown to be proportional to the intracellular levels of bacteria. Therefore, understanding the molecular mechanisms underlying Wolbachia accumulation within organisms is extremely important for future epidemic control and research. Using Drosophila as a model insect, our lab has previously observed Wolbachia tropism to stem cell niches. Current work has identified polar cells as an additional site of Wolbachia tropism and demonstrated that Wg/Wnt signaling is important for Wolbachia intracellular accumulation in these somatic cells. In this thesis, we first observed that the Wg/Wnt pathway protein Armadillo also controls Wolbachia levels in the germline cells, indicating the possibility of having a conserved molecular mechanism controlling Wolbachia. Using RNAi and small molecule inhibitors of Shaggy, another component of the canonical Wg/Wnt pathway, we demonstrate that the canonical Wg/Wnt signaling is essential for Wolbachia intracellular accumulation. Our investigation provides fundamental insights into the mechanisms of Wolbachia intracellular accumulation. Furthermore, it offers novel strategies to modulate Wolbachia in non-model insect species, including various disease transmitting Anopheles, Culex, and Aedes. These findings potentially will increase the effectiveness of a Wolbachia-based vector transmitted disease suppression. / 2017-02-28

Master of Science / Department of Diagnostic Medicine/Pathobiology / A. Sally Davis / Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic Phlebovirus that is a significant threat to ruminants and humans. RVFV is categorized as an overlap Select Agent by the Department of Health and Human Services and US Department of Agriculture. Therefore, the study of RVFV’s pathogenesis and the development of novel diagnostic tools for the prevention and control of outbreaks and virus spread is crucial. RVF is endemic to sub-Saharan Africa but has spread beyond the continent to the Arabian Peninsula indicating the competence of the virus to emerge in new areas. Thus, the high likelihood of RVF’s spread to other non- endemic countries also spurs the need for development and implementation of rapid diagnostic tests and surveillance programs. In the US, RVFV is a Select Agent, requiring BSL-3 enhanced containment practices for research work. First, we developed a method for the detection of RVFV RNA by reverse transcriptase real-time PCR (RT-qPCR) using non-infectious, formalin- fixed, paraffin-embedded tissues (FFPET). The results from FFPET RT-qPCR were compared to prior results for fresh-frozen tissues (FFT) RT-qPCR, as well as immunohistochemistry and histopathology completed on the same FFPET blocks. We developed a novel technique using a rapid and low cost magnetic bead extraction method for recovery of amplifiable RVFV RNA from FFPET. FFPET RT-qPCR can serve as an alternative tissue-based diagnostic test, which does not require a BSL-3 research facility. Second, we assessed the diagnostic accuracy and precision of a recombinant RVFV nucleoprotein based competitive ELISA (cELISA) assay to detect RVFV antibodies. The cELISA results were compared to the virus neutralization test, the gold standard serological assay for RVFV. This prototype cELISA is easy to implement, sensitive, specific, and safe test for the detection of antibodies to RVFV in diagnostic and surveillance applications. RVF is an important transboundary disease that should be monitored on a regular basis. The diagnostic tests developed and validated in this thesis could be used in endemic or non-endemic countries for the early detection of RVF and assist with the implementation of countermeasures against RVFV.

Introdução: Arbovírus são vírus transmitidos por artrópodos, pertencendo, principalmente, aos gêneros Flavivirus (Flaviviridae), Alphavirus (Togaviridae) e Orthobunyavirus (Bunyavirus). Os Flavivirus, em sua maioria, são associados a zoonoses, causando doenças humanas febrís, febres hemorrágicas e encefalites. Inclusive, causam epidemias que são sério problema de saúde pública. Este estudo mostra uma pesquisa de Flavivirus em culicídeos, de diferentes regiões do país, utilizando uma técnica para identificação viral por RT-PCR com primers Flavivirusespecíficos e uma Multiplex-nested-PCR com primers espécie-específicos. Métodos: Culicídeos foram capturados, quantificados, identificados, agrupados em lotes por espécie e congelados. No laboratório, os animais foram macerados e tiveram o RNA extraído. Estes extratos foram submetidos a RT-PCR gênero-específica e à Multiplex-nested-PCR, para detecção e identificação dos vírus a nível de espécie. Resultado: De 3317 culicídios adultos e 571 larvas coletados em 4 diferentes regiões do Brasil, Sul, Sudeste, Norte e Nordeste, fez-se 246 lotes de mosquitos e desses foi possível obter amplicon sugestivo de Flavivirus em 16 (6,5%). Em 3 lotes contendo larvas de Aedes albopictus obteve-se amplicon sugestivo de vírus do dengue tipo 3. Também, em 13 lotes contendo Haemagogus leucocelaenus, Aedes aegypti e Aedes albopictus foi possível obter amplicons sugestivos de vírus do dengue tipos 1 e 2. Dos amplicons obtidos, 4 tiveram nucleotídios seqüenciados o que permitiu confirmar a presença dos vírus do dengue tipo 3 e Cacipacoré. Conclusão: O trabalho permitiu concluir que: a metodologia de RT-PCR para Flavivirus seguida de Multiplex-nested-PCR espécie-específica foi adequada para detecção e identificação destes vírus em culicídios; amplificaram-se genomas de Flavivirus em 6,5% dos lotes de culicídios estudados; vírus do dengue tipo 1 e tipo 2 foram encontrados infectando Aedes aegypti de Santos em 1999, Manaus em 2005-2006 e Foz do Iguaçu; vírus do dengue tipos 2 e 3 foram encontrados em Aedes albopictus de Santos em 1999 e Manaus em 2005-2006, sugerindo que este mosquito participe na transmissão de dengue; vírus do dengue tipo 3 foi encontrado em larvas de Aedes albopictus mostrando transmissão vertical do vírus; vírus do dengue tipo 1 foi encontrado infectando Haemagogus sp. sugerindo existência de ciclo silvático deste vírus; Aedes aegypti do Amazonas estavam infectados com o vírus Cacipacoré. / Introduction: Arbovirus are rodent-borne viruses mostly from Flavivirus (Flaviviridae), Alphavirus (Togaviridae) e Orthobunyavirus (Bunyavirus) genus. Flavivirus, are commonly zoonotic and can cause febrile illness, haemorrhagic fever and encephalitis. Flavivirus outbreaks occur in Brazil and are a major public health problem. We show here a research looking for Flavivirus infections in Culicidae by a RT-PCR using Flavivirus-especific primers and a Multiplex-nested-PCR using specie-specific primers for virus identification. Methods: Culicidae were captured, quantified, identified, pooled based on the specie and frozzen. In the laboratory, the animals were crushed and had the RNA extracted. These extracts were tested by a Flavivirus genus-specific RT-PCR followed by a specie-specific Multiplex-nested-PCR. Results: From 3317 captured adult Culicidae and 571 collected larvae in 4 different regions of Brazil, 246 pools were obtained and from these, Flavivirus indicative amplicons were obtained in 16 (6.5%). Amplicons of dengue type 3 were obtained from 3 pools of Aedes albopictus larvae. It was also possible to obtain indicative amplicons of dengue types 1 and 2 in 13 pools of Haemagogus leucocelaenus, Aedes aegypti and Aedes albopictus. Besides, 4 amplicons had the nucleotides sequenced, confirming the mosquito infection by dengue type 3 and Cacipacoré viruses. Conclusion: The technique combining a Flavivirus genus-specific RT-PCR followed by a specie-specific Multiplex-nested-PCR was suitable for detection of these viruses in the mosquitoes; Flavivirus infecting Culicidae were detected in 6.5% of the analyzed mosquito pools; dengue virus type 1 and type 2 were found infecting Aedes aegypti from Santos (1999), Manaus (2005-2006) and Foz do Iguaçu cities; dengue type 2 virus was found in Aedes albopictus from Santos city (1999) and Manaus city (2005-2006), suggesting that this mosquito could be participating on dengue transmition; dengue type 3 virus was found in Aedes albopictus larvae showing the vertical transmission of this virus; dengue type 1 virus was found infecting Haemagogus sp. what suggests on the existence of a sylvatic maintenance cycle of this virus; Aedes aegypti from Amazonas state were found infeted by Cacipacoré virus.

Introdução - No litoral sul do Estado de São Paulo, no período de 1975 a 1978, ocorreu uma epidemia, a Encefalite pelo arbovírus Rocio. A região foi objeto de estudo de diversos investigadores. Altas taxas de morbidade e mortalidade devido ao processo epidêmico foram observadas e causaram impacto socioeconômico. A maioria dos indivíduos infectados, no início da epidemia, era do sexo masculino e estava em idade produtiva, sendo trabalhadores rurais da região. Diante das limitações hospitalares e inespecificidade do tratamento na época, houve desde uma lenta convalescença, seqüelas até a ocorrência de óbitos, afetando a economia da região, que repercutiu principalmente na queda no turismo. Mediante este fato, justifica-se a importância deste estudo histórico-documental da encefalite por arbovírus Rocio. Objetivo - Objetivou-se relatar acontecimentos sociais e naturais, medidas clínicas, impactos midiáticos e avanços científicos relacionados à doença, verificando, a trajetória do Rocio. Método - Foi feita uma revisão de literatura de trabalhos publicados desde o início da epidemia até os dias atuais. As fontes consultadas foram teses, dissertações, livros, periódicos, bancos de dados de jornais e revistas, bases de dados cooperativas, relatórios de instituições públicas, contato com especialistas e comunicações em eventos. Resultados - Foi possível analisar como a mídia impressa relatou os acontecimentos sociais relacionados à epidemia e como foi a reação popular às notícias veiculadas, além de discutir a possibilidade de o homem voltar a ser acometido pelo Rocio, diante das atuais mudanças climáticas, acelerada urbanização e pressão sobre a cobertura vegetal no litoral sul do Estado, o que altera a ecologia das populações dessa região. Conclusões - Houve o desencontro entre informações veiculadas pela mídia e dados científicos fornecidos por pesquisadores e autoridades sanitárias, o que dificultava a aceitação da epidemia pela população e viabilizava a distorção de informações e criação de barreiras aos métodos de combate ao possível vetor. Ainda não se sabe como o vírus Rocio tornou-se emergente no litoral sul do Estado em 1975 e o porquê do seu silenciamento, entretanto é conhecido que esse arbovírus ainda mantém atividade, possibilitando o retorno da epidemia no país. / Introduction - On the south coast of São Paulo State, Brazil, there was an encephalitis epidemic due to the arbovirus Rocio from 1975 to 1978. Thus, the region has been the object of study by researchers. High rates of morbidity and mortality caused an enormous social impact. The most infected individuals at the beginning of the epidemic was male and was in the productive age. Facing the limitations of the hospital and nonspecific treatment at the time, there was since a slow convalescence, sequels and even the occurrence of deaths, affecting the regional economy, which mainly reflected the drop in local tourism. In view of this fact, it is the importance of this historical and documentary study of encephalitis caused by arbovirus Rocio during the epidemic period. Aim - The aim of this study was to report social and natural events, clinical measurements, media impacts and scientific advances related to the disease, checking the trajectory of virus Rocio. Method It was made a literature review of studies published since the beginning of the epidemic until the present day. The sources which were consulted: theses, dissertations, books, periodicals, databases of newspapers and magazines, electronic databases, reports from public institutions, contact with specialists in communications and events. Results - In this study, it was possible to analize how the written press communicated the social reactions related to the epidemic and how the population reacted the news communicated, in addition was possible to discuss the possibility of the human being be affected by Rocio again due to the current climate change, accelerated urbanization and pressure on the vegetation on the coast south of the State, thus changing the ecology of the population of that region. Conclusions - There was a contradiction between the information carried by the media and scientific data provided by researchers and health authorities, which hindered the acceptance of the epidemic by the population and facilitated the distortion of information. This fact created barriers to methods of combating the possible vector. It is not known how the virus Rocio has become emerging on the south coast of the State in 1975 and why it became silent, however it is know that the arbovirus still has activity, what allows the return of the epidemic in the country.