A dengue é a mais importante arbovirose no mundo; aproximadamente 50 milhões de infecções ocorrem anualmente acarretando 500.000 casos de dengue hemorrágica e 22.000 mortes. A febre amarela é uma doença hemorrágica viral com elevada mortalidade que é transmitida por mosquitos. Vacinas eficazes contra a febre amarela já estão disponíveis há quase 70 anos e são responsáveis por uma redução significativa de ocorrências da doença no mundo, no entanto, cerca de 200.000 casos de febre amarela ainda ocorrem anualmente, principalmente na África. Dessa forma, o desenvolvimento de fármacos antivirais contra essas viroses é uma prioridade de saúde pública. Os produtos naturais sejam de origem vegetal ou animal, possuem uma extensa diversidade química, sendo uma fonte inesgotável de compostos com promissoras atividades biológicas. No Brasil, é grande a incidência de animais venenosos ou peçonhentos, tais como serpentes, sapos e escorpiões. Os venenos desses animais são fontes de diversas substâncias químicas que ainda não possuem a sua atividade biológica e farmacológica completamente estudada. Neste trabalho avaliamos a potencial ação antiviral de peçonhas de serpentes (Crotalus durissus terrificus, Bothrops jararacussu, Bothrops jararaca, Bothrops pirajai, Bothrops moojeni, Bothrops brasili e Bothrops fonseca) e escorpião (Tityus serrulatus) contra os virus da febre amarela e dengue usando diferentes estratégias metodológicas (pré-tratamento, pós-tratamento, virucida, adsorção e internalização). Primeiramente realizamos um screening com as peçonhas brutas, observando que a peçonha de Crotalus durissus terrificus inibiu a replicação viral apresentando os maiores índices de seletividade (IS). Crotoxina, crotamina, crotapotina, convulxina, giroxina, PLA2-CB e PLA2-IC, isoladas de Crotalus durissus terrificus, foram então testadas nas diferentes estratégias metodológicas contra os vírus dengue e febre amarela. Foi possível verificar que crotoxina, PLA2-CB e PLA2-IC inibiram a replicação viral com altos índices de seletividade (IS). A ação verificada ocorreu na fase inicial do ciclo de replicação viral (pré-tratamento, virucida, adsorção). A ação antiviral verificada neste estudo foi atribuida a ação da PLA2, visto que a crotoxina é um complexo protéico composto pela crotapotina e pela PLA2-CB. Posteriormente avaliamos uma fosfolipase sem atividade catalítica isolada de Bothrops jararacussu, a BthTX-I. Essa fosfolipase apresentou baixa inibição da replicação viral, sugerindo que a atividade catalítica da fosfolipase é importante, mas possivelmente não a única responsável pela ação antiviral. Os resultados obtidos permitem sugerir também que as fosfolipases apresentam ação tanto sobre a partícula viral quanto sobre receptores celulares, o que justifica os altos índices de seletividade observados. / Dengue is the most important arbovirus disease in the world; nearly 50 million infections occur annually resulting in 500,000 cases of DHF and 22,000 deaths. Yellow fever is a viral haemorrhagic fever with high mortality that is transmitted by mosquitoes. Effective vaccines against yellow fever have been available for almost 70 years and are responsible for a significant reduction of the disease worldwide. However, about 200,000 cases of yellow fever still occur annually, mainly in Africa. Thus, the development of antiviral drugs against these viruses is a public health priority. Natural products of plant or animal origin have an extensive chemical diversity, and an inexhaustible source of compounds with promising biological activities. In Brazil, there is a high incidence of poisonous or venomous animals such as snakes, frogs and scorpions occur. The venoms of these animals are a source of several chemicals that does not possess biological and pharmacological activity completely studied. In this study, we assess the potential antiviral action of snake venom (Crotalus durissus terrificus, Bothrops jararacussu, Bothrops jararaca, Bothrops pirajai, Bothrops moojeni, Bothrops brasili and Bothrops fonseca) and Scorpion (Tityus serrulatus) against yellow fever and dengue viruses using different methodological strategies (pre-treatment, post-treatment, virucidal, adsorption and internalization). First, we performed a screening with the crude venoms, founding that the venom of Crotalus durissus terrificus inhibited viral replication showing the highest selectivity index (SI). Crotoxin crotamin, crotapotin, convulxin, gyroxin, PLA2-CB and PLA2-IC isolated from Crotalus durissus terrificus, were then tested in the different methodological strategies against dengue and yellow fever viruses. We found that crotoxin, PLA2-CB and PLA2-IC inhibited viral replication with high SI. The action of these compounds against the virus was at the first steps of the replication cycle (pre-treatment, virucidal, adsorption). The antiviral action observed in this study was attributed to the action of PLA2, since crotoxin is a protein complex composed of crotapotin and PLA2-CB. Afterwards, we evaluated a phospholipase without catalytic activity isolated from Bothrops jararacussu, the BthTX-I. This phospholipase showed low inhibition of viral replication, showing that the catalytic activity of phospholipase is important, but perhaps not the only one responsible for the antiviral action. Our results also suggest that phospholipases have action on the viral particle and on cell receptors, which explains the high levels of selectivity observed.

The proposed public history project, La Fièvre Jaune, will be one component of a larger exhibit sponsored by the Archdiocese of New Orleans, Office of Archives and Records entitled Song of Farewell: Catholic Cemeteries of New Orleans, focusing on New Orleans’s historic Catholic cemeteries, funeral chapels, relics, and burial rights. Using cemetery and death records, La Fièvre Jaune documents many of the Catholic, largely Irish immigrants struck by yellow fever in 1853 and the role of St. Patrick’s cemetery as the burial site for this population. The epidemic took the lives of some 8,000 people. This project will provide insight into the ways that the Catholic Church in New Orleans responded to the 1853 yellow fever epidemic using photographs, official correspondence, as well as cemetery and death records. The entire exhibit will be housed at the Old Ursuline Convent Museum in the French Quarter.

In the yellow fever mosquito, Aedes aegypti (L.), hemolymph-circulating diuretic hormones act upon the renal organs (Malpighian tubules) to regulate primary urine composition and secretion rate; however, the molecular endocrine mechanisms underlying rapid water elimination upon adult eclosion and blood feeding are not fully understood. Bioinformatic analysis of the current Aedes aegypti genome assembly reveals only a single predicted corticotropin releasing factor (CRF)-like diuretic hormone 44 (DH44) gene, but two DH44 receptor genes. The tissue expression profiles of the DH44 receptor(s), and specifically the identity of the DH44 receptor(s) in the Malpighian tubule, are undetermined in any mosquito species. This dissertation shows that Vectorbase gene ID AAEL008292 encodes a DH44 receptor (AaegGPRdih1) transcribed in Malpighian tubules. Sequence analysis and transcript localization indicate that AaegGPRdih1 is the co-ortholog of the Drosophila melanogaster DH44 receptor (CG12370-PA). The presence of conserved amino acid residues between AaegGPRdih1 and vertebrate CRF receptors suggests this mosquito receptor modulates multiple G protein-dependent intracellular signaling pathways. Quantitative PCR analysis of a time course of Malpighian tubule cDNA reveals AaegGPRdih1 abundance increases paralleling periods of observed urination. This suggests that target tissue receptor biology is linked to the known periods of release of diuretic hormones from the nervous system, pointing to a common up-stream regulatory mechanism. Higher relative abundance of AaegGPRdih1 transcript in female Malpighian tubules 24 hours after blood feeding suggests a role for AaegGPRdih1 in the excretion of nitrogen waste. RNA-mediated silencing to establish the significance of AaegGPRdih1 to mosquito Malpighian tubule physiology was inconclusive.

Focusing on the 1851 International Sanitary Conference, this dissertation analyses an important episode in the international regulation of health, trade, passengers, and cargo in a period of epidemic crisis. It argues that a group of diplomats and physicians appointed to represent 12 European nations instituted a new international forum that extended – and occasionally rivalled – national and local agencies for epidemic governance. Together, delegates endeavoured to establish a common sanitary policy in Europe and in the Orient. By creating shared surveillance and judicial mechanisms – while standardising definitions and practices – delegates aimed to engineer the flow of people, vessels, cargo, and diseases in the Mediterranean region. As a transnational forum, the Conference was a platform where doctors and diplomats reinterpreted models of public health and sanitary administration while creating institutions that challenged conventional concepts of borders, national policy, and state sovereignty. As a multinational event, the Conference marked the unprecedented transition from local, national and, bilateral public health policies into a coherent transnational project for the governance of epidemics. The dissertation is based on extensive research conducted in hitherto largely unexplored medical, diplomatic, and national collections in Britain, France, Italy, Portugal, Spain, and the United States of America. Sources ranging from diplomatic correspondence to medical publications and personal diaries, tie together multiple national and professional perspectives while untangling a diversity of personal and state agendas that fundamentally shaped the foundation of international public health mechanisms and contributed towards the crystallisation of medical concepts. Chapter one demonstrates how economic and political concerns about the impact of quarantine on international trade led to calls for international regulation and the standardization of quarantine practices in the Mediterranean region. Drawing on medical reports, pamphlets and diplomatic correspondence, the chapter exposes the multitude of quarantine practices in the Mediterranean region and a growing international demand for prophylactic reform. These exchanges, it is shown, culminated with the organization of the 1851 International Sanitary Conference in Paris. Chapter two argues that the Conference challenged previous diplomatic and medical protocols by including two professional groups in the process of regulating international public health. The lack of precedent allowed diplomatic and medical delegates to establish new rules for the conduct of the conference, which gave them a relatively high level of autonomy from the states they represented. Chapter three focuses on the problems of constructing a shared aetiological classification and regulating quarantine practices. It shows that, although doctors gained progressive control over the Conference, ultimately diplomatic agendas shaped the final outcome. In addition, it demonstrates that, rather than defending the elimination of quarantines, liberal states supported the continuation of quarantine practice in the Mediterranean; albeit that they managed to severely limit its operation in practice. Finally, chapter four examines how European and Oriental sanitary institutions were uniformly redesigned and new international judicial mechanisms created. These measures variously affected the sovereignty of the participating states by limiting their independent capacity to set national epidemic policies. However, the chapter argues that these negotiations took the shape of sovereignty bargains: by loosening control over specific elements of their sovereignty, states managed to advance their political, economic and sanitary agendas. By looking at the International Sanitary Conference of 1851, this dissertation shows how the foundations of international public health had consequences not only for the control of epidemic diseases and the circulation of goods and people in the Mediterranean region, but also for the authority and status of the nation states. By doing so, it reveals that international public health governance resulted from the amalgamation of a particular configuration of expert and diplomatic struggles and compromises. Moreover, the dissertation shifts the traditional local and national focus in the history of medicine to a wider and international context where local and national traditions struggled to produce coherent discourses and practices.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 274-319).

Thesis--Universidad de Sevilla. / "Apéndice demografico": p. 134-180. Includes bibliographical references (p. 132-134).

This thesis is a social history of disease and mortality in the American Deep South before the Civil War. Yellow fever attacked the region at epidemic levels every two or three years between 1800 and 1860, killing about eight percent of the urban population, and as many as 20 or 30 percent of recent migrants from Europe. With little epidemiological understanding of mosquito-borne viruses-and almost no public health infrastructure to ameliorate disease-the only real protection from this scourge was to "get acclimated": fall sick with, and survive, yellow fever. About half of all people would die in the acclimating process. By placing the Deep South within an Atlantic disease diaspora uncontained by continental boundaries, the project shifts the fault-lines of the Southern past from North-South political conflicts onto similarly formative but overlooked ecological processes in the Greater Caribbean. Yellow fever and mass mortality are largely absent from the recent historiography on the cotton kingdom and "slave racial capitalism." But as well as being a “slave society,” this thesis suggests the Deep South was also a "disease society": Deep Southerners discussed yellow fever obsessively, worked according to its seasonal schedule, and judged others based on their perceived vulnerability to the disease. Yellow fever, and immunity to it, profoundly shaped the asymmetrical hierarchies of Deep Southern society, with acclimated "immunocapitalist" creoles on top, and unacclimated "foreigners" below. Slavers and their allies argued only intellectually-inferior but naturally-resistant black people could perform the arduous labour of sugar and cotton cultivation in the Deep South, as whites too frequently died. This became the region's chief argument for permanent racial slavery. However, almost every slave revolt in Louisiana coincided with a particularly bad epidemic, suggesting slaves found disease politically intriguing and understood that yellow fever left white society chaotic and vulnerable to attack.

Avec plus de 20% de morts annuels dus aux maladies infectieuses, celles-ci restent un sujet majeur de santé publique. Des maladies d’origine virale (ré)émergent suite aux changements environnementaux, climatiques et sociétaux : le virus Ebola, la Dengue ou, plus récemment, le virus Zika. Dans ce contexte, il est donc aujourd’hui crucial de développer des vaccins efficaces et sûrs contre les infections virales émergentes. Ce projet de thèse vise à mettre en place une nouvelle stratégie de production de vaccins vivants atténués ciblant les virus à ARN en travaillant sur le virus de la fièvre jaune (genre Flavivirus). Après une analyse génomique qui a permis d’approfondir une technique de modification des virus appelée « ré-encodage », des mutants de la fièvre jaune ont été produits puis caractérisés in vitro et in vivo. En parallèle, un modèle rongeur de la fièvre jaune a été développé et a permis de tester in vivo à la fois l’innocuité et l’efficacité vaccinale des virus ré-encodés. / Despite recent considerable improvements, infectious diseases remain a major issue for public health, with an estimated 20% of annual deaths caused by infections. Among them, viral diseases (re)emerge following environmental, climatic and societal changes: Ebola, Dengue and Zika viruses have recently been the object of special attention. The development of safe and efficient vaccines against emerging viruses is a major challenge for global public health. This thesis work is in line with this issue. Using the yellow fever virus (YFV, genus Flavivirus) as a model, we tried to define new strategies for the design of live-attenuated vaccines for viral infections prevention. After a genomic analysis that allowed to go further into a procedure for virus modification named “re-encoding”, we generated and characterised both in vitro and in vivo mutant strains of YFV. In parallel, a rodent model was set up to test in vivo both the safety and the protective efficiency of the re-encoded viruses.

Made available in DSpace on 2014-11-10T11:09:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-05-23Bitstream added on 2014-11-10T11:57:42Z : No. of bitstreams: 1 000790896.pdf: 1624782 bytes, checksum: 86f691c9d97bad42fa2588df159f183a (MD5) / O Vírus da Febre amarela (YFV) causa doença com considerável morbidade e mortalidade nas regiões tropicais. Diversos vírus possuem estratégias para a alteração dos processos celulares. Mecanismos de splicing celulares são essenciais para diversificar a expressão dos genes e podem aumentar seu potencial de gerar proteínas. A replicação de YFV e as interações entre proteínas virais e celulares não são totalmente conhecidas. A proteína celular hSlu7 possui sinal de localização nuclear e tem um papel importante nas reações catalíticas do segundo passo do splicing. Estudos demonstram que sob infecção de YFV hSlu7 transloca para o citoplasma. A translocação de proteínas entre o citoplasma e o núcleo pode representar um mecanismo viral da regulação da expressão gênica. Este estudo teve como objetivo a caracterização da interação entre a proteína hSlu7 e NS5 viral, bem como estudar os efeitos de interações sobre os mecanismos de splicing alternativo após a infecção de YFV. Para identificar interação de NS5 de YFV com hSlu7 foi realizado ensaio de co-imunoprecipitação. Para verificar alteração de splicing celular foram utilizados replicons pEGFP-ADAR, pI12-IL7R, pEFGP-FGFR2, bem como a alteração de isoformas de XBP-1 endógenos. Os resultados indicam que NS5 de YFV interage com proteína hSlu7 e que sua interação pode influenciar no metabolismo RNA celular. YFV demonstrou exercer modulação no splicing celular, a avaliação de replicons sugerem que em splicing dependente de hSlu7, bem como a independente ocorre uma regulação viral atuando sobre sítios de splicing fraco e que a interação hSlu7-NS5 pode alterar direta e indiretamente a regulação trans-acting / Yellow fever virus (YFV) causes disease with significant morbidity and mortality in tropical regions. Several viral strategies are avail for recruitment and alteration of the biochemical cellular processes. Cellular splicing mechanisms are essential to diversify the gene expression and increase it’s proteomic potential. Replication of YFV and the interactions between viral and cellular proteins are unknown. The cellular protein hSlu7 has an nuclear localization and an important role in the second catalytic reaction step of the alternative splicing. In our study group demonstrated that under YFV infection hSlu7 translocates to the cytoplasm. The translocation of proteins between nucleus and cytoplasm may represent a viral mechanism of cellular gene expression regulation, interference in the protein availability of the alternative splicing and viral replication control. This study aimed to characterize the interaction between the viral protein hSlu7 and NS5, as well as studying the effects of interactions on the mechanisms of alternative splicing after YFV infection. To identify interaction with YFV NS5 hSlu7 was conducted co-immunoprecipitation assay. To verify changes in cellular splicing replicons were used pEGFP-ADAR, PI12-IL7R, pEFGP-FGFR2 as well as the change of isoforms of XBP-1 endogenous.The results indicated that YFV NS5 protein interacts with hSlu7 and that their interaction may influence cellular metabolism RNA. YFV perform modulation on cellular splicing, the evaluation of replicons suggest that in hSlu7 splicing dependent and independent regulation occurs viral acting on weak splice sites and that the interaction hSlu7-NS5 can change directly or indirectly to regulate trans- acting

abstract: Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age, whom are particularly prone to Dengue severe infection and death, cannot be immunized using current approved DV vaccine. The most important issues documented so far are the lack of efficiency and enhancement of the disease in young seronegative recipients, as well as uneven protection against the four DV serotypes. Based on data from clinical trials that showed enhanced performance of dengue vaccines when the host has previous anti-flaviviral immunity, I proposed here an attractive solution to complement the current vaccine: a recombinant measles vaccine vectoring dengue protective antigens to be administered to young infants. I hypothesized that recombinant measles virus expressing Dengue 2 and 4 antigens would successfully induce neutralizing responses against DV2 and 4 and the vaccine cocktail of this recombinant measles can prime anti-flaviviral neutralizing immunity. For this dissertation, I generated and performed preclinical immune assessment for four novel Measles-Dengue (MV-DV) vaccine candidates. I generated four MVs expressing the pre membrane (prM) and full length or truncated (90%) forms of the major envelope (E) from DV2 and DV4. Two virus, MVvac2-DV2(prME)N and MVvac2-DV4(prME), expressed high levels of membrane associated full-length E, while the other two viruses, MVvac2-DV2(prMEsol)N and MVvac2-DV4(prMEsol)N, expressed and secreted truncated, soluble E protein to its extracellular environment. The last two vectored vaccines proved superior anti-dengue neutralizing responses comparing to its corresponding full length vectors. Remarkably, when MVvac2-DV2/4(prMEsol)N recombinant vaccines were combined, the vaccine cocktail was able to prime cross-neutralizing responses against DV 1 and the relatively distant 17D yellow fever virus attenuated strain. Thus, I identify a promising DV vaccination strategy, MVvac2-DV2/4(prMEsol)N, which can prime broad neutralizing immune responses by using only two of the four available DV serotypes. The current MV immunization scheme can be advantageus to prime broad anti-flaviviral neutralizing immunity status, which will be majorly boosted by subsequent chimeric Dengue vaccine approaches. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016