Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-16T11:43:38Z No. of bitstreams: 1 mauro-franca-da-silva.pdf: 1113117 bytes, checksum: 4721a993f32ab60140b4d0cf8c0f704f (MD5) / Made available in DSpace on 2012-11-16T11:43:39Z (GMT). No. of bitstreams: 1 mauro-franca-da-silva.pdf: 1113117 bytes, checksum: 4721a993f32ab60140b4d0cf8c0f704f (MD5) Previous issue date: 2007 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O vírus atenuado da febre amarela, subcepa 17DD, é utilizado por Bio-Manguinhos para a produção da vacina contra a febre amarela. Esta vacina tem sido utilizada para a imunização humana com um excelente histórico de eficácia e segurança. Entretanto, nos últimos anos, devido à ocorrência de alguns casos de eventos adversos associados ao vírus vacinal cepa 17D e subcepa 17DD, apontou-se a necessidade de desenvolvimento de uma vacina inativada. Para a implementação desta nova vacina torna-se necessário o desenvolvimento de métodos de quantificação de antígenos virais. Diferentes metodologias de quantificação podem ser utilizadas na produção de vacinas inativadas, sendo as mais comuns o teste imunoenzimático (ELISA) e o teste de dose-resposta. O presente estudo teve como objetivo o estabelecimento de um ELISA visando à detecção do antígeno do vírus da febre amarela inativado. Para este propósito, foram obtidos estoques de partículas virais da subcepa 17DD, a partir de culturas de células Vero, os quais foram purificados e quantificados por métodos bioquímicos e virológicos clássicos, respectivamente. Para o desenvolvimento do teste utilizamos diferentes anticorpos como capturana fase sólida. Os resultados obtidos para os testes utilizando o anticorpo 2D12 como captura mostraram um limite de detecção do antígeno no ELISA foi de 2,21 log 10 PFU/0,1mL e (1,55 µg/0,1mL). A partir deste valor, foi estabelecido um controle positivo contendo o vírus 17DD atenuado com título de 3,06 log10 PFU/mL e (29µg/0,1mL). Os resultados mostram, também, que o ELISA foi capaz de detectar o vírus 17DD inativado por formaldeído até a diluição 1:16 (52,9 µg/0,1mL). Baseado nos resultados obtidos acredita-se que o desenvolvimento de um teste de ELISA para detecção e quantificação do antígeno 17DD possa representar umimportante avanço tecnológico no controle da produção de uma vacina inativada contraa febre amarela. / The attenuated 17DD substrain of yellow fever virus is used in Bio-Manguinhos for yellow fever vaccine production. This vaccine has been used for human immunization with an excellent history of efficacy and safety. However, in the latest years, the occurrence of adverse events associated with 17D and 17DD substrain pointed to the necessity of developing technologies for the production of an inactivated vaccine. The implementation of this new vaccine will require methods for antigen quantification. Different methodologies of quantification can be used, being the most commonlyused the Enzyme-Linked Immunosorbent Assay (ELISA) and dose response test.The aim of this study was the establishment of an ELISA for the detection of inactivated yellow fever virus antigen. For this purpose, 17DD virus was obtained from Vero cell cultures, purified and quantified by biochemical and virological classical methods, respectively. The results showed that ELISA test using the 2D12 capture antibody presented a limit of 2,21 log10PFU/0.1mL of viral titer and (1,55 µg viral protein/0.1mL). Based on this value, a positive control was established which contained the attenuated 17DD substrain of yellow fever virus with a titer of 2,95log 10 PFU/mL and (29 µg/0.1mL). The results also showed that the ELISA was able to detect 17DD virus inactivated by formaldehyde up the dilution 1:16 (52,9 µg protein/0,1mL). The development of an ELISA test for the detection and quantification of 17DD antigen can represent an important step in the production control of the inactivated vaccine against of yellow fever.

This thesis describes how a solution can be built to detect human flu-like symptoms. Flu-like symptoms are important to detect to prevent Covid-19 [6]. As people are returning to work there is a need for a simple way of detecting flu-like symptoms to prevent the spread of Covid-19. Other than a solution, this thesis concluded how human flu-like symptoms can be detected, with cameras specifically. This is to know what symptoms are most likely to work for a prototype. The technique of cameras and thermal cameras made this project possible as well as the technique of a single-board computer. The technique of cloud-based services is also an important part of this project. This project has resulted in a novel prototype using a single-board computer, cameras, and various cloud-based services to detect and inform a person if he or she has a human flu-like symptom.

Le virus Lassa entraine une fièvre hémorragique endémique en Afrique de l’Ouest et représente un problème de santé publique. Les connaissances sur la pathogénèse et les réponses immunitaires associées à la maladie sont partielles. Nous avons suivi les paramètres pathologiques, virologiques et immunologiques associés aux infections létales et non létales du LASV chez le singe cynomolgus. Le tableau clinique a été caractérisé par une dépression, une anorexie, une perte de poids et une asthénie chez les animaux survivants, tandis que ces mêmes symptômes ont été accompagnés de fièvre, de difficultés respiratoires et d’épistaxis chez les animaux infectés par une dose létale. Seuls ces derniers ont montré une perturbation des paramètres de coagulation, une rhabdomyolyse et une hausse des marqueurs de lésions rénales. Nous avons observé un tropisme radicalement différent en fonction de la sévérité de la maladie, avec une dissémination virale dans les organes plus importante et plus rapide chez les animaux décédés, la présence de particules infectieuses plus nombreuses et des modifications anatomopathologiques plus sévères. Une réponse immunitaire innée et adaptative précoce et puissante a été associée avec le contrôle de l’infection et la survie tandis que les infections fatales ont été caractérisées par une réponse inflammatoire ressemblant au choc septique, une défaillance de la réponse immunitaire ainsi qu’une réplication virale incontrôlée. Cette étude permet d’améliorer nos connaissances de la pathogénèse de la fièvre de Lassa et d’apporter des marqueurs d’infection prédictifs de la maladie / Lassa virus causes a hemorrhagic fever endemic in West Africa and represents a threat for civilians. The pathogenesis and the immune responses associated with the disease are poorly understood. We followed pathological, virological and immunological parameters associated with fatal and non-fatal Lassa virus infection in the cynomolgus monkey. The clinical picture was characterized by depression, anorexia, weight loss and asthenia in survivors whereas the same symptoms were supported by fever, respiratory difficulties and epistaxis in animals infected with the lethal dose. Only fatalities have shown coagulation parameters dysfunction, rhabdomyolysis and an increase of renal function markers. We observed a different viral tropism in a function of the disease severity, with viral dissemination in organs that was more important and faster in fatalities, the appearance of numerous infectious particles number and more severe pathologic changes. Early and robust innate and adaptive immune response has been associated with the control of infection and recovery whereas fatal infections were characterized by a sepsis like inflammatory response, defective immune response as well as uncontrolled viral replication. This study sheds light on the pathogenesis of Lassa fever and reveals infection markers predictive of the disease outcome

Dengue fever (DF) and dengue hemorrhagic fever (DHF) are emerging infectious diseases which are endemic in many regions of the globe, many of which are resource-poor areas. DHF and DF impose a severe economic health burden in tropical and subtropical areas. Dengue virus causes an acute febrile illness that can be a self-limited febrile illness, as seen in most cases of DF, or a life-threatening illness with plasma leakage and shock, as seen in cases of DHF. A systematic review of the literature revealed gaps in the knowledge base of clinical laboratory findings of dengue illness with regards to longitudinal dynamics and classification and predictive modeling of disease severity. The objective of this thesis was to investigate the utility of clinical laboratory variables for classification and prediction of disease outcomes. The data used in this investigation was derived from a prospective study of Thai children presenting to either of two study hospitals within 72 hours of onset of an acute febrile illness. Systematic data collection, including clinical laboratory parameters, and routine clinical management continued each day until 24 hours after the fever had subsided. A final diagnosis of DHF, DF, or other febrile illness (OFI) was assigned by an expert physician after chart review. The first research objective of this study was to describe the temporal dynamics of clinical laboratory parameters among subjects with DHF, DF, or OFI. Data were analyzed using lowess curves and population-average models. Quadratic functions of clinical variables over time were established and demonstrated significantly divergent patterns between the various diagnostic groups. The second research objective was to establish and validate tools for classification of illness severity using easily obtained clinical laboratory measures. Bivariate logistic regression models were established using data from one hospital in an urban area of Thailand as a training data set and validated with a second data set from a hospital in a rural area of Thailand. The validated models maintained a high sensitivity and specificity in distinguishing severe dengue illnesses without using the hallmark indicators of plasma leakage. The third research objective used classification and regression tree (CART) analysis to established diagnostic decisions trees using data obtained on the day of study enrollment, within the first 3 days of acute illness. Decision trees with high sensitivity were established for severe dengue defined either as: 1) DHF with evidence of shock (dengue shock syndrome, DSS); or 2) DSS or dengue with significant pleural effusion. This study expands existing knowledge of the potential utility of clinical laboratory variables during different phases of dengue illness. The application of the results of these studies should lead to promising opportunities in the fields of epidemiological research and disease surveillance to reduce the health burden, and improve the clinical management, of dengue illness. Future directions involve application of these algorithms to different study populations and age groups. Additionally, other analytical techniques, such as those involving CART analysis, can be explored with these data.

The public health and veterinary importance of Gulf Coast ticks, Amblyomma maculatum Koch (1844) have become more apparent during the last several decades. In addition, new records of this three-host ixodid tick presently show a geographic distribution throughout much of the southern United States. Rickettsia parkeri, a spotted fever group rickettsia (SFGR) that is commonly found infecting the Gulf Coast tick, was only recently recognized as a human pathogen. Over the last decade, more than 20 human cases of disease caused by R. parkeri have been recognized in the Americas, all of which were similar in presentation to mild Rocky Mountain spotted fever. In addition, a novel, poorly characterized SFGR, “Candidatus Rickettsia andeanae”, was recently identified in A. maculatum from Peru, United States, Chile and Argentina. As the recognition of R. parkeri as a pathogen and “Ca. R. andeanae” as an additional SFGR in A. maculatum only recently occurred, a general gap exists in our understanding of the biology of these SFGRs. The overall objective of this dissertation was to contribute to our knowledge of SFGR infecting A. maculatum. In Chapter 3, we present a prevalence study of R. parkeri, and “Ca. R. andeanae” in A. maculatum from Mississippi where we detected 15.2% R. parkeri-singly infected ticks and 3.1% total “Ca. R. andeanae” infected ticks of which 1.7% were co-infected with R. parkeri. In Chapter 4, we discuss finding four genetically different populations of A. maculatum from Mississippi infected with a homogenous population of R. parkeri, using Single Strand Conformation Polymorphism analysis. Those initial data relating to “Ca. R. andeanae” provided a foundation for studies described in Chapters 5 and 6. We report the first morphological study of “Ca. R. andeanae” using transmission electron microscopy in Chapter 5 and isolation of this SFGR in ,A. maculatum cell co-culture in Chapter 6. We anticipate that results presented in this dissertation will contribute to our understanding of the ecology of ,A. maculatum as a vector for the human pathogen, R. parkeri, and increase the current understanding of both R. parkeri and “Ca. R. andeanae” in A. maculatum.

Le paludisme compte parmi les plus lourds fardeaux qui pèsent sur l’Afrique subsaharienne en termes de maladie, dans un contexte international de grande mobilisation pour son contrôle. Le paradoxe est que les moyens thérapeutiques (préventifs et curatifs) adéquats pour son contrôle sont disponibles et largement diffusés. Plusieurs travaux ont montré qu’à la base de cet échec se trouvent de nombreux facteurs culturels, sociaux, économiques, environnementaux, etc. qui sont nécessaires en plus des moyens thérapeutiques traditionnels pour un contrôle efficace du paludisme. Les expériences menées dans le sens de la prise en compte de ces facteurs se sont souvent situées dans la logique des professionnels de la santé sans véritablement tenir compte de la logique des communautés. Au Bénin, le paludisme demeure la première cause de morbidité et de mortalité dans la population générale et surtout celle des enfants de moins de cinq ans. Sur les dix dernières années (1996-2005) et malgré les importants efforts engagés pour son contrôle, le paludisme est resté la première cause de consultation et d’hospitalisation dans la population des enfants de moins de cinq ans avec une tendance à la hausse. La présente étude a choisi de voir dans quelle mesure l’empowerment communautaire et la participation appropriative des parents dans l’élaboration et la mise en œuvre des projets de lutte contre le paludisme des enfants de moins de cinq ans contribue à la réduction de la morbidité et de la mortalité de ces derniers ainsi qu’à l’amélioration de la qualité de vie des populations. Pour ce faire, il a été mis en place un dispositif quasi-expérimental pré-post, pilote et témoin. L’intervention de promotion de la santé et d’éducation pour la santé a été un accompagnement du village pilote dans l’identification des priorités et la mise en œuvre des solutions propres à lutter contre le paludisme de l’enfant reconnu comme problème prioritaire de santé. Le projet a été mis en place suivant la logique de la communauté pilote qui a identifié sept différents micro-projets, suivant ses besoins prioritaires, pour efficacement lutter contre le paludisme de l’enfant. Parmi ces sept micro-projets, cinq ont été pleinement mis en œuvre, le sixième partiellement et, le dernier non encore entamé avant le terme provisoire du projet qui a été de 27 mois. L’ensemble de ces activités a impliqué environ 80% de la population de la communauté pilote. Au terme des 27 mois, la grande majorité des parents interrogés trouvent qu’il s’agit d’un projet qui leur a apporté d’éléments nouveaux dans l’appréciation de la situation de la fièvre et de sa prise en charge et, par conséquent, il devrait continuer. L’analyse des représentations, les entretiens de groupe, le dépistage actif, le dépouillement des registres dans les formations sanitaires fréquentées par les membres de la communauté pilote ainsi que le recensement des décès d’enfants pendant la période de l’intervention, comparés aux données d’avant intervention ont permis de noter un changement statistiquement significatif dans les pratiques de recours aux soins adéquats, une réduction de la morbidité et de la mortalité palustres (respectivement 42% et 87% des taux observés avant l’intervention) avec également une différence statistiquement significative comparativement au village témoin. Une confirmation de ces résultats est obtenue à partir des registres d’approvisionnement et de cession des médicaments anti-palustres utilisés dans le cadre du projet dans le village pilote ainsi que les données d’interviews individuelles approfondies. Les femmes, dont les fonctions traditionnelles dans ce milieu leur confèrent un statut de l’ombre, ont joué un rôle prépondérant dans l’initiation des micro-projets et la prise en charge adéquate des cas de fièvre des enfants. D’autres aspects du développement du village pilote ont vu le jour à partir de ce projet, tels que la mise en place d’une fontaine d’eau potable, l’élaboration et la mise en œuvre d’un nouveau modèle d’habitat permettant une meilleure protection contre les moustiques vecteurs du paludisme, etc. Il est à noter que le projet se poursuit sous la direction du comité villageois mis en place pour la circonstance et avec un contact régulier maintenu entre ce dernier et le chercheur principal.

African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication. SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production. The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

L'objectif de cette thèse est le développement d'un vaccin recombinant capripoxvirus protégeant contre la variole des ruminants, la Fièvre de la Vallée du Rift (FVR) et la Peste des Petits Ruminants (PPR) comme modèle vaccinal destiné aux pays atteints par ces infections. Une première partie de ce travail a consisté en une enquête sérologique en Tunisie pour évaluer les prévalences PPR et FVR. L'enquête menée a montré une séroprévalence PPR de 7,6% et l'absence de FVR. Le risque lié à une infection par le virus de la fièvre de la vallée du Rift n'est pas nul en raison de l'identification des vecteurs compétents Culex theileri et Culex pipiens dans les zones échantillonnées. L'élaboration du vaccin capripoxvirus FVR-PPR porte sur l'expression des gènes NSmGN-FVR et H-PPR où chacune des valences est insérée dans le site de la thymidine kinase et le site d'un analogue du récepteur à l'interleukine 8 respectivement. Le vecteur choisi pour la souche vaccinale Kenya Sheeppox-1. Bien que nos travaux aient conduit à l'obtention du capripoxvirus double recombinant, ce dernier n'a pu être purifié. L'alternative a donc été d'évaluer l'effet protecteur et l'immunogénicité induits par le simple recombinant capripoxvirus- NSmGN-FVR, qui est un produit de l'étape intermédiaire dans l'élaboration du double recombinant FVR-PPR. L'effet protecteur de notre construction a été validé par deux expérimentations chez des souris Mus m. musculus MBT/Pas, avec épreuve infectieuse. Le nombre de doses administrées, les voies d'administration ont été déterminants dans cette protection justifiée par l'obtention d'anticorps neutralisants anti-FVR. L'étude de l'immunogénicité a été réalisée sur un modèle caprin sans épreuve infectieuse, une séroconversion FVR a été observée. La lymphoprolifération et le typage des sous populations lymphocytaires ont été analysés. / The aim of this thesis was to develop a capripoxvirus based recombinant vaccine against ruminant pox, Rift Valley fever (RVF) and peste des petits ruminants (PPR) considered as a vaccine model for countries affected by these infections. The first part of the work consisted in a serological survey conducted in Tunisia to detect the PPR and RVF presence. A PPR seroprevalence of 7.6% has been found and no antibodies against RVF were detected. However, the risk of infection with rift valley fever virus persists since competent vectors such as Culex pipiens and Culex theileri has been identified in the sampled areas. The development of the RVF-PPR vaccine candidate is based on the NSmGN-FVR and H-PPR gene expression - where each of the genes is inserted into the thymidine kinase and the Interleukin 8 receptor analogue genes, respectively. The vector chosen is the vaccine strain Sheeppox Kenya-1. Although the double recombinant RVF-PPR has been produced, it could not be purified. The alternative was to evaluate the protection and the immunogenicity of the single recombinant capripoxvirus NSmGN-FVR, which is a product of an intermediate step of the process of the double recombinant preparation. The protection of our vaccine candidate has been performed by two mice experiments in Mus m. musculus MBT/Pas, with challenge. The number of doses, the route of administration played a key role in the protection confirmed by the presence of neutralizing anti-RVF antibodies. The study of the immunogenicity of the vaccine candidate was conducted in goats without challenge, RVF seroconversion has been shown. Lymphoproliferation studies and lymphocytes subpopulations typing have been analysed.

Enteric fever caused by Salmonella enterica serovars Typhi and Paratyphi A are a major public health concern in Kathmandu. The aim of this thesis was to identify and assess the population most at risk by investigating epidemiologic trends of enteric fever within a subset population of Kathmandu. Therefore,the burden and incidence of enteric fever within the study population and the seasonal and gender distribution of enteric fever was assessed. Considerable burden of enteric fever, unrelated to population density, correlating with the seasonal fluctuations in rainfall was observed. This thesis also aimed to improve the understanding of enteric fever transmission by identifying probable transmission routes,hence various water and food samples were analysed and the extent of faecal contamination in them was determined. S. Typhi isolates were sequenced and genotyped and combined with GPS data to longitudinally study the local distribution and infer transmission of this human restricted bacterial pathogen. Extensive clustering of typhoid independent of population size and density and existence of an extensive range of genotypes within typhoid clusters including individual households with multiple cases was observed. These observations predict that indirect transmission had an overwhelming contribution for disease persistence, potentially through contaminated water. Consistent with this hypothesis, S. Typhi and S. Paratyphi A were detected in water supplies and it was observed that typhoid was spatially associated with public water sources and low elevation. A concurrent case-control study was also conducted which allowed for the determination of risk factors in the population at risk. These studies imply that resources should be allocated toward controlling the most important vectors of enteric fever, including food sold by vendors, chlorination of drinking water, construction of proper water distribution and sewage networks,vaccination campaigns and hygiene education.

Objective: The purpose of this investigation was to describe the long-term effects and functional outcomes of patients in Virginia who were reported to the Virginia Department of Health with West Nile virus (WNV) non-neuroinvasive and neuroinvasive disease. The study identified the duration of symptoms after initial illness, the number of persons who fully recovered versus the number who continue to be symptomatic and how patients' quality of life differed after illness.Methods: The study population was drawn from 60 human cases that met the surveillance case definition for non-neuroinvasive and neuroinvasive WNV illness in Virginia between 2002-2004. Information was collected during personal interviews using a standard questionnaire. The questionnaire included questions on demographics, clinical signs and symptoms, existing medical conditions and the respondents' personal assessment of health. Statistical analysis were used to compare pre and post illness symptoms, respondents vs. non-respondents, and non-neuroinvasive respondents vs. neuroinvasive respondents. Results: Thirty-four patients were enrolled in the study. Five (14.7%) respondents had non-neuroinvasive disease and 29 (85.2%) had neuroinvasive disease. Thirty respondents (88.2%) reported being hospitalized. Respondents with non-neuroinvasive disease spent a median of 3.5 (range, 0-7) days in the hospital and were unable to resume normal activities for a median of 17 (range, 7-365) days. Respondents with neuroinvasive disease spent a median of 7.5 (range, 0-82) days in the hospital and were unable to resume normal activities for a median of 127.50 days (range, 0-1023). Two (40%) of the respondents that suffer from non-neuroinvasive illness were unable to resume normal activities for at least 90 days. Fifteen (51.7%) respondents with neuroinvasive disease were unable to resume normal activities for at least 90 days. At the time of the interview, 20% of respondents with non-neuroinvasive disease reported fatigue, tremors, arthralgia, paralysis and memory problems. Respondents with neuroinvasive disease reported fatigue (58.5%), weakness (51.7%), myalgias (37.9%), confusion (41.4%), and memory loss (55.2%). Conclusion: WNV illness, including non-neuroinvasive illness, may be more serious and prolonged than generally thought. Neuroinvasive disease resulted in long-term morbidity and non-neuroinvasive disease resulted in work absenteeism and extended recovery periods. The mortality rates and potential long-term effects associated with non-neuroinvasive and neuroinvasive illness emphasizes the importance of continuing to develop effective methods of targeting preventive education to high-risk populations while continuing to pursue longer-term solutions such as vaccines to prevent emerging infection. Further research is needed to document the long-term effects of WNV, especially in areas with a high number of WNV human cases with more non-neuroinvasive patients. WNV is an emerging infectious disease with a wide clinical spectrum and variable long-term effects; thus a public health concern.