Kyasanur Forest disease virus (KFDV) of the Flaviviridae virus family has caused seasonal infections and periodic outbreaks in Karnataka, India. First identified in 1957, KFDV annually infects 400-500 people and has a fatality rate of 3-5%; there are no approved antivirals and the existing licensed vaccine’s effectiveness appears to be questionable. Many tools for KFDV research are limited and this work sought to develop methods for analysing antivirals, including interferon (IFN)-α/β species. The BHK-21 (ATCC) cell line allowed for high virus propagation and distinguishable cytopathic effects (CPE) for determining antiviral effectiveness. The additional tool of a reverse genetics system expressing a full-length cDNA KFDV genome with a GFP reporter failed to propagate, despite numerous GFP genome-insertion strategies. The clinically approved IFN-α2a or IFN-α2b has had variable success at combatting flavivirus diseases in people, especially in the immuno-compromised. The continued passaging of KFDV-infected cells with repeated IFN-α2a treatment did not eliminate KFDV and had little effect on infectious particle production. IFN-αspecies, αWA and α were more effective than IFN-α2a and α2b at reducing KFDV; however dose ranges indicated that while low concentrations could limit CPE, higher concentrations were needed to inhibit virion release. Avoidance of IFN-α/β through Jak/STAT signalling repression was attributed to the NS5 protein, specifically the RdRp domain based on data obtained with luciferase and vesicular stomatitis virus (VSV) recovery assays. However, the mechanism appears to act subsequently to STAT1/2 activation without NS5 binding to any Jak/STAT components. A non-infectious, replicative system serving as a platform for antiviral drug testing against KFDV in a high throughput manner could only provide luciferase signals when the NS proteins capable of driving replication, were supplied in cis (subgenomic) but not in trans (antigenome). To conclude, IFN-α species such as IFN-αWA may be better suited than the licensed IFN-α2a for treatment of KFDV infections; however, IFN effects appear to be subdued in vitro due to the actions of the NS5 protein. While IFN may not be a successful antiviral against KFDV, the work in this thesis provides a foundation for evaluating other potential anti-KFDV therapeutics. / February 2016

MY-24 is an aristeromycin derivative previously shown to protect AG129 type I and II interferon receptor knockout mice from lethal challenge with Tacaribe virus (TCRV). TCRV is nonpathogenic to humans, but is closely related to the highly pathogenic New World arenaviruses that cause often-fatal viral hemorrhagic fever syndromes. Remarkably, MY-24 prevented mortality without reducing TCRV burden in the circulation or tissues. To investigate the mechanism by which MY-24 protects AG129 mice against TCRV infection, we first characterized the natural history of disease in the model with an emphasis on cytokine responses and vascular integrity to establish the best times to evaluate the effects of MY-24 treatment on host responses believed to contribute to pathogenesis and fatal outcome. We found that viral replication in the blood and in various tissues precedes a hyperproduction of proinflammatory mediators that may lead to the destabilization of the endothelial barrier and increased vascular leakage believed to contribute to terminal shock associated with severe cases of hemorrhagic fever. We also found slightly reduced virus titers in certain tissues from MY-24-treated mice, suggesting that there may be a weak antiviral effect; however, TCRV was not cleared from lung, spleen, brain or kidney in recovering animals out to 40 days post-infection, indicative of the establishment of chronic infection in mice that are able to survive the initial challenge. Neutralizing antibodies do not appear to play a major role in the antiviral effect of MY-24, whereas reductions in several key proinflammatory cytokines in mice treated with MY-24 may serve to reduce vascular leakage caused by TCRV infection.

Introduction: Recent Ebola epidemic in West Africa have put viral hemorrhagic fever diseases in the spotlight. Uganda has had several outbreaks throughout the years, which have successfully been managed. Nurses’ patient education plays an important role in the work to increase public awareness about viral hemorrhagic fever diseases. Objectives: To assess how nurses at the emergency department educate the patients and relatives about the viral hemorrhagic fever diseases. Methods: An explorative and descriptive qualitative study with qualitative approach have been used. In-depth interviews with 18 open-ended questions have been conducted with nurses in the emergency department. Data was analyzed by qualitative content analysis and analyzed with Peplau’s theory of interpersonal relationship. Results: Through data analysis four categories were developed; Wide variety of educational techniques, Experienced obstacles, How to attain wider audience and Preferable characteristics as an educating nurse. Nurses used many different approaches when educating about viral hemorrhagic fever diseases, often uniquely combined. Lack of time and too unstable patients in the emergency department were seen as the main obstacles to educate. Methods to reach the community and employing a special education-nurse on the ward were suggestions to improve the preventive work against viral hemorrhagic fever diseases. Conclusion: Nurses are aware of the importance of patient education and use the educational methods they believe to be the most effective. However, patients in the emergency department often come in too late and priority should be on preventive measures. Training the nurses in educational techniques and patient education could be a key in decreasing the risk of coming outbreaks. / Bakgrund: Den senaste ebolaepidemin i västafrika har gjort att blödarsjukdomar hamnat i rampljuset. Uganda har genom åren drabbats av flera utbrott som framgångrikt hanterats. Sjuksköterskors patientutbildning spelar en viktig roll i arbetet för att öka allmänhetens medvetenhet om blödarsjudkomar. Syfte: Att undersöka hur sjuksköterskor på akutmottagningen utbildar patienter och anhöriga om blödarsjukdomar. Metod: En explorativoch deskriptiv kvalitativ studie med kvalitativ ansats användes. Sjuksköterskor som arbetar på akutmottagningen har djupintervjuats med 18 öppna frågor. Data har analyserats med en kvalitativ innehållsanalys och analyserats utifrån Peplaus ”interpersonal relationship theory”. Resultat: Genom dataanalysen utvecklades fyra kategorier; Stor variation på utbildningstekniker, Upplevda hinder, Hur man ska nå en bredare publik och Önskvärda egenskaper som utbildande sjuksköterska. Sjuksköterskorna använder många olika metoder för utbilda patienter och anhöriga om blödarsjukdomar, ofta i unika kombinationer. Tidsbrist och alltför instabila patienter på akutmottagningen sågs som de största hindren för att utbilda. Metoder för att nå allmänheten och att anställa en speciell utbildningssjuksköterska på avdelningen var några av förslagen för att förbättra det förebyggande arbetet mot blödarsjukdomar. Slutsats: Sjuksköterskorna är medvetna om vikten av patientutbildning och använder de pedagogiska metoder de anser vara mest effektiva. Då patienterna kommer till akutmottagningen är det dock ofta för sent och preventiva åtgärder borde prioriteras. Att öka sjuksköterskornas kunskap i utbildningstekniker och patientutbildning kan vara en viktig del i det preventiva arbetet för att minska risken för kommande utbrott.

Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.

Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.

Thesis (M.P.H.M.(Primary Health Care Management))--Mahidol University, 2006.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Orientador: Wilson Castro Ferreira Junior / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Matemática, Estatística e Computação Científica / Made available in DSpace on 2018-08-16T08:15:58Z (GMT). No. of bitstreams: 1 Vilamiu_RaphaelGustavod'Almeida_D.pdf: 17502226 bytes, checksum: af859cc18d3a4c5a74c324d3e6c0865a (MD5) Previous issue date: 2010 / Resumo: Nesta tese, desenvolvemos uma técnica para gerar grafos à partir de conjuntos de séries temporais considerando a correlação entre estas e uma extensão do Método de Decomposição Empírica (EMD) para grafos (GEMD). Tal trabalho se justifica pelo fato de que uma grande gama de sinais formados por conjuntos de séries temporais não possuem uma localização bem definida em nenhum espaço n-dimensional. Desta forma, as relações entre as séries temporais só são satisfatoriamente representadas com o uso de grafos. Contudo, o desenvolvimento do GEMD é dependente do uso de algum método de interpolação em grafos. Tais métodos são escassos e não produzem propriedades satisfatórias para o uso no GEMD. Para esta finalidade, estendemos a interpolação por Funções de Base Radial (RBF) em Grafos (GRBF), onde a norma euclidiana no cálculo da matriz de interpolação por RBF é substituída pela norma induzida pelo grafo. Testes numéricos sugerem que a extensão possui boas propriedades de convergência e uma técnica é desenvolvida para garantir a existência e unicidade da solução. Finalmente, aplicamos o GEMD em um conjunto de dados de incidência de Dengue Hemorrágica na Tailândia. Os modos intrínsecos encontrados desta forma não apresentam nenhuma onda viajante emanando de nenhuma das províncias, contrastando com o resultado utilizando o EMD original [5]. Além disso, os períodos médios dos modos intrínsecos de [5] são claramente distintos dos encontrados pela decomposição por GEMD / Abstract: In this thesis, we developed a technique to generate a graph from a set of temporal series, which are then decomposed trough an extension of the Empirical Mode Decomposition (EMD) on Graphs (GEMD) created by us. This procedure is justified by the fact that a huge amount of signals cannot be properly localized on an n-dimensional space which can only be properly represented by a graph. The development of the GEMD is dependent on some graph interpolation method. Such methods are scarce in the literature and do not have the necessary properties to accomplish the GEMD decomposition. For this goal, we extend the Radial Basis Functions (RBF) interpolation to graphs (GRBF), where the euclidean norm used in the calculation of the RBF interpolation matrix is substituted by a graph induced norm. Numerical tests suggests that GRBF have good convergence properties and we present a technique which guarantees the existence and uniqueness of the solution. We finally apply the GEMD decomposition to a data set of Dengue Hemorrhagic Fever incidence in Thailand. The intrinsic modes found in this way do not show any traveling wave emanating from any of the provinces, contrasting with the results found using the original EMD [5]. Moreover, the mean period for the intrinsic modes in [5] are clearly diverse of those found by GEMD decomposition / Doutorado / Doutor em Matemática Aplicada

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-11-20T11:26:09Z No. of bitstreams: 2 Tese - Solomar Martins Marques - 2013.pdf: 2691124 bytes, checksum: 04ee7837e335b7d3d617e5d8fe652956 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2014-11-20T11:26:37Z (GMT) No. of bitstreams: 2 Tese - Solomar Martins Marques - 2013.pdf: 2691124 bytes, checksum: 04ee7837e335b7d3d617e5d8fe652956 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-11-20T11:26:37Z (GMT). No. of bitstreams: 2 Tese - Solomar Martins Marques - 2013.pdf: 2691124 bytes, checksum: 04ee7837e335b7d3d617e5d8fe652956 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-04-26 / Introduction: In the 21st century, Brazil has become the country with the highest number of cases of dengue in the world, with a wide distribution of Aedes aegypti in all regions and simultaneous circulation of the four serotypes. The identification of the four serotypes is confirmed in Goiânia, which is among the municipalities and cities with the highest concentration of reported cases. Objective: To identify clinical and laboratory markers associated with complications and severe forms of dengue in Goiania, from 2001 to 2011. Methods: A case-control study in which the cases are severe forms (DHF and CCD) and the controls are patients with CD. Secondary data from the Information System for Notifiable Diseases (Sinan) was used, Brazilian Ministry of Health. Verification of consistency, checking and data analysis was performed by the SPSS 20.0 program. Results: 3359 patients aged under 15 years were laboratory confirmed as dengue, with 349 cases and 3010 controls. It did not differ by gender. It is noted an increased tendency to severity, over the years (X2 for trend = 19.03, p <0.0001). Regarding age, the greatest risk of severe forms strata was 5-9 years old (OR 2.32, 95% CI 1.28 to 4.03) and 10 to 14 years old (OR 2.08, 95% CI 1.50 to 2.89). Discussion: Abdominal pain, hypotension and hemorrhagic phenomena such as petechiae, epistaxis and gastrointestinal bleeding were significantly associated with severity after logistic regression. Regarding the laboratory tests available, hemoconcentration, thrombocytopenia and tourniquet test had statistically significant association with CCD and HDF. We identified 10 patients with neurological complications, including 2 deaths. Conclusions: detailed clinical assessment and collection of blood counts are strongly suggested for all patients in the pediatric age group. This study contributed to consolidate the classical signs and ordinary laboratories as important instruments in both the diagnosis, the prognosis of children and adolescents with dengue disease. / Introdução: No século 21, o Brasil se tornou o país com maior número de casos relatados de dengue no mundo, com ampla distribuição do Aedes aegypti em todas as regiões e circulação simultânea dos quatro sorotipos. Goiânia tem confirmada a identificação dos quatro sorotipos e se encontra entre os municípios e localidades com maior concentração de casos notificados. Objetivo: Identificar marcadores clínicos e laboratoriais associados a formas graves de dengue em menores de quinze anos de idade, na cidade de Goiânia, de 2001 a 2011. Métodos: estudo caso-controle, onde Casos são definidos como formas graves (DCC e FHD) e os Controles, pacientes com DC. Utilizados dados secundários do Sistema de Informação de Agravos de Notificação (Sinan). Feita verificação da consistência, checagem e análise de dados, pelo programa SPSS 20. Resultados: 3.359 menores de 15 anos foram confirmados laboratorialmente como dengue, sendo 349 casos e 3.010 controles. Não apresentaram diferenças quanto ao sexo. Nota-se incremento da tendência de gravidade, ao longo dos anos (X2 p/tendência= 19,03; p<0,0001). Quanto à idade, o maior risco de formas graves foi nos estratos de 5 a 9 anos (OR 2,32; IC95% 1,28 - 4,03) e 10 a 14 anos (OR 2,08; IC95%1,50 - 2,89). Discussão: Dor abdominal, hipotensão e fenômenos hemorrágicos como petéquias, epistaxe e sangramento gastrointestinal tiveram associação estatisticamente significante com gravidade após regressão logística. Respectivo aos exames laboratoriais disponíveis, hemoconcentração, plaquetopenia e prova do laço tiveram associação estatisticamente significante com FHD e DCC. Foram identificados 10 pacientes com complicação neurológica, sendo dois óbitos. Conclusões: avaliação clínica minuciosa e a coleta de hemograma são fortemente sugeridas em todos os pacientes na faixa etária pediátrica. Este trabalho contribuiu para consolidar os sinais clínicos clássicos e os laboratoriais básicos como importantes ferramentas auxiliares, tanto no diagnóstico, quanto no prognóstico de crianças e adolescentes com dengue.

Le virus de la fièvre hémorragique de Crimée-Congo (VFHCC) est un virus transmis par des tiques, appartenant au genre Nairovirus de la famille des Bunyaviridae. Il est réparti sur plus d’une trentaine de pays de plusieurs continents : Europe, Asie et Afrique ; avec une mortalité moyenne estimée de 30%. Le VFHCC peut causer à l’homme une maladie hémorragique très sévère et parmi divers symptômes, il peut induire une inflammation aigüe et des lésions hépatiques. Les phagocytes mononucléaires, les hépatocytes et les cellules endothéliales ont été décrits comme étant des cellules cibles lors d’études cliniques et post-mortem ainsi que lors d’études en modèle murin. Nous avons analysé, lors d’étude in vitro, la réponse cellulaire de cellules présentatrices de l’antigène (CPAs) et d’hépatocytes humains. Afin de mieux comprendre la pathogenèse du VFHCC, nous avons comparé la réponse de ces cellules avec celle du virus Dugbe (DUGV), un nairovirus génétiquement le plus proche de VFHCC considéré comme faiblement pathogénique. Finalement, afin améliorer la détection de DUGV in vitro et lors d’études épidémiologiques de terrain, nous avons développé un test moléculaire en temps réel pour détecter et quantifier DUGV.Nous avons observé que le VFHCC induisait une réponse inflammatoire chez les CPAs, en revanche, DUGV induisait une réponse plus forte, ce qui suggère que VFHCC induirait une inhibition sélective de certains médiateurs de la réponse pro-inflammatoire. Lors de l’infection in vitro des hépatocytes par le VFHCC, nous avons observé que le virus induisait du stress du RE, l’activation de l’IL-8 un médiateur pro-inflammatoire, et la modulation des deux voies pro-apoptotiques. Quand nous avons comparé cette réponse à celle induite par DUGV nous avons trouvé que la différence majeure était l’absence d’apoptose. Ces différences pourraient, en partie, expliquer le rôle du foie dans la pathogenèse induite par le VFHCC. / Crimean-Congo hemorrhagic fever virus (CCHFV) is a widely distributed tick-borne member of the Nairovirus genus (Bunyaviridae) inducing an average mortality rate of 30% in humans. CCHFV induces a severe hemorrhagic disease in infected patients that includes, among other bleeding symptoms, acute inflammation and liver lesions. The mononuclear phagocytes, the hepatocytes and the endothelial cells were described to be the main target cells in both human clinical studies and animal model in vivo studies.We analysed the in vitro cellular response of host antigen presenting cells (APC) and hepatocytes. Then, to better elucidate the pathogenesis of CCHFV, we compared the response of these cells after infection with Dugbe virus (DUGV), a mild pathogenic virus genetically close to CCHFV. In order to improve DUGV detection in vitro and in field studies, we also developed a molecular real-time quantitative tool to detect and quantify DUGV.We found that CCHFV induced an inflammatory response in both APCs tested; however DUGV induced a higher cytokine/chemokine response in these target cells than CCHFV. Our results suggest that CCHFV was able to selectively inhibit the activation of some inflammatory mediators in the in vitro infection and that CCHFV/DUGV cellular response differences could be relevant in pathogenesis. On the other hand, when we in vitro infected hepatocytes with CCHFV, we observed that it was able to induce ER-stress, activate IL-8 secretion and modulate both mitochondrial and death receptor pathways of apoptosis. When we compared this cellular response with that induced by DUGV, we found that the most striking difference was the absence of apoptosis. These differences could, in part, explain the role of the liver in the pathogenesis induced by CCHFV.