Made available in DSpace on 2019-03-30T00:07:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-10-29 / Dengue is an infectious, tropical and endemic disease. Dengue demands numerous efforts aimed at its prevention and control. The lack of an effective vaccine as well as specific treatment to combat the virus has encouraged the development of research to verify products with antiviral activity, especially the use of natural products. This study aimed to evaluate the antiviral activity in vitro of medicinal plants of Ceará flora against serotypes 2 and 3 of the dengue virus. In search of ethanol extracts were used Acrocomia aculeata (Jacq.) Lood. ex Mart, Azadirachta indica A. Juss, Cajanus cajan (L.) Millsp., Caryocar coriaceum wittm, Mormodica charantia L., Plectranthus amboinicus (Lour.), Schinus terebinthifolius Raddi, Ximenia americana L. and the essential oil of Eugenia jambolana (Lam.) and Vanillosmopsis arborea Baker (1000; 500; 250; 125; 62,5 e 31,25 ?g/mL). In the early stages of evaluation tests were performed cytotoxicity of samples in Vero cells. Then the screening of antiviral activity was performed with the non-cytotoxic samples and used three methodological strategies (pre-treatment, post-treatment and virucidal) and different time points were tested (1000, 500 and 250 ?g/mL) compared to viral infection. Only the Azadirachta indica (CC50 197.0 ?g/mL), Cajanus cajan (CC50 156.5 ?g/mL) and Caryocar coriaceum (CC50 199.4 ?g/mL) showed cytotoxic potential. The remaining plants even at the highest concentration tested (1000 ?g/mL), did not exert cytotoxicity at least 50% of the monolayer and were used in subsequent stages of the search. The screening of antiviral activity found that the ethanol extracts of Acrocomia aculeata (Jacq.) Lood. ex Mart and Plectranthus amboinicus (Lour.) developed antiviral action against DENV-2 in all valuation techniques performed in this study, a fact that may be related to the presence of tannins and flavonoids. The essential oil Vanillosmopsis arborea Baker, rich in alpha-bisabolol, inhibited the virus through all valuation techniques, except for pre-treatment against DENV-3, suggesting viral inhibition in the early and late stages of the cycle replicative. These plants here identified as potential antiviral against dengue virus have already been studied in other surveys which cited its antimicrobial and antiviral actions. The findings of this screening revealed promising plants in the investigation of an antiviral drug against dengue virus, especially the ethanol extracts of Acrocomia aculeata (Jacq.) Lood.; the Plectranthus amboinicus (Lour.) and the essential oil of Vanillosmopsis arborea Bake. The essential oil of Vanillosmopsis arborea Baker and Plectranthus amboinicus (Lour.) are nontoxic to mammalian last and detaining potential inhibition of DENV cells, suggesting the feasibility of further research as its antiviral activity and more precise elucidation of mechanism of action. / A dengue é uma doença infecciosa, tropical e de caráter endêmico a qual demanda inúmeros esforços visando sua prevenção e controle. A ausência de uma vacina disponível para a população, bem como um tratamento específico no combate ao vírus tem incentivado a realização de pesquisas buscando a verificação de produtos com atividade antiviral, destacando-se a utilização de produtos naturais. O presente estudo objetivou avaliar a atividade antiviral in vitro de plantas medicinais da flora cearense contra os sorotipos 2 e 3 do vírus da dengue. Na pesquisa foram utilizados extratos etanólicos de Acrocomia aculeata (Jacq.) Lood. ex Mart, Azadirachta indica A. Juss, Cajanus cajan (L.)Millsp., Caryocar coriaceum wittm, Mormodica charantia L., Plectranthus amboinicus (Lour.), Schinus terebinthifolius Raddi, e Ximenia americana L. e o óleo essencial de Eugenia jambolana (Lam.) e Vanillosmopsis arborea Baker (1000; 500; 250; 125; 62,5 e 31,25 ?g/mL). Nas fases iniciais foram realizados os testes de avaliação da citotoxicidade das amostras em células Vero. Em seguida, a triagem da atividade antiviral foi realizada com as amostras não citotóxicas e foram utilizadas três estratégias metodológicas (pré-tratamento, pós-tratamento e virucida), variando o momento do tratamento das células com as amostras (1000; 500 e 250 ?g/mL) em relação à infecção viral. Somente a Azadirachta indica (CC50 197,0 ?g/mL), Cajanus cajan (CC50 156,5 ?g/mL) e Caryocar coriaceum (CC50 199,4 ?g/mL) apresentaram potencial citotóxico. As demais plantas, mesmo na maior concentração testada (1000 ?g/mL), não exerceram citotoxicidade a pelo menos 50% da monocamada e foram utilizadas nas etapas seguintes da pesquisa. A triagem da atividade antiviral identificou que os extratos etanólicos de Acrocomia aculeata (Jacq.) Lood. ex Mart e Plectranthus amboinicus (Lour.) desenvolveram ação antiviral contra o DENV-2 em todas as técnicas de avaliação realizadas no presente estudo, fato este que pode estar relacionado à presença de taninos e flavonóides. O óleo essencial de Vanillosmopsis arborea Baker (candeeiro), rico em alfa-bisabolol, inibiu o vírus por meio de todas as técnicas de avaliação, exceto por prétratamento contra o DENV-3, sugerindo inibição viral nas fases iniciais e tardias do ciclo replicativo. Estas plantas aqui identificadas com potencial antiviral contra o vírus da dengue já foram estudadas em outras pesquisas as quais citaram suas ações antimicrobianas e antivirais. Os achados deste screening revelaram plantas promissoras na investigação de um fármaco antiviral contra o vírus da dengue, destacando-se os extratos etanólicos da Acrocomia aculeata (Jacq.) Lood. ex Mart, a macaúba; do Plectranthus amboinicus (Lour.), o malvariço, e do óleo essencial de Vanillosmopsis arborea Baker, o candeeiro. Sendo os dois últimos não tóxicos para células mamíferas e detendo potencial de inibição do vírus DENV em ambas as fases de replicação avaliadas, sugerindo a viabilidade de aprofundar as pesquisas quanto a sua atividadeantiviral e elucidação mais precisa do mecanismo de ação.

Made available in DSpace on 2019-03-30T00:18:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-09-27 / Guillain-Barré Syndrome is a polyneuropathy characterized by weakness, often started in an acute way, which bilaterally affects the limbs in a symmetrical manner, although there are some cases with asymmetry. In two-thirds of the patients, the start of the symptoms is preceded by an infectious disease of the upper respiratory or gastrointestinal tract. In literature, there are reports showing that the dengue virus is the causal agent of this syndrome; that way, this research was to identify a presence of the dengue virus as an etiological agent in participants suspected of Guillain-Barré Syndrome in the State of Ceará. The study was performed in a tertiary hospital in the period from April 2016 to March 2017 by means of an active search for participants with clinical suspicion of Guillain-Barré Syndrome. Data collection took place during the hospital stay of the individuals through structured interviews, analysis of medical charts and collection of serum and/or cerebrospinal fluid samples from the participants. In this study, serological diagnoses for dengue (IgG and IgM) and molecular diagnoses were accomplished by using the RT-PCR technique. Of the 23 individuals with clinical suspicion of Guillain-Barré Syndrome, 22 were analyzed according to the inclusion criteria of the research. Of these 22 participants, 11 were diagnosed with Guillain-Barré Syndrome. After performing an IgM and RT-PCR serological test for dengue, four individuals showed positive IgM for dengue, two of them with diagnosis of Guillain-Barré Syndrome and one of them showed positive RT-PCR. The individual who showed positivity for dengue through the RT-PCR method during hospital stay concomitantly with neurological symptoms evolved with severity of the clinical picture, and then died. Therefore, we conclude that, probably, the dengue virus infection seems to be present in a higher proportion than expected, thereby suggesting a possible neglect of this etiology in the investigation of the cases of Guillain-Barré Syndrome in the State of Ceará. KEYWORDS: Guillain-Barré Syndrome; Dengue fever. / A Síndrome de Guillain-Barré é uma polineuropatia que se caracteriza por fraqueza, geralmente, de início agudo, acometendo bilateralmente os membros de forma simétrica, embora alguns casos apresentem assimetria. Em dois terços dos pacientes, o início dos sintomas é precedido por uma doença infecciosa do trato respiratório superior ou gastrointestinal. Há relatos na literatura do vírus dengue ser o agente causador da síndrome, dessa forma, o objetivo desta pesquisa foi identificar a presença do vírus dengue como agente etiológico em participantes suspeitos da Síndrome de Guillain-Barré no Estado do Ceará. O estudo foi realizado em um hospital terciário, durante os meses de abril de 2016 a março de 2017, através de busca ativa de participantes com suspeita clínica da Síndrome de Guillain-Barré. A coleta de dados ocorreu durante a internação do indivíduo, através de entrevista estruturada, por meio de análise de prontuários e coleta de amostras de soro e/ou líquido cefalorraquidiano dos participantes. Neste estudo, foram realizados diagnóstico sorológicos para dengue (IgG e IgM) e diagnóstico molecular através da técnica de RT-PCR. Dos 23 indivíduos com suspeita clínica da Síndrome de Guillain-Barré, 22 foram analisados conforme os critérios de inclusão da pesquisa. Desses 22 participantes, 11 foram diagnosticados com a Síndrome de Guillain-Barré. Após realizar teste sorológico para dengue IgM e RT-PCR, 4 indivíduos apresentaram IgM positivo para dengue, 2 desses com diagnóstico da Síndrome de Guillain-Barré e um deles apresentou RT-PCR positivo. O indivíduo que apresentou positividade para dengue, através do método RT-PCR, durante o internamento hospitalar concomitante aos sintomas neurológicos, evoluiu com gravidade do quadro e óbito. Portanto, concluímos que, provavelmente a infecção pelo vírus dengue parece estar presente em uma proporção maior que esperada, sugerindo possível negligência desta etiologia na investigação dos casos de Síndrome de Guillain-Barré em nosso Estado. PALAVRAS-CHAVE: Síndrome de Guillain-Barré; Dengue.

Made available in DSpace on 2019-03-29T23:50:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-12-11 / Leptospirosis is a zoonosis of great incidence throughout the world, affecting tropical and temperate regions like Brazil. Its symptoms may vary depending on the particular serotype, the immune status of the host and age. Depending on these factors, the disease may appear from a mild infection to more severe forms. This syndrome may evolve in a way that compromises the renal, lung, and liver system, and may even lead to the patient s death. Therefore, it becomes difficult to differentiate leptospirosis from other diseases of acute febrile syndrome only through clinical and epidemiological criteria. Indeed, dengue is an endemic disease that affects tropical regions. Faced with this problem, we aimed to investigate anti-dengue antibodies in patients with clinical suspicion of leptospirosis. The study used 154 serum samples from patients with suspected leptospirosis, from January to July 2012, listed in the database of the Central Laboratory of Public Health of the State of Ceará (LACENCE). As regards to leptospirosis, we found 42 positive samples, 104 negative and 8 inconclusive. In the 112 samples without leptospirosis diagnosis, emerged the interest of investigating cases of dengue by detecting specific antibodies through the IgM-ELISA technique. Of the patients analyzed for dengue, 82 were positive, 25 were negative and 5 inconclusive. Therefore, the study identified a significant share of confirmed cases of dengue in patients with clinical suspicion of leptospirosis and who were not notified for dengue during the study period. Thus, there is the need for further studies of this nature in order to identify possible sub-reports of dengue or other diseases with undifferentiated febrile illness (UFI). After performing the diagnostic tests for dengue, 30 patients remained without specific laboratory diagnosis, with the possibility of other diseases or the same ones investigated and that did not have a positive result in the test performed. Therefore, the specific diagnosis of each infection becomes important to enable the development of epidemiological surveillance measures and specific treatment for every disease. / A leptospirose é uma zoonose de grande incidência mundial e que acomete regiões de clima tropical e temperado como o Brasil. Seus sintomas podem variar de acordo com o sorovar específico, estado imunológico do hospedeiro e idade. Dependendo desses fatores a doençapode se apresentar como uma infecção leve até formas mais graves. Essa síndrome pode evoluir de modo a comprometer o sistema renal, pulmonar e hepático, podendo, inclusive, ocasionar a morte do paciente. Por conseguinte, torna-se dificil diferencia a leptospirose de outras doenças de síndrome febril aguda somente por meio de criterios clínicos e epidemiológicos. De fato, a dengue é uma doença endêmica e acomete as regiões de clima tropical. Diante dessa problemática, objetivou-se pesquisar anticorpos anti-dengue em pacientes com suspeita clínica de leptospirose. O estudo teve como objeto 154 amostras de soro de pacientes,no período de janeiro a julho de 2012, coletados no banco de dados do Laboratório Central de Saúde Pública do Estado do Ceará (LACEN-CE). Das amostras, verificou-se que, no pertinente à leptospirose, 42 foram positivas, 104 negativas e 8 inconclusivas. Das 112 amostras não diagnosticadas para leptospirose, investigou-se a presença ou não de casos de dengue, por meio da técnicaIgM-ELISA para detecção de anticorpos específicos. Dos pacientes analisados, 82 foram positivos, 25, negativos e 5, inconclusivos. Com efeito, o estudo detectou porcentagem significativa de casos confirmados de dengue em pacientes com suspeita clínica de leptospirose, mas que não foram notificados para dengue no período estudado. Após a realização dos testes diagnósticos para dengue, 30 pacientes permaneceram sem diagnóstico laboratorial especifico. Daí se infere que esses pacientes podem estar acometidos de doenças diferentes das estudadas ou das mesmas investigadas, mas que não apresentaram positividade no teste realizado.Portanto, é fundamental o diagnóstico específico para cada infecção a fim de possibilitar a elaboração de medidas de vigilância epidemiológica e tratamento próprio para cada doença. Pois, do contrário, o paciente poderá receber um tratamento médico-hospitalar inadequado, com risco de complicações de seu estado de saúde, podendo ocasionar perigo de morte ao paciente.

Abstract not available

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: TNF-alpha; Type-I IFN; Dengue virus; differential display method. Includes bibliographical references (p. 64-74).

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2008.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Dengue is the most common mosquito-borne human disease, leading to 2.5 billion people at risk, 50-100 millions infections each year worldwide and among them, 500 000 severe dengue cases (dengue hemorrhagic fever, DHF/ dengue shock syndrome, DSS) plus more than 20 000 deaths. It can be caused by any of four dengue virus serotypes, which are antigenicly distinct and belong to the Flaviviridae family, genus Flavivirus. However, up till now there is no specific drug and vaccine against dengue. Understanding mechanisms developed by dengue virus to exploit host cells during all stages of the replication cycle is a first step towards the rationale design of anti-viral strategies. Very little is known about the late stages, which consist of assembly, budding and secretion of the virus. It is therefore very important to develop tools in order to study the egress of the virus. In this study, I investigated a stable cell line named Hela-prME that expresses serotype 1 dengue virus (DENV-1) prM and E native structural envelope proteins and constitutively produces dengue recombinant subviral particles (RSPs). Biochemical characterization of DENV-1 RSPs has validated that this cell line is a potential tool to study the dengue viral late-stage. Indeed, the maturation process observed with RSPs is similar to the pathway described for real virus (cleavage of prM fragment, homodimerization of E, acquisition of complex sugars). To better understand and depict the dengue virus late-stage secretion, I combined various electron microscopy (EM) techniques e.g. classical transmission electron microscopy (TEM), negative staining, immunogold labeling on cryo-ultrathin sections (Tokuyashu method) and tomography (ET) with such RSPs tool. The EM results obtained illustrate that electron dense particles and tubules labeled by antibodies directed against E and prM proteins were abundantly found in the lumen of endoplasmic reticulum (ER)-related cisternae of HeLa prME cells. Epositive particles were also found in other structures such as Golgi stacks and vesicles nearby as well as in aggregates with electron dense materials inside and surrounded by membrane. These particles are most likely corresponding to DENV-1 RSPs whereas the tubules may be other structures induced by assembly of prM and E proteins. This study has clearly shown that DENV-1 RSPs assemble in the ER and transport through the secretory pathway before being released. This work further validates the use of dengue RSPs and RSPs-producing cells as a model to study viral egress. / published_or_final_version / Pathology / Master / Master of Philosophy

Dengue fever poses a constant serious risk and continues to be a major public health threat in Saudi Arabia, particularly in the city of Jeddah where, since 2006, despite formally introduced Control Strategies, there has been a significant increase in the number of cases. International literature suggests that a range of variables can influence the persistence of dengue, including climatic conditions, the quality of the urban environment, socioeconomic status and control strategies. The overall aims of this research are to understand neighbourhood influences on the pattern of dengue fever across Jeddah City and to make a preliminary determination of the enabling factors for, and barriers to, the effective implementation of the Control Strategies for dengue fever in Jeddah City. A mixed methods research design using quantitative and qualitative data was used. Quantitative data were obtained from administrative sources for dengue fever cases and some of the spatial and temporal variables associated with them, but new variables were created for neighbourhood status and the presence of surface water. Qualitative data are drawn from key informant interviews with 15 people who were, or who had been, working on dengue fever Control Strategies. A qualitative descriptive analysis was based on pre- determined and emergent themes. The spatial and temporal analysis of the variables related to dengue fever in Jeddah City neighbourhoods revealed that neighbourhood status has a direct relationship with dengue fever cases, which is mediated through population density and the presence of non- Saudi immigrants. While there was no relationship with the presence of swamps, seasonal variations in the incidence of dengue were most pronounced in neighbourhoods of low socioeconomic status. The qualitative review of dengue Control Strategies indicated five themes: (1) workforce characteristics and capability, (2) knowledge about dengue fever in Saudi Arabia and Jeddah City, (3) operational strategies for dengue fever control in Jeddah City, (4) the progress of implementation, and (5) overall view of the Government strategies in Jeddah City. This analysis found that the Strategies were well regarded but that aspects of implementation were not always effective. Nevertheless, both quantitative and qualitative results showed the persistence dengue fever problems in Jeddah City neighbourhoods and suggested how cases might be controlled. The number of dengue fever cases in Jeddah City neighbourhoods could continue to rise if the direct and indirect variables affecting dengue fever at the neighbourhood level are not well controlled. Careful attention to the further monitoring of patterns of dengue and specific neighbourhood Control Strategies are recommended, and established Control Strategies need to be implemented as designed. Nonetheless, there is still a need to develop new approaches that can examine and address neighbourhood level issues of dengue fever control.

The aims of this study were to investigate the role of the diversity of dengue virus populations in changing patterns of virus transmission and disease. Prior to the commencement of this study, dengue 2 virus (DENV-2) had been associated most frequently with severe disease, so the study commenced with this serotype. Because it was not possible to quantitate diversity in the entire 11 kb of the viral genome, the study focussed on the envelope (E) gene, because the E protein is the major protein on the surface of the virion and thus might be under strong selective pressure from the host immune system and from the requirement to engage specific receptors on host cells. This study was the first direct quantification of the diversity of dengue virus populations in individual hosts. The nucleotide sequences of more than 70 per cent of the E genes in each virus population differed from the consensus nucleotide sequence for the population. In the course of quantitating genetic diversity in DENV-2 virus populations in patients and in mosquitoes, recombinant DENV-2 and both parental virus populations were detected in a single mosquito. This was the first such report. In 2001, just after the commencement of this study, Myanmar had the largest outbreak of dengue on record. Unlike previous outbreaks, 95 per cent of dengue viruses isolated from patients were of a single serotype, DENV-1. Despite the large number of cases of dengue, the proportion of patients with severe dengue was low. In the light of these observations, the direction of this study changed to focus on DENV-1. Phylogenetic analysis of the E genes of DENV-1 collected before and after the 2001 dengue outbreak suggested that some time before 1998, an early lineage of DENV-1 had become extinct and had been replaced by two new lineages. There was no evidence that these changes were due to selection or to recombination within the E protein genes of the old clade of viruses and the newly introduced viruses. A more detailed analysis was undertaken, of the entire genome of 11 human DENV-1 isolates and of 4 from mosquitoes recovered in Yangon between 1971 and 2002, to determine whether the extinction of the pre-1998 lineage of DENV-1 (clade A) and the appearance of the two new lineages (clades B and C) could have been due to selective pressures acting on genes other than E. Evidence of only weak selection was found in the NS5 gene (at amino acids 127,135 and 669) but the resultant amino acid changes did not distinguish all recent viruses from viruses belonging to the extinct clade. The phylogenetic relationships between individual genes from these viruses and between the open reading frames were similar. No evidence was found of recombination that might have given rise to two new clades of virus with enhanced fitness. Collectively, these data suggested that the extinction of clade A viruses and their replacement by the two new clades, between 1998 and 2000 was a stochastic event in an inter-epidemic period when rates of virus transmission were low. This was the first report of such an extinction of a lineage of DENV-1 and its replacement by new lineages. At about the same time as the 2001 outbreak of DENV-1 infection in Myanmar, an outbreak of DENV-1 began in the Pacific. A comparison of the nucleotide sequences of the E genes of viruses from the Pacific with those of viruses from throughout south-east Asia suggested that the outbreak in the Pacific was due to the introduction of multiple genotypes of DENV-1 from Asia and that some of these DENV-1 could have originated in Myanmar. The principal observations from this study are: - (a) Dengue virus populations in individual hosts are extremely heterogenous and may contain a significant proportion of non-infectious genomes. (b) Intra-serotypic recombination between dengue viruses may be far more common than the literature suggests but it may not be detected because of the almost universal use of consensus nucleotide sequences. (c) Significant changes in dengue virus genotypes that occur at single localities may be due to genetic bottlenecks rather than to selection or to recombination. (d) Dengue viruses can be transported more than 10,000 km to cause outbreaks in non-endemic areas. Key words: Dengue viruses, diversity, recombination, selection, genetic bottleneck