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Avalia??o do potencial antiviral da Annona muricata (Graviola) e Spondias mombin (Caj?) contra o v?rus dengue-2 em cultura de c?lulasLima, T?bata Lo?se Cunha 13 March 2015 (has links)
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Previous issue date: 2015-03-13 / A dengue ? uma doen?a de notifica??o compuls?ria e cerca de 50 a 100 milh?es de casos s?o
registrados anualmente. Possui amplo espectro cl?nico e ? transmitida ao homem atrav?s da
picada dos mosquitos do g?nero Aedes, tendo como principal vetor a esp?cie Aedes aegypti. O
agente etiol?gico da doen?a ? o v?rus dengue (DENV) pertencente ao g?nero Flavivirus,
fam?lia Flaviviridae e s?o conhecidos quatro sorotipos antigenicamente distintos (DENV-1,
DENV-2, DENV-3 e DENV-4). Atualmente o tratamento da dengue ? apenas de suporte, feito
atrav?s de intensa hidrata??o. Ainda n?o existe uma vacina comprovadamente eficaz ou
tratamento espec?fico, o estudo de poss?veis antivirais que possam diminuir a viremia no
paciente ? de alt?ssima relev?ncia, uma vez que a carga viral ? um dos fatores associado ao
aparecimento das formas graves da doen?a (febre hemorr?gica da dengue e s?ndrome do
choque da dengue). No presente estudo n?s avaliamos o potencial antiviral de extratos brutos
obtidos a partir das folhas das plantas do Nordeste brasileiro Annona muricata (graviola) e
Spondias mombin (caj?) contra o DENV-2 em cultura de c?lulas C6/36 e Vero. A avalia??o
da a??o dos extratos brutos foi feita por meio da quantifica??o da carga viral atrav?s da PCR
em Tempo Real (qRT-PCR) e pela t?cnica de contagem de unidades formadoras de placa
(PFU). As concentra??es dos extratos de ambas as plantas utilizadas foram: 0,01, 0,1 e
1mg/mL. As culturas de c?lulas infectadas foram submetidas ao tratamento com os extratos
durante os per?odos de 24-168h horas (7 dias). C?lulas Vero tratadas com o extrato da S.
mombin n?o apresentaram redu??o na carga viral. Em contrapartida, quando estas c?lulas
foram tratadas com o extrato da A. muricata, uma hora ap?s infec??o, observou-se uma
redu??o significativa na carga viral nas primeiras horas (24h), quando comparadas com as
c?lulas n?o tratadas utilizadas como controle positivo. Ao serem tratadas em intervalos de 24
horas apresentaram uma redu??o na carga viral nos dias subsequentes (at? o s?timo dia). N?o
foi observada redu??o na carga viral em c?lulas C6/36 tratadas com ambos os extratos. De
acordo com os nossos resultados, o extrato da planta A. muricata possui potencial antiviral
promissor contra a infec??o pelo DENV-2 em cultura de c?lulas Vero. / Dengue is a reportable disease and about 50 to 100 million cases are reported annually. It has
a wide clinical spectrum and is transmitted to humans through the bite of Aedes mosquitos,
the main vector the Aedes aegypti species. The causative agent of disease is dengue virus
(DENV) belonging to the genus Flavivirus, family Flaviviridae and are known four
antigenically distinct serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). Currently the
treatment of dengue is supportive, made by intense hydration. Although there is no proven
effective vaccine or specific treatment, the study of potential antiviral drugs that can reduce
viremia in patients is very high importance, since the viral load is one of the factors associated
with the development of severe forms of the disease (hemorrhagic fever dengue and dengue
shock syndrome). In the present study we evaluated the antiviral potential of crude extracts
obtained from the leaves of plants in Northeastern Brazil Annona muricata (soursop) and
Spondias mombin (caja) against DENV-2 in cultured C6/36 and Vero. The evaluation of the
activity of the crude extracts was performed by the quantification of viral load by RT-PCR
(qRT-PCR) and counting technique of plaque forming units (PFU). The concentrations of
extracts of both plants used were 0.01, 0.1 and 1 mg/mL. The infected cell cultures were
subjected to treatment with the extracts during periods of 24-168h hours (7 days). Vero cells
treated with the S. mombin extract showed no reduction in viral load. In contrast, when these
cells were treated with the extract of A. muricata, one hour after infection, significant
reductions in viral load in the first hour was observed (24 h) when compared to untreated cells
used as positive control. When they are treated at 24 hour intervals showed a reduction in
viral load in subsequent days (until day). There was no reduction in viral load in C6/36 cells
treated with both extracts. According to our results, the plant extract has antiviral A. muricata
promising potential against infection by DENV-2 in Vero cell culture.
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Silver Nanoparticles Inhibit the Binding and Replication of Dengue VirusWilliams, Kelley J. 18 May 2015 (has links)
No description available.
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Safety and Stability of Samples Stored on Filter Paper for Molecular Arbovirus DiagnosisBringeland, Emelie January 2021 (has links)
Expanding urbanization, climate change, and population growth contribute to increased transmission and spread of arthropod-borne viruses (arboviruses), many of which cause severe disease in humans. Pathogenic arboviruses include dengue, Zika, tick-borne encephalitis, and sindbis viruses, which together threaten more than half the global population. Thus, there is a constant need for safe, specific, and sensitive molecular tests to identify early-stage infections for accurate diagnosis and molecular epidemiological data for disease prevention and control. The study tested the biosafety of using FTA™ cards when working with pathogenic arboviruses by conducting an infectivity assay using sindbis virus. Conditions for RNA extraction and storage of arboviruses on FTA were analyzed by measuring viral RNA (vRNA) stability using a SYBR-Green, Pan-Flavi RT-qPCR method composed of degenerate primers able to detect a variety of flaviviruses. Data from a Pan-Flavi RT-qPCR study comprising of 222 clinical blood and serum samples collected from a 2018 dengue virus outbreak in Hanoi (Vietnam) was analyzed to establish applicability of FTA for molecular epidemiology and diagnosis. Results showed that sindbis virus infectivity was inhibited by FTA-adsorption. FTA-adsorbed arboviruses were extracted with the highest yield using Trizol extraction and were preserved at storage at 4-20ºC for up to 30 days. The results showed that clinical blood samples acquired higher yields of vRNA for molecular testing than serum samples and that it may be possible to perform sequencing for genomic analysis. The study suggests that FTA cards may facilitate the storage and transportation of adsorbed arboviruses for downstream molecular epidemiological and diagnostic tests.
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Role of the 17-beta-hydroxysteroid dehydrogenase type 12 (HSD17B12) in hepatitis C and related flaviviruses replication.Mohamed, Bassim 08 1900 (has links)
Dans le monde entier, les infections virales causent des problèmes de santé majeurs et récurrents, engendrant de sérieux problèmes socio-économiques. Notamment, les virus de la famille Flaviviridae qui représentent un fardeau considérable sur la santé mondiale et font partie des domaines prioritaires de la virologie médicale selon le rapport 2016 du ‘Global Virus Network’. Bien que le traitement actuel contre le virus de l’hépatite C (VHC) ait un taux de guérison dépassant 98%, d’autres comme le virus de la dengue (DENV) et le virus zika (ZIKV) n’ont pas encore de traitement spécifique autorisé. En prenant avantage de la grande expertise de notre laboratoire dans l’étude du VHC, nous avons utilisé des données d’une étude de biologie des systèmes visant à identifier l’interactome des différentes protéines virales. Les techniques utilisées ont combiné l’immunoprécipitation des protéines virales suivie de l’identification des protéines interacteurs humaines par spectrométrie de masse. Des études de génomique fonctionnelle par ARN interférent (ARNi) ont permis d’étudier l’effet de la diminution de l’expression des protéines identifiées sur la réplication du VHC. Cette étude a conduit à la découverte de l’interactant spécifique 17-bêta-hydroxystéroïde déshydrogénase de type 12 (HSD17B12 ou DHB12) de la protéine virale Core comme facteur cellulaire requis à la réplication du VHC. HSD17B12 est une enzyme cellulaire dont l’activité catalytique est requise pour l’élongation des acides gras à très longue chaîne (VLCFA) lors de la deuxième des quatre réactions du cycle d’élongation. Dans cette étude, nous avons déterminé que les cycles de réplication du VHC, ZIKV et DENV dépendent de l’expression et de l’activité métabolique du facteur cellulaire HSD17B12. Ainsi, nous avons étudié les effets de l’inhibition de l’expression génique par ARNi et de façon pharmacologique sur la réplication de plusieurs flavivirus dans une approche antivirale à large spectre. Nous avons démontré que le silençage de HSD17B12 diminue significativement la réplication virale, l’expression des protéines virales et la production de particules infectieuses de cellules Huh7.5 infectées par la souche JFH1 du VHC. L'analyse de la localisation cellulaire de HSD17B12 dans des ii cellules infectées suggère une colocalisation avec l'ARN double brin (ARNdb) aux sites de réplication virale, ainsi qu’avec la protéine Core (et les gouttelettes lipidiques) aux des sites d’assemblage du virus. Nous avons également observé que le silençage de HSD17B12 réduit considérablement le nombre et la taille des gouttelettes lipidiques. En accord avec ces données, la diminution de l’expression de HSD17B12 par ARNi réduit significativement l’acide oléique et les espèces lipidiques telles que triglycérides et phosphatidyl-éthanolamine dans l'extrait cellulaire total. Ces travaux suggèrent une contribution de la capacité métabolique de HSD17B12 lors de la réplication du VHC. De même, nous avons démontré que le silençage de HSD17B12 réduit significativement les particules infectieuses de cellules infectées par DENV et ZIKV. Ces études supportent le rôle de HSD17B12 dans l’efficacité des processus de la réplication de l'ARN viral et de l’assemblage de particules virales. De plus, l'inhibiteur spécifique de HSD17B12, INH-12, réduit la réplication du VHC à des concentrations pour lesquelles aucune cytotoxicité notable n'est observée. Le traitement avec 20 μM d'INH-12 réduit jusqu'à 1,000 fois les particules infectieuses produite par des cellules Huh-7.5 infectées par DENV et ZIKV lors de plusieurs cycles de réplication, et bloque complètement l'expression des protéines virales. En conclusion, ces travaux ont conduit à une meilleure compréhension du rôle de HSD17B12 lors de la synthèse de VLCFA et de lipides requise à la réplication du VHC, permettant d’explorer l’inhibition de HSD17B12 et de l’élongation d’acides gras à très longue chaîne comme nouvelle approche thérapeutique pour le traitement à large spectre des infections par les virus de la famille Flaviviridae. / Infections with viruses are major recurrent socio-economical and health problems worldwide. These include infections by viruses of the Flaviviridae family, which present a substantial global health burden and are among the priority areas of medical virology according to the Global Virus Network 2016 report. While the current treatment regimens for hepatitis C virus (HCV) infection have cure rates of more than 98%, other important members of Flaviviridae like dengue virus (DENV) and zika virus (ZIKV) have no specific licensed treatments. By taking advantage of the most-studied HCV, which our lab has developed a vast expertise in the last 20 years, we used proteomics data of an HCV interactome study, combining viral protein immunoprecipitation (IP) coupled to tandem mass spectrometry identification (IP-MS/MS) and functional genomics RNAi screening. The study uncovered the 17-beta-hydroxysteroid dehydrogenase type 12 (HSD17B12, also named DHB12), as a specific host interactor of core that promotes HCV replication. HSD17B12 catalytic activity is involved in the synthesis of very-long-chain fatty acids (VLCFA) upon the second step of the elongation cycle. In this study, taking HCV as a virus model, we elucidated the dependency of HCV, dengue virus (DENV) and zika virus (ZIKV) replication on expression and metabolic capacity of the host factor HSD17B12. We investigated the effects of the inhibition of gene expression by RNAi and of its pharmacological enzymatic inhibition on flavivirus replication in a broad-spectrum antiviral approach. We showed that silencing expression of HSD17B12 decreases viral replication, viral proteins and iv infectious particle production of the JFH1 strain of HCV in Huh7.5 cells. The cellular localization analysis of HSD17B12 showed a co-staining with double-stranded RNA (dsRNA) at viral replication sites and with core protein (and lipid droplets) at virus assembly sites. Furthermore, HSD17B12 gene silencing drastically reduced the number and size of lipid droplets. In association, the reduced expression of HSD17B12 by RNAi decreases oleic acid levels and lipids such as triglycerides (TG) and phosphatidylethanolamine (PE) in whole-cell extract. The data suggested the requirement of the metabolic capacity of HSD17B12 for HCV replication. Similarly, we provide evidence that HSD17B12 silencing significantly reduces DENV and ZIKV infectious particles. The studies support a role of HSD17B12 for effective viral RNA replication and particle assembly processes. Moreover, the specific HSD17B12 inhibitor, INH-12, reduces HCV replication at concentrations for which no appreciable cytotoxicity is observed. The treatment of DENV- and ZIKV-infected Huh- 7.5 cells with 20 μM of INH-12 dramatically reduces production of infectious particles by up to 3-log10 in infection assays, and completely block viral protein expression. In conclusion, these studies extends our understanding of the role of HSD17B12 in VLCFA synthesis required for the replication of HCV, allowing to explore the inhibition of HSD17B12 and elongation of VLCFA as a novel therapeutic approach for the treatment of a broad-spectrum of viruses of the Flaviviridae family.
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