Avaliação da atividade antioxidante do extrato aquoso de Ilex paraguariensis (erva mate) na peroxidação lipídica e na aterosclerose experimental em coelhos

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em FarmáciaO estresse oxidativo está envolvido na etiologia e/ou progressão de várias patologias, inclusive a aterosclerose. O extrato aquoso de I. paraguariensis (erva-mate) é uma bebida rica em compostos antioxidantes amplamente consumida na América do Sul. Neste estudo foi verificado o efeito antioxidante do extrato aquoso de erva-mate contra a lipoperoxidação sérica induzida por CuCl2 in vitro e questionado se o mate poderia reduzir a progressão da aterosclerose in vivo em coelhos submetidos à dieta hipercolesterolêmica. Soro humano foi incubado com CuCl2 na ausência ou presença de diferentes concentrações do extrato de erva-mate. A lipoperoxidação sérica, avaliada pelas substâncias reativas ao ácido tiobarbitúrico (TBARS), foi significativamente inibida pelo extrato aquoso de erva-mate. O potencial antiaterogênico do extrato de erva-mate foi avaliado em coelhos machos Nova Zelândia, com 8 semanas de idade, alimentados com dieta rica em colesterol (1%). Os animais foram divididos em quatro grupos: controle (grupo C), controle-I. paraguariensis (grupo CI), hipercolesterolêmico (grupo HC) e hipercolesterolêmico-I. paraguariensis (grupo HCI). O consumo diário do extrato de erva-mate (grupos CI e HCI) foi de 400 mL. Após 2 meses de tratamento, o colesterol total sérico dos animais hipercolesterolêmicos foi aproximadamente 11 vezes maior do que o dos controles (p0,05). O perfil lipídico dos animais dos grupos hipercolesterolêmicos não foi afetado (p>0,05). O conteúdo de colesterol hepático foi aproximadamente 3 vezes maior nos grupos hipercolesterolêmicos em relação aos coelhos controles (p0.05). Em conclusão, os resultados mostraram que o extrato aquoso de erva-mate inibiu a lipoperoxidação sérica in vitro e pode atenuar a progressão da aterosclerose in vivo em coelhos submetidos à dieta hipercolesterolêmica, sem contudo, diminuir os níveis séricos de colesterol. Aparentemente, o efeito antiaterosclerótico não esteve associado à inibição da lipoperoxidação. No momento, outras explicações não podem ser excluídas, tais como, o efeito do extrato sobre fatores imunológicos, agregação plaquetária ou adesão monocitária

Construção e caracterização de um replicon e clone infeccioso de uma cepa brasileira de dengue sorotipo 3 (BR Den3 290-02)

Orientadora : Dra. Claudia Nunes Duarte dos SantosTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-graduação em Biologia Celular e Molecular. Defesa : 26/05/2009Bibliografia: f.88-105Área de Concentração: Biologia MolecularResumo: Os vírus da dengue pertencem ao gênero Flavivírus, família Flaviviridae e são vírus ARN simples fita com um genoma de ~11kb. O seu genoma é flanqueado por regiões não codificadoras e codifica três proteínas estruturais (C, prM e E) e sete não-estruturais (NS1, NS2A, NS2B, NS3, NS4A, NS4B e NS5) as quais são traduzidas em uma poliproteína e processadas pela protease viral e proteases do hospedeiro. Atualmente a dengue constitui a arbovirose mais comum que afeta os seres humanos, no entanto até o presente não existem tratamento específico ou vacinas disponíveis. As manifestações clínicas abrangem desde estados assintomáticos até a dengue hemorrágica/síndrome do choque por dengue. Os mecanismos envolvidos na patogênese da doença não são bem compreendidos e ainda representam um desafio. O desfecho parece ser o resultado de uma combinação de fatores virais e do hospedeiro. Nesse contexto o uso do sistema subgenômico de replicon e do clone infeccioso representam instrumentos de pesquisa poderosos, pois permitem o estudo de marcadores de virulência podendo contribuir para um melhor entendimento da patogênese da dengue e eventualmente dirigir intervenções tais como terapias antivirais. Eles possibilitam definir o papel de mutações nas proteínas virais, estudar o processo de replicação viral e inclusive testar drogas antivirais. Nosso objetivo foi gerar e caracterizar biologicamente o replicon e o clone infeccioso de um isolado clínico recente de dengue sorotipo 3. A fim de alcançar nosso objetivo primeiramente clonamos diferentes segmentos do genoma viral amplificados por RT-PCR em um vetor pGEM®-T-easy. Todos os clones foram sequenciados e utilizados na montagem do replicon e clone infeccioso em vetores de baixa cópia (pBAC e pAC) contendo o promotor da T7 ARN polimerase e uma sequência de ribozima. A maioria dos procedimentos de clonagem foi realizada na cepa Top10 de E. coli. Após a obtenção de um clone estável do replicon, esse foi utilizado na transcrição in vitro e transfecção de células C6/36 de mosquito. As células foram analisadas por imunofluorescência indireta com o uso de anticorpos policlonais anti-dengue 3 em diferentes tempos (48, 72, 96 e 120h) póstransfecção. Os resultados mostraram que o replicon foi eficientemente transfectado e replicou nas células. Também foi possível observar que proteínas virais foram reconhecidas pelo anticorpo policlonal, no entanto nenhuma partícula viral foi produzida. Infelizmente nós ainda não tivemos sucesso na montagem do clone infeccioso, porém esse trabalho assim como a inserção de marcadores moleculares no sistema de replicon ainda estão em progresso.Abstact: Dengue viruses belong to the genus Flavivirus, family Flaviviridae and are single stranded RNA viruses with a genome of ~11kb. Its genome is flanked by non-coding regions and codes for three structural (C, prM and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) which are translated into a polyprotein and processed by viral and host proteases. Currently dengue is considered the most common arboviral disease afflicting men, however there are no specific treatment or efficient vaccines available yet. The clinical manifestations can range from asymptomatic to dengue hemorrhagic fever/dengue shock syndrome. The mechanisms involved in the disease pathogenesis are not well understood and are still a challenge. The outcome seems to be the result of a combination of viral and host factors. In this context the use of the subgenomic replicon system and the infectious clone system represent powerful research instruments as they allow the study of molecular markers of virulence, contributing to a better understanding of dengue pathogenesis and eventually directing interventions such as antiviral therapy. They can be used: to define the role of mutations in the viral proteins, to study viral replication and even to test antiviral drugs. Our objective was to generate and characterize biologically a replicon and infectious clone from a recent clinical isolate of dengue virus serotype 3. To achieve our objective we first cloned different RT-PCR fragments from the viral genome in the pGEM®-T-easy vector. All clones were sequenced and used to assemble the replicon and the infectious clone in low copy vectors (pBAC and pAC) containing a T7 RNA polymerase promoter and a ribozyme sequence. Most cloning procedures were done using Top10 E. coli strain. After the generation of a stable replicon clone, it was used for in vitro transcription and transfection in C6/36 mosquito cells. Cells were analyzed by indirect immunofluorescence using anti-dengue 3 polyclonal antibodies at different time points (48, 72, 96 and 120h) posttransfection. The results showed that the replicon was efficiently transfected and replicated in the cells. We also observed that the viral proteins could be recognized by the polyclonal antibody, but no virus particle was produced. Unfortunately we have not yet succeeded to assemble the infectious clone, but this as well as the insertion of molecular markers in the replicon system, are works still in progress

Genetic and biological characterization of a densovirus isolate that affects dengue virus infection

Brevidensoviruses have an encapsidated, single-stranded DNA genome that predominantly has a negative polarity. In recent years, they have received particular attention due to their potential role in the biological control of pathogenic arboviruses and to their unnoticed presence in cell cultures as contaminants. In addition, brevidensoviruses may also be useful as viral vectors. This study describes the first genetic and biological characterization of a mosquito densovirus that was isolated in Brazil; moreover, we examined the phylogenetic relationship between this isolate and the other brevidensoviruses. We further demonstrate that this densovirus has the potential to be used to biologically control dengue virus (DENV) infection with in vitro co-infection experiments. The present study provides evidence that this densovirus isolate is a fast-spreading virus that affects cell growth and DENV infection

A glance at subgenomic flavivirus RNAs and microRNAs in flavivirus infections

Submitted by Luciane Willcox ([email protected]) on 2016-10-07T17:57:24Z No. of bitstreams: 1 A glance at subgenomic flavivirus RNAs.pdf: 1116086 bytes, checksum: ad309753dad5211fbf1e8351e5c01fe9 (MD5)Approved for entry into archive by Luciane Willcox ([email protected]) on 2016-10-07T18:07:41Z (GMT) No. of bitstreams: 1 A glance at subgenomic flavivirus RNAs.pdf: 1116086 bytes, checksum: ad309753dad5211fbf1e8351e5c01fe9 (MD5)Made available in DSpace on 2016-10-07T18:07:41Z (GMT). No. of bitstreams: 1 A glance at subgenomic flavivirus RNAs.pdf: 1116086 bytes, checksum: ad309753dad5211fbf1e8351e5c01fe9 (MD5) Previous issue date: 2016-05-28FiocruzFundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.The family Flaviviridae comprises a wide variety of viruses that are distributed worldwide, some of which are associated with high rates of morbidity and mortality. There are neither vaccines nor antivirals for most flavivirus infections, reinforcing the importance of research on different aspects of the viral life cycle. During infection, cytoplasmic accumulation of RNA fragments mainly originating from the 3' UTRs, which have been designated subgenomic flavivirus RNAs (sfRNAs), has been detected. It has been shown that eukaryotic exoribonucleases are involved in viral sfRNA production. Additionally, viral and human small RNAs (sRNAs) have also been found in flavivirus-infected cells, especially microRNAs (miRNAs). miRNAs were first described in eukaryotic cells and in a mature and functional state present as single-stranded 18-24 nt RNA fragments. Their main function is the repression of translation through base pairing with cellular mRNAs, besides other functions, such as mRNA degradation. Canonical miRNA biogenesis involves Drosha and Dicer, however miRNA can also be generated by alternative pathways. In the case of flaviviruses, alternative pathways have been suggested. Both sfRNAs and miRNAs are involved in viral infection and host cell response modulation, representing interesting targets of antiviral strategies. In this review, we focus on the generation and function of viral sfRNAs, sRNAs and miRNAs in West Nile, dengue, Japanese encephalitis, Murray Valley encephalitis and yellow fever infections, as well as their roles in viral replication, translation and cell immune response evasion. We also give an overview regarding other flaviviruses and the generation of cellular miRNAs during infection

Development of a quantitative NS1-capture enzyme-linked immunosorbent assay for early detection of yellow fever virus infection

Submitted by Manoel Barata ([email protected]) on 2018-02-09T12:42:33Z No. of bitstreams: 1 taissaDevelopment.pdf: 2289648 bytes, checksum: e06564731f7c627430412daf18e48364 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2018-05-02T14:59:59Z (GMT) No. of bitstreams: 1 taissaDevelopment.pdf: 2289648 bytes, checksum: e06564731f7c627430412daf18e48364 (MD5)Made available in DSpace on 2018-05-02T14:59:59Z (GMT). No. of bitstreams: 1 taissaDevelopment.pdf: 2289648 bytes, checksum: e06564731f7c627430412daf18e48364 (MD5) Previous issue date: 2017Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Yellow fever is an arboviral disease that causes thousands of deaths every year in Africa and the Americas. However, few commercial diagnostic kits are available. Non-structural protein 1 (NS1) is an early marker of several flavivirus infections and is widely used to diagnose dengue virus (DENV) infection. Nonetheless, little is known about the dynamics of Yellow fever virus (YFV) NS1 expression and secretion, to encourage its use in diagnosis. To tackle this issue, we developed a quantitative NS1-capture ELISA specific for YFV using a monoclonal antibody and recombinant NS1 protein. This test was used to quantify NS1 in mosquito and human cell line cultures infected with vaccine and wild YFV strains. Our results showed that NS1 was detectable in the culture supernatants of both cell lines; however, a higher concentration was maintained as cell-associated rather than secreted into the extracellular milieu. A panel of 73 human samples was used to demonstrate the suitability of YFV NS1 as a diagnostic tool, resulting in 80% sensitivity, 100% specificity, a 100% positive predictive value and a 95.5% negative predictive value compared with RT-PCR. Overall, the developed NS1-capture ELISA showed potential as a promising assay for the detection of early YF infection

Genetic and biological characterization of a densovirus isolate that affects dengue virus infection

Brevidensoviruses have an encapsidated, single-stranded DNA genome that predominantly has a negative polarity. In recent years, they have received particular attention due to their potential role in the biological control of pathogenic arboviruses and to their unnoticed presence in cell cultures as contaminants. In addition, brevidensoviruses may also be useful as viral vectors. This study describes the first genetic and biological characterization of a mosquito densovirus that was isolated in Brazil; moreover, we examined the phylogenetic relationship between this isolate and the other brevidensoviruses. We further demonstrate that this densovirus has the potential to be used to biologically control dengue virus (DENV) infection with in vitro co-infection experiments. The present study provides evidence that this densovirus isolate is a fast-spreading virus that affects cell growth and DENV infection

First report of autochthonous transmission of Zika virus in Brazil

In the early 2015, several cases of patients presenting symptoms of mild fever, rash, conjunctivitis and arthralgia were reported in the northeastern Brazil. Although all patients lived in a dengue endemic area, molecular and serological diagnosis for dengue resulted negative. Chikungunya virus infection was also discarded. Subsequently, Zika virus (ZIKV) was detected by reverse transcription-polymerase chain reaction from the sera of eight patients and the result was confirmed by DNA sequencing. Phylogenetic analysis suggests that the ZIKV identified belongs to the Asian clade. This is the first report of ZIKV infection in Brazil

Dengue virus type 3 isolated from a fatal case with visceral complications induces enhanced proinflammatory responses and apoptosis of human dendritic cells

O artigo encontra-se disponível em acesso aberto no site do Editor.Submitted by Manoel Barata ([email protected]) on 2018-09-18T19:33:15Z No. of bitstreams: 1 JournalVirolj5374.pdf: 1684849 bytes, checksum: ad2c1970d09d920e97d0fac732573033 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2018-09-19T16:45:53Z (GMT) No. of bitstreams: 1 JournalVirolj5374.pdf: 1684849 bytes, checksum: ad2c1970d09d920e97d0fac732573033 (MD5)Made available in DSpace on 2018-09-19T16:45:53Z (GMT). No. of bitstreams: 1 JournalVirolj5374.pdf: 1684849 bytes, checksum: ad2c1970d09d920e97d0fac732573033 (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, BrasilFundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, BrasilFundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil / Universidade de la Republica. Facultad de Ciencias. Sección Virologia, Montevideo, Uruguai.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Instituto de Previsión Social. Laboratorio Central de Salud Publica. Servicio de Pediatria, Asuncion, Paraguay.Instituto de Previsión Social. Laboratorio Central de Salud Publica. Servicio de Pediatria, Asuncion, Paraguay.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil.Universidade Federal de Santa Catarina. Laboratório de Imunofarmacologia e Doenças Infecciosas. Florianópolis, SC, BrazilFundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.A recent (2007 to 2009) dengue outbreak caused by dengue virus (DENV) in Paraguay presented unusual severe clinical outcomes associated with 50% mortality rates. Although it has been reported that inflammatory responses influence the severity of dengue virus infection (T. Pang, M. J. Cardosa, and M. G. Guzman, Immunol. Cell Biol. 85:43-45, 2007), there remains a paucity of information on virus-innate immunity interactions influencing clinical outcome. Using human dendritic cells from a major innate immune cell population as an in vitro model, we have investigated signature cytokine responses as well as infectivity-replicative profiles of DENV clinical isolates from either a nonfatal case of classical dengue fever (strain DENV3/290; isolated in Brazil in 2002) or a fatal case of dengue fever with visceral complications isolated in Paraguay in 2007 (strain DENV3/5532). Strain DENV3/5532 was found to display significantly higher replicative ability than DENV3/290 in monocyte-derived dendritic cells (mdDCs). In addition, compared to DENV3/290 results, mdDCs exposed to DENV3/5532 showed increased production of proinflammatory cytokines associated with higher rates of programmed cell death, as shown by annexin V staining. The observed phenotype was due to viral replication, and tumor necrosis factor alpha (TNF-α) appears to exert a protective effect on virus-induced mdDC apoptosis. These results suggest that the DENV3/5532 strain isolated from the fatal case replicates within human dendritic cells, modulating cell survival and synthesis of inflammatory mediators