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Oliveira, Anibal Silva de. "Clonagem e expressão das proteínas recombinantes NS1 e NS3 do vírus da dengue tipo 3." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-21062013-141504/.
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A dengue é uma doença infecciosa com grandes taxas de morbimortalidade, causada pelo vírus da dengue (DENV). Segundo a Organização Mundial de Saúde, cerca de 50 a 100 milhões de pessoas são infectadas anualmente em mais de 100 países tropicais e subtropicais de todos os continentes. O espectro clínico da infecção pelo DENV pode incluir formas assintomáticas ou sintomaticas que variam desde uma febre indeterminada e autolimitada, passando pela febre clássica da dengue (FD) até quadros graves denominados febre hemorrágica da dengue/síndrome do choque da dengue (FHD/SCD). Recentemente, ocorreu um dramático aumento do número de casos de FHD/SCD nas Américas, e este aumento coincidiu com a introdução do dengue sorotipo 3, genótipo III. No presente trabalho, objetivou-se a clonagem e a expressão das proteínas NS1 e NS3 do vírus da dengue tipo 3. As proteínas NS1 e NS3 do DENV-3 foram clonadas e expressas com sucesso em sistema procarioto. A amplificação dos genes das proteínas NS1 e NS3 foi realizada por RT-PCR, o qual gerou amplicons de cerca de 1050 e 1850 pb, respectivamente. Em seguida, os genes foram clonados por inserção dos amplicons no vetor plasmidial pCR-XL. Os genes de NS1 e NS3 foram subclonados no vetor de expressão pQE-30 através de sítios de restrição para as enzimas BamHI e HindIII. A expressão proteica foi obtida em sistema procarioto utilizando a cepa BL21(DE3) de E. coli, resultando em proteínas de 45 e 70 kDa as quais foram confirmadas por análises em Western blot utilizando como anticorpo primário fluido ascítico imune de camundongos e soro de pacientes com dengue. Estas proteínas virais podem ser utilizadas para estudos relacionados à patogênese, replicação e mecanismos de escape do sistema imune do DENV, além disso, podem ser potencias antígenos em métodos de diagnóstico.Dengue is an infectious disease with high morbidity and mortality rates caused by dengue virus (DENV). According to the World Health Organization, about 50 to 100 million people are infected annually in more than 100 tropical and subtropical countries from all continents. The clinical spectrum of DENV infection can includes asymptomatic or symptomatic forms ranging from undetermined and self-limited fever, through dengue fever (DF) to severe disease called dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Recently, there has been a dramatic increase in the number of cases of DHF/DSS in the Americas, and this increase coincided with the introduction of dengue virus type 3 (DENV-3), genotype III. The present study aimed to clone and express NS1 and NS3 proteins of DENV-3. The NS1 and NS3 proteins of DENV-3 was successfully cloned and expressed in a prokaryotic system. Amplification of NS1 and NS3 genes was carried out by RT-PCR, which yielded amplicons of approximately 1050 and 1850 bp, respectively. Then, the genes were cloned by inserting the amplicons into the plasmid vector pCR-XL. NS1 and NS3 genes were subcloned into the expression vector pQE-30 through the restriction sites for BamHI and HindIII enzymes. The protein expression was obtained in a prokaryotic system using the strain BL21 (DE3) of E. coli, resulting in 45 and 70 kDa proteins, which were confirmed by Western blot analysis using immune mouse ascitic fluid and serum of patients with dengue as primary antibody. These viral proteins can be used to study the pathogenesis, mechanisms of replication and immune escape of DENV, moreover, can be potential antigens in diagnostic methods.
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Marozin, Sabrina. "Interferon Escape of Respiratory Syncytial Virus: Functional Analysis of Nonstructural Proteins NS1 and NS2." Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-54265.
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Zwart, Lizahn. "Investigating two AHSV non-structural proteins : tubule-forming protein NS1 and novel protein NS4." Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/62198.
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African horse sickness is an equid disease caused by African horse sickness virus (AHSV). AHSV produces seven structural proteins that form the virion and four non-structural proteins with various roles during replication. The first part of this study investigated the intracellular distribution and co-localisations of NS1 with other AHSV proteins to facilitate its eventual functional characterisation. Confocal microscopy revealed that NS1 formed small cytoplasmic foci early after infection that gradually converged into large fluorescent NS1 tubule bundles. Tubule bundles were more organised in AHSV-infected cells than in cells expressing NS1 alone, suggesting that tubule bundle formation requires the presence of other AHSV proteins or regulation of NS1 expression rates. NS1 occasionally co-localised with VP7 crystalline structures, independently of other AHSV proteins. However, when NS1-eGFP, a modified NS1 protein that contains enhanced green fluorescent protein (eGFP) near the C-terminus, was co-expressed with VP7, co-localisation between these proteins occurred in most co-infected cells. It is not clear how the addition of eGFP to NS1 induces this co-localisation and further investigation will be required to determine the function of NS1 during viral replication.
The second part of the study focused on characterising the novel non-structural AHSV protein NS4. The NS4 open reading frame (ORF) occurs on segment 9, overlapping the VP6 ORF in a different reading frame. In silico analysis of segment 9 nucleotide and NS4 predicted amino acid sequences revealed a large amount of variation between serotypes, and two main types of NS4 were identified based on these analyses. These proteins differed in length and amino acid sequence and were named NS4-I and NS4-II. Immunoblotting confirmed that AHSV NS4 is translated in AHSV infected insect and mammalian cells, and also in Sf9 insect cells infected with recombinant baculoviruses that overexpress the genome segment 9 proteins, VP6 and NS4. Confocal microscopy showed that NS4 localised to both the cytoplasm and nucleus, but not the nucleolus, in AHSV-infected cells and recombinant baculovirus infected Sf9 cells. Nucleic acid protection assays using bacterially expressed purified NS4 showed that both types of NS4 bind dsDNA, but not dsRNA. This was the first study to focus on AHSV NS4. Future work will focus on determining the role of non-structural proteins in viral pathogenesis, and will involve the use of a reverse genetics system for AHSV.Dissertation (MSc)--University of Pretoria, 2013.
Genetics
MSc
Unrestricted
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Costa, Simone Morais da. "Vacinas de DNA contra o vírus da dengue utilizando como antígenos as proteínas NS1 e NS3." reponame:Repositório Institucional da FIOCRUZ, 2008. https://www.arca.fiocruz.br/handle/icict/12179.
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Previous issue date: 2015-04-14Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil
O vírus da dengue (DENV) consiste de quatro sorotipos antigenicamente relacionados: DENV-1, DENV-2, DENV-3 e DENV-4. Apesar dos diversos esforços para o desenvolvimento de uma vacina contra dengue, ainda não há nenhuma comercialmente disponível. As proteínas não estruturais 1 e 3 (NS1 e NS3) são indicadas como antígenos promissores para o desenvolvimento de uma vacina contra DENV. Segundo alguns estudos, a proteína NS1 é capaz de induzir uma resposta protetora de anticorpos com atividade de fixação do complemento. A proteína NS3, que realiza reações enzimáticas essenciais para a replicação viral, parece ser imunogênica, contendo um predomínio de epítopos para linfócitos T CD4+ e CD8+. No presente trabalho nós avaliamos o potencial de vacinas de DNA baseadas nas proteínas NS1 e NS3 de DENV-2. Foram construídos cinco plasmídeos, pcTPANS3, pcTPANS3H, pcTPANS3P, pcTPANS3N e pcTPANS3C, contendo a seqüência que codifica o peptídeo sinal do ativador de plasminogênio de tecido humano (t-PA) fusionado ao gene NS3 inteiro ou partes destes. Todos estes plasmídeos mediaram a expressão das proteínas recombinantes in vitro em células eucarióticas Camundongos foram inoculados com estes plasmídeos e desafiados com DENV-2 por via intracerebral (i.c.). Nenhuma destas construções induziu níveis satisfatórios de proteção. Além dos plasmídeos com NS3, foram construídas quatro vacinas de DNA baseadas no gene NS1: 1 - pcENS1, que codifica a região C-terminal da proteína E fusionada à NS1, 2 - pcENS1ANC, similar ao pcENS1 com a adição da porção N-terminal da NS2A (ANC), 3 - pcTPANS1, que codifica o peptídeo sinal t-PA fusionado à NS1 e 4 - pcTPANS1ANC, semelhante ao pcTPANS1 com a adição da seqüência ANC. A proteína NS1 recombinante foi detectada nos extratos celulares e sobrenadante das culturas de células BHK transfectadas com pcTPANS1, pcENS1 e pcENS1ANC. Tais resultados indicam que as seqüências sinais t-PA e E direcionaram a NS1 para secreção. A proteína NS1 também foi observada associada à membrana plasmática de células transfectadas com pcENS1ANC, demonstrando a importância da seqüência ANC para o seu ancoramento. Todos os camundongos imunizados com pcTPANS1 ou pcENS1 produziram altos níveis de anticorpos, direcionados principalmente para epítopos conformacionais da NS1, enquanto que somente metade dos animais inoculados com pcENS1ANC apresentaram níveis detectáveis de anticorpos A resposta de anticorpos se mostrou duradoura (até 56 semanas após a primeira dose das vacinas), e os animais apresentaram uma rápida resposta secundária após um reforço de DNA. Camundongos imunizados com os plasmídeos pcTPANS1 e pcENS1 se mostraram protegidos contra desafios com DENV-2 por via i.c., sendo o pcTPANS1 levemente mais protetor. Estes dois plasmídeos ativaram a produção de diferentes subclasses de IgG específicas contra NS1. Não foi observada proteção interespecífica quando camundongos imunizados com pcTPANS1 foram desafiados por via i.c. com DENV-1. Os animais imunizados com o pcTPANS1 foram desafiados com DENV-2 por via intraperitoneal e também se mostraram protegidos. Neste modelo de desafio, foi observada uma diminuição dos efeitos histopatológicos do vírus no fígado dos animais vacinados. Resultados preliminares sugerem à lise de células infectadas com DENV-2, dependente do complemento, na presença dos anticorpos direcionados contra NS1
Dengue virus (DENV) consists of four antigenically related serotypes: DENV-1, DENV-2, DENV-3 and DENV-4. Although considerable research has been conducted towards the development of a DENV vaccine, no vaccine is yet commercially available. The non-structural proteins 1 and 3 (NS1 and NS3) have been identified as promising antigens for the development of vaccines against DENV. According to some reports, NS1 can elicit a protective antibody response with complement-fixing activities. NS3, a protein that carries out enzymatic reactions essential for viral replication, appears to be immunogenic, presenting a preponderance of the CD4+ and CD8+ T cell epitopes. In the present work we investigate the potential of DNA vaccines based on the DENV-2 NS1 and NS3 proteins. We constructed five recombinant plasmids, pcTPANS3, pcTPANS3H, pcTPANS3P, pcTPANS3N and pcTPANS3C, which contain the sequence that codes the signal peptide derived from the human tissue plasminogen activator (t-PA) fused to the full or partial length of the DENV-2 NS3 gene. Results indicated that these plasmids promoted the expression of recombinant proteins in eukaryotic cells. Mice were inoculated with these plasmids and challenged by the intracerebral (i.c.) route with DENV-2. None of these constructs induced acceptable protection. Moreover, we constructed four DNA vaccines based on the DENV-2 NS1 gene: 1 - pcENS1, coding the C-terminal of the E protein fused to NS1, 2 - pcENS1ANC, similar to pcENS1 with the addition of the N-terminal of NS2A (ANC), 3 - pcTPANS1, coding the t-PA signal sequence fused to NS1 and 4 - pcTPANS1ANC, similar to pcTPANS1 with the addition of the ANC sequence. The recombinant NS1 protein was detected in cell extracts and culture supernatants from pcTPANS1-, pcENS1- and pcENS1ANC-transfected BHK cells. Such results indicated that the E and t-PA sequences targeted NS1 to secretion. NS1 was also observed in association with plasma membrane of pcENS1ANC-transfected cells, which demonstrated the importance of the ANC sequence for cell anchoring. High levels of antibodies, mainly recognizing surface-exposed conformational epitopes of NS1, were induced in all mice immunized with pcTPANS1 and pcENS1, while only half of pcENS1ANC-inoculated animals presented detectable antibody levels. Long-term antibody response was observed in pcTPANS1 and pcENS1 immunized animals (56 weeks after the first vaccine inoculation) and there was a rapid secondary response after a DNA booster. Protection was elicited in pcTPANS1- and pcENS1-immunized mice challenged with DENV- 2 by the i.c. route and the pcTPANS1 seemed to generate a slightly higher protection. Moreover, these two plasmids induced different NS1-specific IgG subclasses. No protection was displayed when pcTPANS1-immunized animals were i.c. challenged with DENV-1. Animals inoculated with pcTPANS1 were also protected when they were challenged with DENV-2 by the intraperitoneal route. Liver tissue from vaccinated animals presented a remarkable decrease of hepatic damages in this challenge mouse model. Preliminary results suggested the complement-mediated lyses of DENV-2 infected cells in the presence of the NS1-specific antibody.
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Figueiredo, Alessandra. "Imunossensores potenciométricos para a detecção da proteína NS1 do vírus da dengue." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-13082013-164540/.
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A dengue é uma doença negligenciada que carece de métodos diagnósticos rápidos nos primeiros dias de infecção. São quatro sorotipos diferentes, cuja monitoração é essencial para o controle da ocorrência de casos graves como a dengue hemorrágica. É urgente o desenvolvimento e disponibilização de um dispositivo capaz de suprir essa demanda, de modo que propomos a utilização de imunossensores potenciométricos, devido a facilidade de miniaturização e produção dos dispositivos e seu baixo custo, além da possibilidade de detecção direta (sem marcadores) e simplicidade de manuseio. Dispositivos sensores de pH, como o transistor de efeito de campo de porta estendida e separada (SEGFET) e amplificadores de instrumentação (AI) podem ser utilizados como transdutores de sinal para a reação antígeno-anticorpo, a partir da utilização de materiais não nernstianos, como o ouro, como plataforma sensível. A proteína NS1 do vírus da dengue é um excelente marcador da infecção, pois é secretada em altas concentrações pelo vírus no sangue de pessoas infectadas logo nos primeiros dias, de modo que o sistema preza pelo diagnóstico precoce da doença. Sua detecção é realizada através da imobilização de anticorpos anti-proteína NS1 na plataforma sensível, permitindo sua quantificação através da detecção da alteração local de carga. O eletrodo foi caracterizado por diversas técnicas de microscopia, entre elas de varredura, confocal e de força atômica, além da utilização de espectroscopia de impedância eletroquímica, permitindo um amplo conhecimento da superfície da membrana sensível. Os imunossensores desenvolvidos apresentaram alta sensibilidade, com capacidade de detecção da ordem de ng.mL-1. Na região linear da curva analítica, foram obtidos sensibilidade correspondente a (15.7 ± 4.4) .10-4 μA.μg.mL-1 para o SEGFET e (3.2 ± 0.3) mV.μg.mL-1 para o AI, sendo que este último apresenta uma maior estabilidade de sinal e dispensa a utilização de uma fonte variável de tensão, reduzindo o custo no desenvolvimento de um dispositivo diagnóstico comercial. Estes resultados levaram a um pedido de patente e o prosseguimento do projeto através da miniaturização do sistema e detecção em amostras reais.Dengue is a neglected disease that lacks fast diagnosis methods in the first days of infection. There are four different serotypes, which monitoring is essential to the occurrence control of severe cases as dengue hemorrhagic fever. The development of a device capable of fulfilling this demand is urgent, so we propose the use of potentiometric immunosensors, since its ease of miniaturization, mass production, low cost and the possibility of direct detection (label-free). pH sensor devices, as the separated extended gate field effect transistors (SEGFET) and instrumentation amplifiers (AI) can be applied as transducers to the antibody-antigen reaction by using non-nernstian materials such as gold as sensitive membrane. The non-structural 1 (NS1) protein is an excellent marker of infection, since its secreted in high concentration in the blood of infected people by the dengue virus in the first days, prioritizing early diagnosis. Its detection is made by immobilization of anti-NS1 protein antibodies, allowing its quantification by local charge changes. The electrode was characterized by many microscopy methods, including scanning electron, confocal and atomic force, besides electrochemistry impedance spectroscopy, providing a wide knowledge of the membrane surface. The developed immunosensors showed high sensitivity with detection capacity in the order of ng.mL-1. In the linear range of the analytic curve, were obtained sensitivities of (15.7 ± 4.4) .10-4 μA.μg.mL-1 for the SEGFET and (3.2 ± 0.3) mV.μg.mL-1 for the AI, whereas the latter has high signal stability sparring the use of a variable voltage source, minimizing the costs in the development of a commercial diagnostic device. These results led to a patent and the project continues by working in miniaturizing and real samples detection.
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Wolff, Michael. "Identifizierung und Charakterisierung von Interaktionen der Nichtstrukturproteine NS1 und NS2 des Respiratorischen Synzytialvirus mit Proteinen der Wirtszelle." Diss., lmu, 2004. http://nbn-resolving.de/urn:nbn:de:bvb:19-24259.
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Evans, Johanna. "Characterisation of the NS1 and the NS2 non-structural protein genes of human respiratory syncytial virus (HRSV)." Thesis, University of Warwick, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283482.
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SHA, Tim Wai. "Functional studies of Influenza A virus NS1 protein." Kyoto University, 2020. http://hdl.handle.net/2433/259078.
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Bossert, Birgit. "Of Mice and Men and Cattle: Functions of the Pneumovirus Nonstructural Proteins NS1 and NS2 in Interferon Escape." Diss., lmu, 2003. http://nbn-resolving.de/urn:nbn:de:bvb:19-7733.
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Smith, Matthew. "Consequences of variation in the influenza virus NS1 protein." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/6969.
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The NS1 protein is a major virulence factor of influenza virus. Although the protein is encoded by all natural influenza viruses, its sequence shows considerable variation suggesting that it interacts intimately with the host. Indeed this multifunctional protein has been described to bind a plethora of cellular factors. The influenza A NS1 protein from A/PR/8/34 was previously shown to enhance translation of a host expressed gene. This is likely a consequence of its ability to counter basally expressed host antiviral strategies that are activated within the cell upon transfection and transient expression of exogenous genes. This work evaluated a panel of NS1 proteins derived from different strains and subtypes of influenza, for their capacity to enhance translation. Although it is possible that all natural NS1 proteins have the capacity for this function, it was found to be often obscured by a dominant inhibitory function that was mapped to the C terminus of the protein. Only NS1 proteins that lack this second function illustrated translational enhancement without prior mutation. Modification of the C terminal domain of NS1 was able to abrogate binding to the CPSF host factor responsible for the maturation of host genes transcribed by polymerase II. This then revealed the potential of most if not all NS1 proteins to act as translational enhancers. A series of NS1 mutants were engineered to test the proposed mechanism of translational enhancement, and this work confirmed that enhancement required NS1 to be present in the cytoplasm of the transfected cell, and retain an intact dsRNA binding site. The intriguing finding that not all natural NS1 proteins bind to the CPSF host factor was investigated to ask whether CPSF interaction sometimes carried a cost to viral fitness. The hypothesis was that some NS1 proteins adopt this global mechanism for the control of interferon to compensate for high levels of PAMP produced by infection more active viral polymerase. The observations deduced from this work lead to the suggestion that NS1 participates in the regulation of interferon at the level of the polymerase complex by modulating the viral polymerase activity, in addition to its previously characterized function to counter the PRR RIG-I, and disruption of CPSF function. Combining the approaches established during this body of work, it was established that the behaviour of the NS1 protein of the newly emerged swine origin H1N1 2009 pandemic virus were unusual. Interestingly this protein was able to strongly enhance translation despite being predominantly localized to the nucleus. Importantly a reverse genetic approach demonstrated that the levels of interferon induced during infection by the pandemic H1N1 strain could be being under represented, and this may explain discrepancies in the literature between different models of pathogenicity of the pandemic virus.
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Silva, Mizia Maria Saboia da. "Contribuição de nanomateriais no desenvolvimento de biossensores para diagnóstico da infecção aguda do dengue." Universidade Federal de Pernambuco, 2014. https://repositorio.ufpe.br/handle/123456789/12330.
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CAPES
O diagnóstico laboratorial da Dengue é fundamental para determinar os cuidados clínicos com o paciente, apoiar os programas de vigilância epidemiológica, pesquisar formulação de vacinas e também para a detecção precoce de uma possível epidemia. A proteína não estrutural 1 (NS1) do vírus Dengue é um marcador utilizado durante a fase aguda da enfermidade e tem sido proposto para o diagnóstico da doença. Atualmente, para diagnóstico da NS1 são usados os ensaios imunoenzimáticos e testes imunocromatográficos. Os imunossensores são dispositivos bioanalíticos que convertem a resposta da interação antígeno-anticorpo em um sinal elétrico, passível de quantificação. Recentemente, a contribuição de nanomateriais a estes dispositivos tem possibilitado aumento na reprodutibilidade e alcance de baixos limites de detecção tornando os imunossensores ferramentas promissoras para diagnóstico clínico. Nesta tese foram desenvolvidos dois imunosensores a base de nanomaterias para a detecção NS1, um marcador importante na infeção aguda da dengue. O primeiro imunossensor, constituído por um eletrodo de carbono vítreo (ECV), foi baseado no uso nanotubos de carbono de parede múltiplas carboxilados (NTCPMs-COOH) recoberto por um filme formado por deposição do Hidrocloreto de Polialilamina (PAH). Anticorpos anti-NS1 foram imobilizados de modo orientado via grupos aminos do PAH. De acordo com os resultados, o imunossensor desenvolvido exibiu uma faixa linear variando entre 0,1 μg mL-1 e 2,5 μg mL-1 de NS1, faixa clínica para diagnóstico precoce na fase aguda da doença. Uma boa correlação foi encontrada entre a concentração de NS1 e a mudança da corrente, mostrando um bom limite de detecção (0.035 μg mL-1). O segundo imunossensor foi baseado em eletrodos impressos usando a transdução eletroquímica, visando o desenvolvimento de testes point-of-care. Os eletrodos impressos foram fabricados com um composto de tinta de carbono-Tiofeno seguidos por um filme de nanopartículas de ouro revestidas com proteína A (AuNP-PtnA) que orientaram a imobilização dos anticorpos anti-NS1. Um imunoensaio direto foi realizado, no qual a captura específica da NS1 foi avaliada através das reações da uma sonda redox com a superfície do eletrodo. De acordo com os resultados, foi observado que o uso do tiofeno na tinta de carbono aumentou significativamente a sensibilidade do eletrodo em 70% em relação ao eletrodo sem modificação. A curva de calibração do sensor mostrou uma faixa de resposta linear entre 0.05 – 0.6 μg mL-1 de NS1 e um limite de detecção de 0.015 μg mL-1. Os imunossensores propostos apresentam-se como tecnologias inovadoras ainda não disponíveis no mercado de sensores. Ambos imunossesores apresentaram o uso combinado de tecnologias eletroquímicas com nanomateriais que contribuiu para uniformização da plataforma sensora, melhorara da estabilidade e reprodutibilidade dos eletrodos.
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Porto, Vanessa Torales. "Acurácia do teste NS1 para dengue no contexto epidemiológico brasileiro." reponame:Repositório Institucional da UnB, 2014. http://repositorio.unb.br/handle/10482/16765.
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Introdução: A dengue é um dos principais problemas de saúde pública no mundo, a forma mais grave da doença é a dengue hemorrágica, fatal se não tratada. No mundo, ocorre em mais de 100 países e territórios – 2,5 bilhões de pessoas estão sob o risco de contraí-la, e anualmente a taxa de incidência atinge 50 milhões de casos (Tauil, 2002). A dengue é uma enfermidade causada por um arbovírus da família Flaviviridae, gênero Flavivírus, que inclui quatro tipos imunológicos: DENV-1, DENV-2, DENV-3 e DENV-4. Um importante alvo dos anticorpos para DENV é a proteína NS1, uma glicoproteína conservada que parece ser essencial para a replicação do vírus. Recentemente, os testes para a detecção do antígeno NS1 no soro humano foram evoluindo. O antígeno NS1 é encontrado juntamente com endotélio, livre ou solúvel no soro de pacientes, a partir de um dia antes do início dos sintomas e pode ser detectado, pelo menos, até cinco dias após o início dos sintomas, o que permite um diagnóstico precoce da doença. Objetivo: Avaliar a acurácia diagnóstica para a detecção do antígeno NS1 Dengue pelo formato ELISA, quando utilizada por laboratórios de saúde pública. Métodos: estudo analítico retrospectivo dos exames de dengue oriundos das unidades sentinelas de dengue e laboratórios de referência nacional e estaduais no período de 2009 a 2010. Foram calculados os valores de sensibilidade, especificidade, Valor preditivo positivo e valor preditivo negativo. Resultados: A sensibilidade média do kit encontrada nos seis estados envolvidos no estudo foi de 94,5% e a especificidade média foi de 61,20%. O valor preditivo positivo de 81,48% no ano de 2009 e em 2010 o valor caiu para 66,17%, e o valor preditivo negativo no ano de 2009 foi de 89,91% e no ano de 2010 o valor aumentou para 94,43%. Conclusão: Considerando a sensibilidade de 94,50% encontrada podemos considerar que o teste é sensível, e raramente deixará de encontrar pessoas com a doença. Em relação a especificidade de 61,20%,considera-se um valor abaixo do esperado, podendo ocasionar resultados falsos negativos. O kit é sensível, porém pouco específico. __________________________________________________________________________________ ABSTRACT
Introduction: Dengue fever is one of the main public health issues in the world. In its most severe form, dengue hemorrhagic fever, it causes gastrointestinal hemorrhaging which, if untreated, can lead to death. It has been detected in more than 100 countries and territories worldwide -approximately 2.5 billion people are at risk of contracting dengue fever and incidence rate is of 50 million cases per year (Tauil, 2002). Dengue fever is caused by an arbovirus of the family Flaviviridae and genus Flavivirus and includes four serotypes: DENV-1, DENV-2, DENV-3 e DENV-4. An important target of antibodies against DENV is the protein NS1, a conserved glycoprotein that appears to be essential for viral replication. Recently, tests for the detection of NS1 antigen in human serum have evolved. This antigen is found in the endothelium, in a free form or soluble in serum of infected patients after one day before symptoms begin, which allows for early diagnosis of the disease. Objective: To evaluate the diagnostic accuracy in the detection of the NS1 antigen in an ELISA format in public health laboratories. Methods: A descriptive retrospective cohort study of dengue laboratory results from sentinel, state and national reference laboratories in 2009 and 2010. Sensitivity, specificity, positive and negative predictive values were determined. Results: The mean sensitivity of the Elisa kit in the six states involved in this study was 94.5% and the mean specificity was 61.2%. Positive predictive value was 81.48% in 2009 and decreased to 66.17% in 2010, whereas the negative predictive value was 89.91% and increased to 94.43% in the years 2009 and 2010, respectively. Conclusion: Considering the mean sensitivity of 94.50%, it can be concluded that the test will rarely fail at detecting contaminated patients. The mean specificity was lower than expected and it can be concluded that mistakes can occur, particularly in assigning false negatives. The kit is sensitive, however non-specific.
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Barberio, Gabriel Salles. "Identificação do biomarcador NS1 na saliva como diagnóstico da dengue." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/25/25145/tde-07112013-105903/.
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Muitas ferramentas de diagnóstico da dengue tornaram-se disponíveis, porém requerem coleta de sangue como amostra para análise. Um método não invasivo de confirmar a infecção por dengue seria de importância considerável para os estudos clínicos e epidemiológicos. Testes de saliva podem encorajar os pacientes a serem mais receptivos para o diagnóstico da dengue. Possíveis problemas com o uso de sangue incluem a exigência de consentimento e a cooperação do paciente. Em muitos casos, flebotomia em indivíduos com fobia de agulhas, portadores de deficiências e discrasias sanguíneas e, especialmente em bebês e crianças, ou ainda devido a razões sociais, religiosas, à necessidade de um enfermeiro treinado e à necessidade de se separar o soro do plasma antes do teste. Objetivo deste trabalho foi identificar RNA viral em amostras de saliva de pacientes infectados com dengue durante o período febril; avaliar o teste rápido para identificar NS1 em amostra de saliva; identificar a presença de NS1 em amostras de saliva por meio do teste ELISA. Foram coletas amostras de saliva de pacientes durante o período febril com e sem dengue. Todos diagnósticos foram confirmados por exame de sangue para IgM/IgG. Foi feito o teste da Reação em Cadeia da Polimerase em Tempo Real em 10 amostras de pacientes com dengue. Em 44 amostras foram testados as tiras de diagnóstico rápido da dengue e ao teste ELISA NS1, para a detecção da proteína NS1. Esse estudo ressaltou a importância da busca pelo diagnóstico laboratorial rápido, prático e financeiramente acessível de doenças febris agudas com sintomas inespecíficos, principalmente em áreas de ocorrência de dengue. Nesse estudo foi encontrada alta especificidade (94%) e média sensibilidade (73%) nos resultados da identificação da proteína NS1 nas amostras de saliva em estágios precoces da infecção por dengue. Esse método, se aprimorado para saliva, poderá ter resultados ainda melhores, por isso mais estudos são necessários.This study brings a literature review about the current dengue status worldwide and in Brazil followed by a systematic review that highlights the dengue diagnosis through saliva. A prospective study was also made, which evaluated the dengue virus infection diagnosis accuracy through the NS1 antigen detection in saliva samples using ELISA assays. The NS1-ELISA results in saliva were compared to the IgM-ELISA and IgG-ELISA serology. A total of 44 saliva samples were obtained from November 2012 to February 2013. The results showed that de NS1-ELISA presented a sensibility of 0.73, specificity of 0.94, Positive Predictive Value of 0.95, Negative Predictive Value of 0.70, Positive Likelihood Ratio of 13.15, Negative Likelihood Ratio of 0.28. Having these findings in mind, it is possible to suggest that the NS1 detection in saliva may be an important diagnostic tool in special cases, such as people that fear needles, with blood dyscrasias, babies, children, as well as to quickly monitor epidemics and their dissemination.
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Bletchly, Cheryl. "Antigenic and structural analysis of the NS1 glycoprotein of dengue virus /." St. Lucia, Qld, 2002. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16420.pdf.
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Fernandes, Pereira Carina. "The influenza A virus NS1 protein and viral mRNA nuclear export." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275570.
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Influenza A virus (IAV) replication and transcription occur in the host cell nucleus; a feature which means both the viral genome (vRNA) and mRNA must be exported from the nucleus to the cytoplasm. The mechanism by which vRNA nuclear export is achieved has been well characterised, but how viral mRNAs are exported is poorly understood. The cellular NXF1-dependent mRNA export pathway has been shown to be involved in the export of some viral mRNAs, but how they are recruited to this pathway is unknown. Prior work from our laboratory showed that segment 7 mRNA was inefficiently exported to the cytoplasm in a sub-viral ‘minireplicon’ system, providing the first indication that there were viral requirements for IAV mRNA nuclear export. Further addition of individual viral polypeptides was tested and the effect on segment 7 mRNA export was analysed by fluorescent in situ hybridization (FISH) and confocal microscopy. This identified the NS1 protein as the viral factor required for efficient segment 7 nuclear export. Mutational studies on NS1 were carried out to unveil the mechanistic role of this protein in viral mRNA nuclear export, by plasmid transfection as well as in the context of recombinant viruses. These approaches indicated that both functional domains of NS1 were necessary to preserve the mRNA export function. Furthermore, these mutant proteins were used to examine the association between NS1 and the NXF1-dependent pathway in the context of mRNA nuclear export. Protein-protein and protein-RNA binding assays indicated that interactions between NXF1 and NS1, and NXF1 and segment 7 mRNA were necessary, but not sufficient to promote segment 7 viral mRNA export. Lastly, the role of NS1 protein in the nuclear export of viral mRNAs from other genome segments was studied. The intracellular localisation of most viral mRNAs was not affected by the absence of NS1 or the presence of an export-incompetent NS1 mutant protein. However, segment 4 mRNA exhibited a similar phenotype to segment 7 mRNA in showing a dependence on NS1 for efficient nuclear export. Overall, the results presented in this dissertation suggest that NS1 acts as an adaptor protein between the viral RNA synthesis machinery and cellular export pathway. This provides deeper insights for the characterization of a recently identified function of the IAV NS1 protein, of being required for the efficient nuclear export of mRNA from “late” kinetic class viral genes.
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Li, Yishan. "Mapping IFN resistance in the NS1 gene of influenza A virus." Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/27266.
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Inhibition of the interferon-mediated antiviral response is a major determinant of virulence in influenza A virus. The NS1 protein of influenza A virus has been identified as the IFN antagonist. However, the specific mechanisms for IFN antagonism are not known. NS1 binds both the PKR as well as single and double stranded RNA to inhibit activation of PKR. Adaptation of human influenza virus to the mouse lung may likewise involve mutations that affect IFN antagonism. The prototype human influenza A virus A/HK/1/68 (H3N2 subtype) was utilized for this project. Six mouse-adapted variants possessing four different point mutations on NS gene were obtained from independently derived mouse-adapted variants. The mutations were located in the RNA binding domain and several sites in an 8 amino acid region from as 98 to 106. Mutant HKMA20c was of interest because it possessed a mutation in common with highly pathogenic avian influenza virus H5N1 (Leucine on AA 103 on NS1 protein). Base on these phenomena, a hypothesis was brought out that this region may encode a site of interaction with a host or viral factor and that mutation(s) on it may enhance the ability of NS1 protein to function as an IFN antagonist. The approach was to first characterize the IFN resistance and IFN induction properties of HK mouse-adapted mutants as well as pathology in mice lung, followed by the generation of defined recombinant viruses possessing desired mutation(s), then analysis of these recombinant viruses for IFN induction and IFN resistance. Rescuing of the HK wild type NS gene and those of HK mouse-adapted NS mutant genes into the backbone of parental HK genome discovered that NS20, NS20c and NS411 produced attenuating phenotypes on their own. Finally, all these mutant NS genes were inserted into backbone of WSN (lab adapted strain, A/WSN/33, H1N1) genome to construct recombinant viruses. Using recombinant viruses that differ due to individual NS1 gene showed that all of the NS1 mutations increased resistance to IFN in mouse cells. The extent of IFN resistance due to individual mutation was influenced by cell types: epithelium versus fibroblast, as well as host type: mouse versus human. Interestingly most of the NS1 mutations attenuated growth of virus which suggests that resistance to IFN involves changes in host interaction that are not optimal for growth in the absence of IFN. Infection assay showed mouse-adapted mutant NS genes conferred resistance to mouse IFN and vulnerability to human IFN. And mutant NS1 protein NSMA20c had the most potent ability to resist human and mouse IFN. IFN assay showed that mutant NS1 protein NSMA20c had the highest ability to induce mouse IFN. Furthermore, all synthetic recombinant viruses induced low amounts of IFN in human cells. Immunopathology of infected lungs showed that mouse adapted progeny virus HKMA20C had acquired a crucial ability to spread to and infected alveoli which may be influenced by IFN resistance. Mouse-adapted variants possess mutations that increase IFN resistance that in some instances leads to higher IFN induction. One of the key discussions was that most of these mutations cluster in a small region that has been previously mapped to involve a region of host protein (eIF4GI) interaction.
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Arsenio, Janilyn. "Development and application of a vaccinia virus based system to study viral proteins modulating interferon expression and interferon induced antiviral activities." Elsevier, 2008. http://hdl.handle.net/1993/5068.
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The interferon (IFN) system is integral to antiviral innate immunity in vertebrate hosts. Inside a cell, viral pathogen associated molecular patterns (PAMPs) trigger the IFN response, comprised of IFN induction and an IFN-induced antiviral state. However, viruses have evolved strategies to counteract the IFN system. The E3 protein of vaccinia virus (VV), encoded by the E3L gene, impedes cytokine expression and suppresses the activation and function of antiviral proteins. Deletion of the E3L gene (VVΔE3L) produces an IFN sensitive mutant virus that is replication defective in most human cell lines. Due to the limited human cell lines available to support VVΔE3L replication, the capacity of E3 inhibition of human IFN-induced antiviral activities is not well defined. In this study, VVΔE3L was generated and characterized to facilitate the study of other viral IFN antagonists at modulating human IFN-induced antiviral responses. A human liver carcinoma cell line, Huh7, was found to support VVΔE3L replication. A comprehensive analysis of VVΔE3L IFN sensitivity revealed E3 inhibits all human type I and type II IFN-induced antiviral activities by modulation of the protein kinase R (PKR) pathway.
Influenza non-structural protein 1 (NS1) is well-known to mediate the suppression of IFN induction and IFN action in influenza virus infections. However, the IFN antagonizing potential of influenza NS1 may be virus subtype and/or isolate specific. VVΔE3L was next applied as an expression vector to study influenza NS1 function in modulating IFN-induced antiviral activities and IFN induction in human cells. Recombinant viruses were generated to express influenza NS1 (from avian H5N1 and pandemic viruses 1918 pH1N1, 1968 pH3N2, and 2009 pH1N1) in replacement of E3. It was found that influenza NS1 inhibits human IFN-induced antiviral activity in a subtype and isolate specific manner. Moreover, influenza NS1 differentially regulates human IFN expression in a virus isolate-dependent manner. Altogether, this work highlights the potential of VVΔE3L as an excellent virus model system to study viral proteins modulating IFN expression and IFN-induced antiviral activities in human cells.
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Velázquez, Flores Anney Lillián. "Identificación del antígeno NS1 y anticuerpo IgM para virus del dengue en estudiantes de Nivel Superior de la UAEMéx." Tesis de Licenciatura, Universidad Autónoma del Estado de México, 2017. http://hdl.handle.net/20.500.11799/70659.
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El dengue es una de las principales enfermedades virales de carácter epidémico. Constituye la arbovirosis más importante a nivel mundial en morbilidad, mortalidad, en México, es una de las principales enfermedades transmitidas por vector1.
En el presente trabajo se realizó una identificación de antígeno NS1 para el virus de Dengue mediante un método inmunoenzimático en una etapa de tipo sándwich, para la detección cualitativa o semicuantitativa del antígeno NS1 y la detección de IgM contra antígenos del Dengue en suero.
MATERIAL Y MÉTODOS
Se analizaron 60 muestras para la detección del antígeno NS1 para DENV y 68 muestras para la detección de inmunoglobulina M mediante ELISA.
RESULTADOS
En el análisis de detección del Antígeno NS1 las 60 muestras procesadas mediante ELISA fueron negativas; el 35.3% de las 68 muestras analizadas para la detección de IgM resultaron positivas. Las cifras muestran que del grupo de personas positivas a la IgM, al menos el 83.3% presenta rash/erupción, el 8.3% además de sentir rash/erupción también presenta petequias y finalmente el 8.4% restante presenta cefalea, fiebre y rash/erupción, el 58.4% se traslada al sur de la República Mexicana, principalmente a Estados considerados zonas endémicas del DENV, como son Guerrero, Michoacán y Veracruz, y a algunas comunidades del Estado de México, principalmente, Valle de Bravo, Acambay, Tejupilco y Malinalco. Amatepec y Villa del Carbón.
CONCLUSIONES
Este estudio mostró que los principales factores de exposición para la IgM positiva son haber viajado a zonas endémicas de la República Mexicana y que el mosquito del género Aedes que transmite el virus del Dengue se está adaptando a nuevas alturas debido a la positividad presentada en las zonas del estado de México donde no se había presentado, lo que constituye un serio problema para la salud en México.
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MENDONÇA, Priscila Dias. "Nano-híbrido de carbono aplicado em imunossensor para detecção da proteína ns1 do vírus da dengue." Universidade Federal de Pernambuco, 2016. https://repositorio.ufpe.br/handle/123456789/18452.
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A dengue é uma doença viral considerada um dos maiores problemas de saúde pública nas regiões tropicais e sub-tropicais do mundo, sendo endemicamente prevalente em cerca de 112 países. Anualmente, afeta cerca de 50 a 100 milhões de pessoas, resultando em taxas de mortalidade entre 0,03% a 1,4%. É uma doença auto-limitante, caracterizada por febre, dor de cabeça, mialgia, entre outros sintomas. Na sua forma severa (síndrome do choque por dengue e febre hemorrágica), a doença pode levar ao óbito, principalmente em crianças. A proteína não estrutural 1 (NS1) do vírus dengue circula abundantemente no sangue durante toda a viremia, estando em níveis maiores na fase aguda; assim esta pode ser utilizada como marcador do estado agudo. Para o controle da infecção estão disponíveis testes diagnósticos baseados em ensaios sorológicos, testes imunocromatográficos e moleculares, entretanto estes apresentam limitações. O desenvolvimento de alternativas mais práticas, quantitativas e econômicas tem resultado na crescente busca por testes baseados em biossensores. Neste trabalho foi desenvolvido um imunossensor para detecção de NS1 baseado em uma plataforma nanoestruturada, constituída de nano-híbrido formado por nanotubos de carbono e filme polimérico de polietilenoimina montado sobre sistema eletroquímico constituído por microeletrodo de ouro. Os anticorpos monoclonais anti-NS1 foram imobilizados sobre a superfície eletródica por ligações covalentes com os nanotubos de carbono, permitindo um alta estabilidade durante as medidas. Todas as etapas de modificações da superfície eletródica foram caracterizadas eletroquimicamente, estrutural e morfologicamente através das técnicas de voltametria cíclica, espectroscopia de infravermelho por transformada de Fourier (FT-IR) e microscopia eletrônica de varredura, respectivamente. A espessura do filme nanoestruturado foi determinada por medidas piezoelétricas, em um sistema de microbalança de cristal de quartzo de acordo com a equação de Sauerbrey. A resposta analítica do imunossensor frente a proteína NS1 foi obtida por amperometria aplicando-se a técnica de voltametria de onda quadrada (VOC). O imunossensor apresentou resposta linear entre 0,1 a 0,6 µg.mL-1 de NS1. Os dados ajustados para a equação de regressão linear exibiu coeficiente de correlação de 0,996 (p << 0,01, n = 7) e um baixo erro relativo (aproximadamente 1%). O imunossensor apresentou limite de detecção de 0,038 µg.mL-1 e limite de quantificação de 0,1 µg.mL-1 de NS1, sendo similar aos obtidos na literatura, porém com a vantagem de não requerer antígenos ou anticorpos marcados (label-free) e utilizar técnica analítica mais simples (VOC). Os resultados indicam que o imunossensor apresenta sensibilidade compatível para detecção de NS1 em níveis sorológicos, permitindo ser uma ferramenta prática, rápida e econômica para o diagnóstico da dengue, sobretudo para detecção precoce da fase aguda.
Dengue is a viral disease considered as a major public health problems in tropical and sub-tropical world, endemically being prevalent in about 112 countries. Annually, it affects 50 to 100 million people, resulting in mortality rates of 0.03% to 1.4%. It is a self-limiting disease characterized by fever, headache, myalgia, among other symptoms. In its severe form (shock syndrome and dengue haemorrhagic fever), the disease can lead to death, especially in children. The nonstructural protein 1 (NS1) of the dengue virus circulates in blood abundantly throughout viremia, with higher levels in the acute phase; thus it can be used as a marker of the acute stage. For control of infection are available diagnostic testings based on serological assays, immunochromatographic and molecular assyas, however these have limitations. The development of more practical, quantitative and economic alternatives has resulted in growing demand for tests based on biosensors. In this work, it was developed an immunosensor for NS1 detection based on a nanostructured platform consisting of nanohybrid comprising carbon nanotubes and polymeric film polyethyleneimine mounted on electrochemical system consisting of gold microelectrode. The anti-NS1 monoclonal antibodies were immobilized on the electrode surface by covalent bonds with carbon nanotubes, allowing for a high stability during the measurements. All steps of electrode modifications were characterized by electrochemical, structural and morphologically techniques through cyclic voltammetry, infrared spectroscopy by Fourier transform (FT-IR) and scanning electron microscopy, respectively. The thickness of the nanostructured film was determined by piezoelectric system using a quartz crystal microbalance, according to the Sauerbrey equation. The analytical response of the immunosensor against NS1 protein was obtained by applying amperometry by square wave voltammetry (SWV). The immunosensor showed a linear response between 0.1 to 0.6 μg.mL-1 NS1. The data set into the linear regression equation showed a correlation coefficient of 0.996 (p << 0.01, n = 7) and a low relative error (about 1%). The immunosensor presented detection limit of 0.038 μg.mL-1 and a limit of quantification of 0.1 μg.mL-1 NS1, being similar to those obtained in literature, but with advantage of not requiring labeled antigen or antibody (label- free) and to require a more simple analytical technique (VOC). The results indicate that the achieved sensitivity was similar NS1 immunosensors, allowing it to be convenient, fast and economical tool for dengue diagnosis, particularly for early detection of acute phase.
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Tai, Hung, and 戴雄. "The role of the non-structural protein, NS1, in influenza virus replication." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44660303.
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Jacobs, Susan Catherine. "Characterisation and analysis of the NS1 gene of tick-borne encephalitis virus." Thesis, Oxford Brookes University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.332598.
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Sanchez, Jonathan L., Zachary Romero, Angelica Quinones, Kristiane R. Torgeson, and Nancy C. Horton. "DNA Binding and Cleavage by the Human Parvovirus B19 NS1 Nuclease Domain." AMER CHEMICAL SOC, 2016. http://hdl.handle.net/10150/622382.
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Infection with human parvovirus B19 (B19V) has been associated with a myriad of illnesses, including erythema infectiosum (Fifth disease), hydrops fetalis, arthropathy, hepatitis, and cardiomyopathy, and also possibly the triggering of any number of different autoimmune diseases. B19V NS1 is a multidomain protein that plays a critical role in viral replication, with predicted nuclease, helicase, and gene transactivation activities. Herein, we investigate the biochemical activities of the nuclease domain (residues 2-176) of B19V NS1 (NS1-nuc) in sequence-specific DNA binding of the viral origin of replication sequences, as well as those of promoter sequences, including the viral p6 and the human p21, TNF alpha, and IL-6 promoters previously identified in NS1-dependent transcriptional transactivation. NS1-nuc was found to bind with high cooperativity and with multiple (five to seven) copies to the NS1 binding elements (NSBE) found in the viral origin of replication and the overlapping viral p6 promoter DNA sequence. NS1-nuc was also found to bind cooperatively with at least three copies to the GC-rich Spl binding sites of the human p21 gene promoter. Only weak or nonspecific binding of NS1-nuc to the segments of the TNF alpha and IL-6 promoters was found. Cleavage of DNA by NS1-nuc occurred at the expected viral sequence (the terminal resolution site), but only in single-stranded DNA, and NS1-nuc was found to covalently attach to the 5' end of the DNA at the cleavage site. Off-target cleavage by NS1-nuc was also identified.
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Gomes, Deriane Elias [UNESP]. "Estudo das interações entre proteína NS1 do hRSV e flavonóides utilizando métodos biofísicos." Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/122135.
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000811761.pdf: 785012 bytes, checksum: d2639de3af3e75addc7def7c6e77f5fb (MD5)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O Vírus Respiratório Sincicial Humano (hRSV) é a principal causa de infecções respiratórias agudas em crianças, como bronquiolite e pneumonia. Este Paramyxovirus tem um RNA de fita simples, com 10 genes que codificam 11 proteínas. Um fator importante que contribui para o sucesso da replicação do hRSV é a evasão do sistema imune, processo no qual a proteína não estrutural 1 (NS1) está envolvida. Esta proteína pode atuar por meio da inibição ou neutralização das etapas da cascata do interferon do tipo I, bem como, pelo silenciamento do complexo ribonucleoproteico do hRSV. O conhecimento da interação da proteína NS1 com ligantes é importante na busca de moléculas que possam interferir na função da proteína e consequentemente resultar na redução da replicação do hRSV dentre os quais os flavonóides têm sido descritos como sendo supressores eficazes da replicação viral. Os objetivos deste estudo incluíram a expressão e purificação da proteína NS1, a caracterização da estrutura secundária e estabilidade térmica por dicroísmo circular e a realização do estudo de interação entre NS1 e quercetina por espectroscopia de fluorescência. Foi realizada a purificação da proteína NS1 em resina de afinidade utilizando cromatógrafo líquido ÄKTA (GE HEALTHCARE LIFE SCIENCES). As medidas de dicroísmo circular (CD) permitiram calcular a porcentagem de estruturas secundárias e a temperatura de melting da NS1. A partir das análises das titulações por fluorescência foram calculados a constante de Stern Volmer (Ksv), a constante de ligação (Kb) e o número de ligantes por sítio (n). Os resultados de CD mostraram que a composição da estrutura secundária de NS1 é de 75 % de alfa-hélice, 3% de folha-beta, 10% de voltas e 12% de outras estruturas e a temperatura de melting da NS1 é de cerca de 65°C. Os resultados de fluorescência mostraram que Ksv e Kb foram da ordem de 104 e 105-106M-1, ...
Human Respiratory Syncytial Virus (hRSV) is the major cause of acute respiratory infections in children, like bronchiolitis and pneumonia. This Paramyxovirus has a single strand RNA with 10 genes that codify 11 proteins. An important factor that contributes for the success hRSV replication is the immune system evasion, process wich Non-Structural Protein 1 (NS1) is involved. This protein can act by inhibiting or neutralizing steps of interferon type I pathway, as well as, by silencing the ribonucleoproteic complex of hRSV. The knowledge of NS1 protein interaction with ligands is important in the search for molecules that could interfere with protein function and hence result in a reduction of hRSV replication among them the flavonoids have been reported to be effective in suppressing viral replication. The aim of this study includes expression and purification of NS1 protein, characterization of its secondary structure and thermal stability through circular dicroism and perform fluorescence spectroscopy interaction study between NS1 and Quercetin. NS1 purification was performed using affinity resin coupled to a liquid cromatograph ÄKTA (GE HEALTHCARE LIFE SCIENCES). Circular dicroism spectra and thermal denaturation were carried out to investigate NS1 secondary structure and stability. Through fluorescence spectroscopy titration results it has been calculated the Stern Volmer constant (Ksv), the binding (Kb) constant and number of ligands per site (n). CD analysis shown that secondary structure composition of NS1 is 75% alpha-helix; 3% beta-sheet; 10% turn and 12% others and, melting temperature of NS1 is around 65°C. Fluorescence results shown that Ksv and Kb were in order of 104 and 105-106M-1, respectively. Ksv dependence with temperature indicates that the interaction between NS1 and quercetin is dynamic. The results suggest that this interaction may potentially contribute to block the xiii protein function and thus quercetin may ...
FAPESP: 2010/50211-0
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Gomes, Deriane Elias. "Estudo das interações entre proteína NS1 do hRSV e flavonóides utilizando métodos biofísicos /." São José do Rio Preto, 2014. http://hdl.handle.net/11449/122135.
Full textAbstract:
Orientador: Fátima Pereira de SouzaCoorientador: Marcelo Andrés Fossey
Banca: Flavio Augusto Vicente Seixas
Banca: Fernanda Canduri
Banca: Karina Alves de Toledo
Banca: Fernando Alves de Melo
Resumo: O Vírus Respiratório Sincicial Humano (hRSV) é a principal causa de infecções respiratórias agudas em crianças, como bronquiolite e pneumonia. Este Paramyxovirus tem um RNA de fita simples, com 10 genes que codificam 11 proteínas. Um fator importante que contribui para o sucesso da replicação do hRSV é a evasão do sistema imune, processo no qual a proteína não estrutural 1 (NS1) está envolvida. Esta proteína pode atuar por meio da inibição ou neutralização das etapas da cascata do interferon do tipo I, bem como, pelo silenciamento do complexo ribonucleoproteico do hRSV. O conhecimento da interação da proteína NS1 com ligantes é importante na busca de moléculas que possam interferir na função da proteína e consequentemente resultar na redução da replicação do hRSV dentre os quais os flavonóides têm sido descritos como sendo supressores eficazes da replicação viral. Os objetivos deste estudo incluíram a expressão e purificação da proteína NS1, a caracterização da estrutura secundária e estabilidade térmica por dicroísmo circular e a realização do estudo de interação entre NS1 e quercetina por espectroscopia de fluorescência. Foi realizada a purificação da proteína NS1 em resina de afinidade utilizando cromatógrafo líquido ÄKTA (GE HEALTHCARE LIFE SCIENCES). As medidas de dicroísmo circular (CD) permitiram calcular a porcentagem de estruturas secundárias e a temperatura de melting da NS1. A partir das análises das titulações por fluorescência foram calculados a constante de Stern Volmer (Ksv), a constante de ligação (Kb) e o número de ligantes por sítio (n). Os resultados de CD mostraram que a composição da estrutura secundária de NS1 é de 75 % de alfa-hélice, 3% de folha-beta, 10% de voltas e 12% de outras estruturas e a temperatura de melting da NS1 é de cerca de 65°C. Os resultados de fluorescência mostraram que Ksv e Kb foram da ordem de 104 e 105-106M-1, ...
Abstract: Human Respiratory Syncytial Virus (hRSV) is the major cause of acute respiratory infections in children, like bronchiolitis and pneumonia. This Paramyxovirus has a single strand RNA with 10 genes that codify 11 proteins. An important factor that contributes for the success hRSV replication is the immune system evasion, process wich Non-Structural Protein 1 (NS1) is involved. This protein can act by inhibiting or neutralizing steps of interferon type I pathway, as well as, by silencing the ribonucleoproteic complex of hRSV. The knowledge of NS1 protein interaction with ligands is important in the search for molecules that could interfere with protein function and hence result in a reduction of hRSV replication among them the flavonoids have been reported to be effective in suppressing viral replication. The aim of this study includes expression and purification of NS1 protein, characterization of its secondary structure and thermal stability through circular dicroism and perform fluorescence spectroscopy interaction study between NS1 and Quercetin. NS1 purification was performed using affinity resin coupled to a liquid cromatograph ÄKTA (GE HEALTHCARE LIFE SCIENCES). Circular dicroism spectra and thermal denaturation were carried out to investigate NS1 secondary structure and stability. Through fluorescence spectroscopy titration results it has been calculated the Stern Volmer constant (Ksv), the binding (Kb) constant and number of ligands per site (n). CD analysis shown that secondary structure composition of NS1 is 75% alpha-helix; 3% beta-sheet; 10% turn and 12% others and, melting temperature of NS1 is around 65°C. Fluorescence results shown that Ksv and Kb were in order of 104 and 105-106M-1, respectively. Ksv dependence with temperature indicates that the interaction between NS1 and quercetin is dynamic. The results suggest that this interaction may potentially contribute to block the xiii protein function and thus quercetin may ...
Doutor
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Panthu, Baptiste. "Développement d’un nouveau système hybride de traduction in vitro et étude du rôle traductionnel de la protéine NS1 de l’Influenza A." Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10131/document.
Full textAbstract:
Le virus de l'Influenza A est l'agent étiologique des épidémies de grippe saisonnière. Ce virus a développé des stratégies complexes pour exprimer ses protéines dans les cellules hôtes dès 4 heures après infection. Au départ de cette étude, je me suis intéressé aux événements intervenant dans l'initiation de la traduction des ARN messagers viraux. L'infection par le virus de l'Influenza A perturbe profondément la physiologie cellulaire, et notamment les processus d'expression des gènes au niveau des étapes de transcription, maturation et export des ARN messagers. De ce fait, j'ai donc commencé par développer les outils permettant de m'affranchir de ces événements nucléaires pour pouvoir me focaliser sur les mécanismes viraux spécifiques de l'initiation de la traduction. Ainsi, j'ai conçu et élaboré un nouveau système de traduction in vitro qui dérive du lysat de réticulocytes de lapin dans lequel sont ajoutés des ribosomes isolés de cellules en culture. Ce lysat, dit hybride, présente l'avantage d'être très efficace pour la production de protéines tout en conservant les caractéristiques traductionnelles des cellules dont les ribosomes dérivent. Le second volet de mes travaux porte sur le rôle de la protéine virale NS1 au niveau de la traduction cellulaire et virale. En combinant des infections virales avec des expériences in vitro et ex-vivo, par transfection d'ARN, je montre que NS1 est capable de stimuler la synthèse protéique des ARNm cellulaires et viraux. Par de la mutagénèse dirigée sur cette protéine de 230 acides aminés, j'observe que la région amino-terminale de la protéine (aa 1-81) est responsable de cet effet activateur. Des mutations ponctuelles au sein de ce domaine révèlent l'importance de deux résidus aminés (R38 et K41) dans la stimulation. En résumé, ces travaux ont permis de mettre au point un nouveau système d'expression in vitro et de mieux comprendre comment est contrôlée la synthèse des protéines virales du virus Influenza AInfluenza A belongs to the orthomyxoviridae family and is the causal agent for the seasonal and epidemic Influenza infections. This virus has developed complex strategies to utilize the host cell protein apparatus for viral protein expression. In this study, I have focused on the events involved during the initiation of translation of viral mRNAs. Influenza A infection profoundly disrupts host cell gene expression mainly at the level of transcription, maturation and mRNA export. As such, it is quite difficult to investigate directly translational control of Influenza. Therefore, I have started my project by elaborating experimental tools that can be used for this purpose. This was done by designing and developing a new in vitro translation system derived from the rabbit reticulocyte lysate which is supplemented with exogeneous ribosomes that have been isolated from different cell types. This lysate, called hybrid system, offers the advantage to be very effective in the production of proteins while maintaining the translational characteristics of the cells from which the ribosomes originate. The second part of my work focusses on the role of the viral NS1 protein on cellular and viral translation. By using an experimental approach based on viral infections together with in vitro and ex vivo translational assays, I could show that NS1 is able to stimulate both viral and cellular protein synthesis. Then, the introduction of deletion mutants of this 230 amino acids protein revealed that its amino-terminal domain (aa 1-81) was responsible for this stimulatory effect (aa 1-81). Finally, the introduction of point mutations in this region showed the importance of two conserved positively charged residues (R38 and K41) for stimulation. In summary, these studies have yielded a new in vitro translation expression system and have shed light on how viral proteins synthesis is regulated by Influenza A virus
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Satterly, Neal Gilpin. "The mRNA Nuclear Export Machinery is Targeted by Influenza Virus and Antivirals." Scholarly Repository, 2009. http://scholarlyrepository.miami.edu/oa_dissertations/197.
Full textAbstract:
Proper mRNA nuclear export is essential for harmonious growth and maintenance of a cell. An effective weapon influenza virus employs to hijack a host cell is its ability to inhibit such export. Exactly how influenza virus achieves this inhibition is not fully known. Here, we demonstrate that upon infection, influenza virus degrades two nucleopore proteins (Nup98 and Nup96), which play a key role in mRNA nuclear export. Also, a main virulence factor of influenza virus (non-structural protein 1, NS1) binds directly to NXF1 and E1B-AP5, two key constituents of the mRNA export pathway (NXF1/NXT pathway) responsible for exporting bulk (~70%) mRNA from the nucleus. By increasing the expression levels of members of the NXF1/NXT pathway, we were able to reverse NS1-mediated inhibition of gene expression. On the other hand, by decreasing the levels of members of the NXF1/NXT pathway, we demonstrated that host cells become more sensitive to influenza virus infection and produce more viral particles. These results demonstrate undiscovered influenza-mediated host interactions that may be used to medicinally inhibit influenza virus. To this end, high-throughput screens were designed to identify small molecule antagonists of both NS1-mediated inhibition of gene expression and influenza virus-mediated cell death. Seventy-one compounds were identified, and the most potent molecule (named compound #8) was examined further. We found that compound #8 releases influenza virus-mediated mRNA nuclear export blockage and decreased viral replication and viral gene expression. Thus, the bulk mRNA nuclear export machinery is vital to antiviral response, and compound #8 enhances its ability to fight the cytopathic effects of NS1 and influenza virus. In conclusion, our data demonstrate that the mRNA export machinery is disrupted by influenza virus, and that this machinery also facilitates an antiviral function. We have also shown that these two events can be manipulated chemically to attenuate the negative effect of the virus and enhance the positive antiviral effect of the mRNA export machinery, thereby providing a powerful, new strategy against the ever-present, global threat of influenza virus.
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Yu, Yi-Hsin Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Role of the RNAi pathway in influenza a virus infected mammalian cells." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2008. http://handle.unsw.edu.au/1959.4/41545.
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The interferon (lFN) signalling and RNA interference (RNAi) pathways are the major antiviral pathways in animals and plants, respectively. Although the mechanism of RNAi remains to be completely characterised, the genes that encode the proteins involved in this process have been identified in the plant, fungi and animal kingdoms (Fagard et al., 2000, Grishok et aI., 2000, Hall et al., 2003, Kanellopoulou et al., 2005, Kolb et al., 2005); with comparative analyses indicating that RNAi is an evolutionarily conserved mechanism. Several studies have identified RNAi suppressors encoded by animal viruses, suggesting an antiviral role for the RNAi pathway in animals as well as plants (Andersson et al., 2005, Bennasser et al., 2006, Garcia et al., 2006, Li et al., 2004, Lichner et al., 2003, Lingel et al., 2005, Lu & Cullen, 2004, Wang et al., 2006). However, most of these studies were performed in non-mammalian systems and as yet, there is no direct evidence indicating that the RNAi pathway plays a significant antiviral role during the infection of mammalian cells. Interestingly, several viruses have now been shown to express their own microRNA (miRNA) in infected cells (Grey et al., 2005, Pfeffer et al., 2005, Pfeffer et al., 2004, Samols et al., 2005, Sullivan et al., 2005). Further, in the case of hepatitis C virus (HCV), there is evidence that the virus usurps the host cell miRNAs to enhance viral replication (Jopling et al., 2005). The principal aim of this project was to investigate the role of RNAi in mammalian cells during viral infection, particularly infection with the influenza A virus. This thesis is divided into six major chapters followed by a brief general discussion. Chapter 1 contains a general introduction to the RNAi pathway. It describes the history of the discovery of RNAi and summarizes the known and proposed antiviral roles of the RNAi pathway in plants and mammalian cells. Chapter 2 describes the general materials and methods used for this project. There are four main result chapters, each dealing with a specific experimental system. Each chapter is divided into a brief introduction, specific materials and methods used, followed by presentation of the experimental results and a brief discussion. Chapter 3 describes the development of an in vitro Dicer activity assay to study the effect of viral proteins on the activity of the mammalian Dicer protein. It was demonstrated that crude cell lysates derived from influenza A virus infected cells impaired the activity of Dicer and this observation was not due to degradation of the Dicer protein by virally-induced proteases. Chapter 4 describes the use of a GFP reporter assay for screening potential RNAi suppressors. This assay is suitable for studying viral proteins in isolation. The effect of the influenza NS1 protein on the RNAi pathway in HEK293 cells was investigated and it was shown that NS1 could exert modest, but nevertheless significant, suppression of the RNAi pathway. Northern studies, performed to examine the processing of shRNA in the presence of NS1, demonstrated that NSI suppressed the RNAi mechanism through interfering with the maturation ofshRNA into siRNA. Chapter 5 describes the effect of over-expressing components of the RNAi pathway on influenza A virus infection. In these experiments, Exportin 5, which encodes a protein involved in the transport of pre-miRNA/shRNA into the cytoplasm, was over-expressed during influenza A virus infection. Reduced viral infection was observed in cells over-expressing Exportin 5, suggesting that this treatment protects cells from virus infection. Chapter 6 describes the expressed small RNA profile during influenza A virus infection in MDCK cells. Novel canine miRNA homologues were identified through cloning and sequencing. No definitive evidence for virally-derived siRNA/miRNA was found but a general reduction of endogenous miRNA expression was detected.
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DIAS, Ana Carolina Matos da Silva. "Desenvolvimento de plataformas sensoras para detecção eletroquímica do antígeno NS1 do vírus da dengue." Universidade Federal de Pernambuco, 2015. https://repositorio.ufpe.br/handle/123456789/17374.
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FACEPE
A infecção pelo vírus dengue (DENV) é uma das doenças tropicais mais negligenciadas e de maior importância de saúde pública no mundo. Novos métodos de diagnóstico da doença têm sido estudados através da detecção da proteína NS1 do DENV. O antígeno NS1 é um importante marcador precoce da fase aguda da dengue, secretado em altas concentrações pelo vírus no sangue de pessoas infectadas logo nos primeiros dias, porém, não é muito utilizado na rotina laboratorial para diagnóstico da doença devido ao alto custo dos ensaios. A presente tese descreve o desenvolvimento de duas plataformas sensoras eletroquímicas baseadas em eletrodos impressos (EIs) modificados com nanomateriais para detecção do antígeno NS1 do DENV. Os EIs foram confeccionados utilizando-se a impressão de tinta de carbono sobre o polietileno tereftalato (PET), suporte para impressão dos moldes. Inicialmente, foram estudados os efeitos de nanotubos de carbono e sua contribuição na transferência de elétrons, condutividade e aumento de área eletroativa da plataforma sensora. O estudo foi baseado na incorporação de nanotubos de carbono funcionalizados com grupos carboxílicos à tinta de carbono. Para detecção do NS1, um imunoensaio do tipo “sanduíche” foi realizado, no qual a captura específica do NS1 pôde ser avaliada através das reações redox da enzima peroxidase conjugada ao anticorpo. Uma faixa linear entre 0,04 g/mL e 2 g/mL de NS1 foi obtida, indicando boa performance analítica do imunossensor, com coeficiente de correlação linear de 0,996 (p<0.0001, n=8) e limite de detecção de 0,012 g/mL de NS1. Posteriormente, foi investigada a contribuição de nanopartículas metálicas no desenvolvimento de sensores eletroquímicos livres de marcação. Foram utilizadas nanopartículas de ouro (NPsAu) funcionalizadas com grupos amina para a imobilização covalente de anticorpos. Na síntese das NPsAu, foi utilizado o polietilenoimina como agente redutor e funcionalizante para promover uma ligação amida com o anticorpo anti-NS1. O imunossensor desenvolvido mostrou curva de calibração com faixa de concentração linear entre 0,1 g/mL e 2 g/mL de NS1 (r = 0,995, p<0.0001, n=7) e limite de detecção de 0,03 g/mL de NS1. A contribuição dos nanomateriais para as plataformas sensoras desenvolvidas mostrou-se efetiva na sensibilidade analítica, devido ao aumento de área eletroativa e maior quantidade de anticorpos imobilizados. A aplicação destes nanomateriais nos imunossensores proporciona novas alternativas de diagnóstico para detecção da proteína NS1 do DENV.
Infection by Dengue Virus (DENV) is one of the most neglected tropical diseases and of higher importance of public health worldwide. New methods of diagnosis of the disease have been studied through the detection of NS1 protein of DENV. NS1 antigen is an important early marker of acute dengue infection secreted in high concentrations by the virus in the blood of infected people in first days, however it is not widely used in the laboratory routine for diagnosis of the disease due to high cost of assays. The present thesis describes the development of two electrochemical sensor platforms based on screen-printed electrodes (SPEs) modified with nanomaterials for detection of NS1 antigen of DENV. SPEs were prepared using carbon ink printing on the polyethylene terephthalate (PET), support for molds printing. Initially, the effects of carbon nanotubes and their contribution to the electron transfer, conductivity and increase of electroactive area of the sensor platform were studied. The study was based on the incorporation of carbon nanotubes functionalized with carboxylic groups to the carbon ink. For NS1 detection, a sandwich-type immunoassay was carried out, wherein the specific capture of NS1 may be assessed by redox reactions of peroxidase enzyme conjugated to the antibody. A linear range between 0.04 g/mL and 2 g/mL NS1 was obtained, indicating good analytical performance of the immunosensor, with linear correlation coefficient of 0.996 (p<0.0001, n=8) and limit of detection of 0.012 g/mL NS1. Subsequently, the contribution of metal nanoparticles in the development of label-free electrochemical sensors was investigated. Gold nanoparticles (AuNPs) functionalized with amine groups were used for covalent immobilization of antibodies. In the synthesis of AuNPs, polyethyleneimine was used as a reducing and functionalizing agent to promote an amide bond with anti-NS1 antibody. The developed immunosensor showed calibration curve with linear concentration range between 0.1 g/mL and 2 g/mL NS1 (r = 0.995, p<0.0001, n = 7) and limit of detection of 0.03 g/mL NS1. The contribution of nanomaterials for the sensor platforms developed proved effective in the analytical sensitivity due to the increase of electroactive area and larger amount of immobilized biomolecules. The application of these nanomaterials in immunosensors provides new alternatives of diagnosis for detection of NS1 protein of DENV.
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Rios, Katiúscia Alexandre. "Utilização de peptídeos quiméricos das proteínas de envelope (E) , não estrutural 1 (NS1) e não estrutural 3 (NS3) para produção de insumos diagnósticos da dengue." Universidade Federal do Amazonas, 2014. http://tede.ufam.edu.br/handle/tede/5543.
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CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
Dengue infection has become a public health problem in tropical and sub-tropical areas. It is a viral disease transmitted by arthropods with high mortality rates. Its clinical diagnosis requires laboratory confirmation because several other conditions can also cause similar symptoms with acute fevers as well as the primary use antibodies or antigens for detection of DENV. The continued development of diagnostic tests that are cheap, sensitive, specific, easy to perform, and that are capable of giving early diagnosis of the dengue virus infection is still a needed. Using bioinformatics, we mapped peptides of Envelope protein (E), Nonstructural 1 and 2 which have antiginity, hidrofility and probality to apper on the surface. The first peptide, with molecular weight of the 17KDa, was called E/ENS3 and corresponds to two Envelope protein (E) sequences and a Nonstructural 3 sequence. The second peptide, with molecular weight of the 15Kda, was called ENS1/3 and it corresponds to a Envelope protein (E) sequence, a Nonstructural 1 sequence and Nonstructural 3 sequence. The peptides were expressed in prokaryotic sistem, purified and used in ELISA (Enzyme-Linked Assay Imunoabsorvente). Using Indirect ELISA in the IgM and IgG antibody detection previously characterized, we obtained as results: (1) IgG research using E/ENS3 and ENS1/3 peptides, sensibility was 65% and the specificity was 91,7% and (2) IgM research, sensibility was 75,7% and and the specificity was 91,8%. These results indicate that use of both E/ENS3 and ENS1/3 peptides may be a alternative to the serological diagnosis of dengue.
A infecção pelo vírus dengue continua sendo um grande problema de saúde pública em regiões tropicais e subtropicais do mundo, sendo a principal doença viral transmitida por artrópodes, com alta morbidade e mortalidade. O diagnóstico clínico da dengue requer confirmação laboratorial devido à semelhança dos sintomas com uma série de outras febres agudas e para detecção de infecções virais primárias ou secundárias. O desenvolvimento de testes baratos, sensíveis, específicos e de fácil execução, que sejam capazes de proporcionar diagnóstico precoce da infecção pelo vírus da dengue, é ainda uma necessidade. Neste trabalho mapeamos, por ferramentas de bioinformática, peptídeos das proteínas de Envelope (E), Não Estrutural 1 e 2 que apresentasse como características: elevada antigenicidade, hidrofilicidade e probabilidade de se apresentar na superfície. O primeiro peptídeo, com peso molecular de 17KDa, foi denominado E/ENS3 e corresponde a duas sequências da proteína de Envelope (E) e uma sequência da Não Estrutural 3. O segundo peptídeo, com peso molecular de 15Kda, foi denominado ENS1/3 e corresponde a uma sequência da proteína de Envelope (E), uma sequência da Não Estrutural 1 e uma sequência da Não Estrutural 3. Os peptídeos foram expressos em sistemas em sistema procarioto, purificados e utilizados em teste de ELISA (Enzyme-Linked Assay Imunoabsorvente). Foram realizadas a detecção de anticorpos IgM e IgG de amostras de laboratório, previamente caracterizadas, através de ELISA Indireto e obteve-se os seguintes resultados: (1) na pesquisa de IgG usando os peptídeos E/ENS3 e ENS1/3, a sensibilidade foi de 65% e a especificidade foi de 91,7% e (2) na pesquisa de IgM a sensibilidade foi de 75,7% e especificidade foi de 91,8%. Estes resultados indicam que a utilização dos peptídeos E/ENS3 e ENS1/3 podem ser uma alternativa para o diagnóstico sorológico da dengue
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Jasim, Muhammad, and Muhammad Haidar Zaman. "Molecular cloning and expression of NS1 of the influenza “A” virus from different subtypes." Thesis, Södertörns högskola, Institutionen för naturvetenskap, miljö och teknik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:sh:diva-28643.
Full textAbstract:
Influenza A virus (IAV) subtypes are globally responsible for seasonal pandemics. Subtypes of IAV virus can beclassified because of differences surface antigenic protein. The non structural protein1 (NS1) of IAV is the only protein that encoded by IAV and contributing role in the pathogensis. NS1 is a virulence factors duringviral replication in the host. NS1 of IAV is known to be multifunctional protein which enhances the pathogenicity of the virus duringt he viral life cycle. The current study is aimed to investigate NS1 of IAV subtype H1N1, H7N7, H4N6 and H5N1, which were successfully cloned in to the pcDNA 3.1 vector. Restriction analysis with enzymes BamH1,Xba1 and Kpn1 and sequencing showed that NS1 gene was successfully cloned. My recombinant proteins have been used by Dr. Broniaczyk to successfully express in cell culture.
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Forbes, Nicole E. "Multifunctional Adaptive NS1 Mutations Are Selected Upon Influenza A Virus Evolution in the Mouse." Thesis, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23553.
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Influenza A virus (IAV) can evolve from low virulence in animal hosts to become highly virulent in humans. Pandemic Influenza A viruses such as the 1918 Spanish Influenza caused over 50 million deaths worldwide. However the genetic determinants of IAV host adaptation and virulence are largely uncharacterized. The IAV NS1 protein is a multifunctional interferon antagonist and a known virulence factor. We hypothesized that NS1 mutations selected upon IAV evolution to a novel host contribute to host adaptation by mechanisms involving increased gene expression and IFN antagonism. To this end, I phenotypically characterized the NS1 mutations selected upon adaptation of A/Hong Kong/1/1968 (H3N2) (HK-wt) to increased virulence in the mouse. Sequencing the NS genome segment of mouse-adapted variants revealed eleven mutations in the NS1 gene and four in the overlapping NEP gene. Using the HK-wt virus and reverse genetics to express recombinant HK NS1 mutant viruses, I demonstrated that all NS1 mutations were adaptive and enhanced virus replication (up to 100 fold) in mouse cells and/or lungs. All but one NS1 mutant was associated with increased virulence measured by survival and weight loss in the mouse. Ten of twelve NS1 mutants significantly enhanced IFN-β antagonism to reduce the level of IFN-β production relative to HK-wt in infected mouse lungs at 1 day post infection, where nine mutants induced viral yields in the lung that were ≥ HK-wt (up to 16 fold increase). Eight of 12 NS1 mutants had decreased binding affinity to the cleavage and polyadenylation specificity factor (CPSF30). The majority of mutant NS1 genes demonstrated increased viral polymerase activity and viral protein production in mouse cells. Viral protein production and viral growth were also assessed in human and canine cell lines; however these adaptive phenotypes were more robust in infected mouse cells. Adaptive NS1 mutations also increased cytoplasmic cellular localization of the NS1 protein in infected cells in a host cell-specific manner. Evaluation of phenotypic trends associated with the NS1 mutants demonstrated an inverse correlation between CPSF30 binding affinity and viral polymerase activity enhancement. This study demonstrates that NS1 is a multifunctional virulence factor subject to adaptive evolution.
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Johnson, Benjamin Francis. "An investigation of influenza A virus NS1 gene variation and its relationship to virulence." Thesis, University of Reading, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437128.
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Pearson, James L. "Analysis of transactivation of the capsid gene promoter of MVM by the NS1 protein." free to MU campus, to others for purchase, 1999. http://wwwlib.umi.com/cr/mo/fullcit?p9962554.
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Koliopoulos, Marios Grigorios. "Structural and functional basis for TRIM25 E3 ligase catalytic activity and NS1-mediated suppression." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/10038260/.
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Ubiquitination is a post-translational modification of proteins with broad regulatory roles across cellular biology. This process involves the addition of ubiquitin molecules on target proteins, altering their cellular role and properties. Ubiquitination is performed by an enzymatic cascade consisting of three enzymes: ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). The tripartite motif (TRIM) family of proteins constitutes one of the largest subfamilies of RING E3 ligases and the majority of them are contributing to the regulation of innate immune responses. They are characterized by a conserved tripartite motif in their N-terminal region which comprises a RING domain, one or two B-box domains and a coiled-coil region. Self-association is believed to be crucial for catalytic activity of TRIM proteins, however, the precise molecular mechanism underlying this observation remains elusive. The work presented in this thesis provides insights into the E3 ligase function of TRIM25 and shows how its oligomeric state is linked to its catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and a ubiquitin-loaded E2 identifies the structural and mechanistic features that promote activation of E2~Ub allowing us to propose a model for the regulation of activity in the full-length protein. In the second part of this thesis, the molecular details of Influenza A NS1-mediated TRIM25 inhibition are presented. The crystal structures of NS1 bound to TRIM25 along with biochemical analysis allowed us to identify the interacting domains and propose a model for the inhibition of substrate ubiquitination during viral infection. The results of this project extend our understanding of the mechanism, structure and regulation of TRIM E3 ligases and their substrates, leading to increased chances of targeting specific steps of the ubiquitination pathway during disease.
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Costa, Vivaldo Gomes da. "Acurácia diagnóstica de dois kits comerciais ELISA para captura do antígeno NS1 no diagnóstico precoce do vírus dengue: uma meta-análise." Universidade Federal de Goiás, 2015. http://repositorio.bc.ufg.br/tede/handle/tede/4881.
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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
The diagnosis of dengue virus (DENV) infection still remains a challenge, due to cross-reactivity between serological tests and to traditional methods that capture IgM, which is a late marker of infection. However, NS1 antigen is an early marker. Accordingly, several studies have evaluated the performance of tests that utilize NS1 capture, but the results of individual studies may be limited due to the restricted sample size of the patients recruited. Therefore, our objective was to perform a meta-analysis of the diagnostic accuracy of two commercial NS1 ELISAs (Panbio® and Platelia™). Methods and Results: Studies of interest were found in PubMed, Embase and Google Scholar databases using defined inclusion/exclusion criteria. A total of 30 studies containing 12.105 total enrolled patients were included. The overall estimated sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio were as follows: 66% (95% confidence interval (CI) 61-71), 99% (95% CI 96 -100), 98 (95% CI 20-464) 0.3 (95% CI 0.2-0.4) and 289 (95% CI 59-1412), respectively, for Panbio®, and 74% (95% CI 63-82 ), 99% (95% CI 97-100), 175 (95% CI 28-1099), 0.3 (95% CI 0.2-0.4) and 663 (95% CI 98-4478), respectively, for Platelia™. The lowest sensitivity values were for secondary infections (57% [95% CI 47-67] and 66% [95% CI 53-77] for Panbio® and Platelia™, respectively) and for the detection of DENV4. Regarding clinical manifestations, the sensitivity of Platelia™ was 69% (95% CI 43-86) and 60% (95% CI 48-70) for fever and dengue hemorrhagic fever, respectively. In addition, the sensitivity of both tests was slightly lower for samples from Southeast Asia and Oceania. Conclusion: DENV1 samples gave higher sensitivity results for both tests. We observed that factors negatively influencing the tests, such as the type of infection and viral serotype, require further investigation to optimize the diagnostic accuracy.
O diagnóstico das infecções pelo dengue vírus (DENV) continua um desafio, principalmente devido a ocorrência de reações cruzadas entre os testes sorológicos e devido aos tradicionais métodos para captura de IgM constituírem marcadores tardio da infecção. Todavia, o antígeno NS1 é um marcador precoce. Nesse contexto vários estudos tem avaliado a performance dos testes para a captura do NS1, porém os resultados dos estudos individuais podem ser limitados, por causa do restrito tamanho amostral dos pacientes recrutados. Portanto, nosso objetivo foi realizar uma meta-análise da acurácia diagnóstica de dois ensaios comerciais ELISA NS1 (Panbio® e Platelia™). Métodos e Resultados: Os estudos de interesse foram extraídos das bases de dados PubMed, Embase e Google Acadêmico, com definidos critérios de inclusão e exclusão. Um total de 30 estudos, perfazendo 12.105 pacientes recrutados, foram incluídos na análise estatística. A estimativa global da sensibilidade, especificidade, razão de verossimilhança positiva e negativa, razão de chance diagnóstica foram: 66% (95% intervalo de confiança (CI) 61-71), 99% (95% CI 96-100), 98 (95% CI 20-464), 0.3 (95% CI 0.2-0.4) e 289 (95% CI 59-1412), respectivamente para o kit da Panbio®. Enquanto para o kit da Platelia™, os resultados obtidos foram, respectivamente: 74% (95% CI 63-82), 99% (95% CI 97-100), 175 (95% CI 28-1099), 0.3 (95% CI 0.2-0.4) e 663 (95% CI 98-4478). A menor performance dos testes ocorreram nas infecções secundárias e na detecção do DENV4. Quanto às formas clínicas da dengue, a sensibilidade do Platelia™ foi de 69% (95% CI 43-86) e 60% (95% CI 48-70), para a febre da dengue e febre hemorrágica, respetivamente. A sensibilidade de ambos os testes foram discretamente menores para as amostras provenientes da Ásia e Oceania. Conclusão: As amostras de DENV1 forneceram maior sensibilidade para ambos os testes. Observamos que os fatores influenciando negativamente os testes, tais como o tipo de infecção e o sorotipo viral necessitam de maiores investigações no intuito de melhor aperfeiçoamento da acurácia diagnóstica
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Dankar, Samar. "Identification of Mutations in the NS1 Gene That Control Influenza A Virus Virulence in the Mouse Model." Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23371.
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The genetic requirements for Influenza virus to infect and adapt to new species is largely unknown. To understand the evolutionary steps required by a virus to become virulent, a human virus (A/HK/1/68) (HK), avirulent in mice, was subjected to 20 and 21 serial lung-to-lung passages in mouse. Sequence analysis revealed the emergence of eleven mutations within the NS1 gene of the new virulent strains, many of which occurred in binding sites for transcriptional and translational cellular factors. In the present study we have rescued viruses containing each of the NS1 mouse adapted mutations onto A/PR/8/34 (PR8) backbone. We found 9 of 16 NS1 mutants were adaptive by inducing mortality, body weight loss in BALB/c mice and enhanced virus replication in MDCK cells with properties of host cell interferon transcription inhibition. Sequence comparisons with the highly pathogenic A/Hong Kong/156/1997 (H5N1) and the most severe pandemic A/Brevig Mission/1/1918 (H1N1) NS1 genes showed convergent evolution with some of the mouse adapted viruses for F103L plus M106I and V226I plus R227K mutations respectively. The F103L and M106I mutations in the HK NS1 gene were shown to be adaptive by assessment with respect to replication, early viral protein synthesis, interferon-β antagonism and tropism in the mouse lung. We extended the study and proved increased virulence associated with F103L+M106I mutations in their respective H5N1 NS1 gene on the PR8 and HK backbones, as well as the PR8 NS1 gene and the H9N2 (A/Ck/Bj/1/95) gene in the PR8 and A/WSN/33 backbones respectively. However the V226I and R227K mutations in their respective HK and 1918 NS1 genes slightly enhanced virulence and viral growth at later stages of infection. This study demonstrates that NS1 is a virulence factor; involved in multiple viral processes including interferon antagonism and viral protein synthesis. Furthermore, NS1 mutations acquired during mouse adaptation are proven to be adaptive in human, mouse and avian NS1 genes.
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Café, Lilian Pinheiro. "Frequência e distribuição dos sorotipos da dengue circulantes no estado de Sergipe no primeiro semestre de 2016." Pós-Graduação em Ciências Aplicadas à Saúde, 2016. http://ri.ufs.br/jspui/handle/riufs/8026.
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The diagnosis of dengue cases in Central Laboratories of Public Health (LACEN) of the country includes the research of NS1 protein and only in positive cases, diagnostic
confirmation occurs by identifying the serotype. Data provided by the Epidemiological
Surveillance of Sergipe revealed that the first half of 2016 all suspected cases that reached the LACEN / SE were negative on screening tests, even in the thirty-first epidemiological week of the same year, Sergipe has reached the seventh place in the racking of dengue cases in the Northeast region and the third in the Quick Survey Infestation Index by Aedes aegypti. This study aimed to identify the serotypes and the epidemiological profile of suspected dengue cases in the first half of 2016. The 437 suspected dengue patients from January to June of this year were registered in LACEN / SE. After exclusion criteria, 382 serum samples of these patients diagnosed and negative for dengue by ELISA NS1 Antigen Platelia kit method were analyzed by RT-PCR in real time on the multiplex system for confirmation of suspected cases of dengue. The distribution of patients according to epidemiological and demographic variables revealed that there was a predominance of cases in the south central region of the state and large Aracaju (53.5%) inferring the ease in access to outpatient and laboratory service in the region the most affected gender was female (62.8%) and the age group of highest incidence was 20-59 years (59.3%). The highest rate was in urban areas (84.9%) and the first three days of symptoms (63.9%) with higher sampling rate. The prevalence of dengue in cases initially presented as a suspect was 22.5% (86) distributed among serotypes DENV4 82.5% (71), DENV1 9.3% (8) and DENV3 8.1% (7). Compared to official data reported that there were no cases of dengue in the period, this study reveals the positive for dengue with cocirculation
of three distinct serotypes being DEV4 the predominant. As also, reports the
first evidence of the last ten years of DENV3 serotype circulating in the state. Data
analysis enabled to view an underreporting of cases screened and thus suggest a
reassessment of the methods used as diagnostic screening so that they can take control measures to the potential for severe disease in a population.O diagnóstico de casos de dengue nos Laboratórios Centrais de Saúde Pública (LACEN) do país contempla a pesquisa da proteína NS1 e, somente em casos positivos, a confirmação diagnóstica ocorre pela identificação do sorotipo. Dados disponibilizados pela Vigilância epidemiológica de Sergipe revelaram que no primeiro semestre de 2016 todos os casos suspeitos que chegaram ao LACEN/SE foram considerados negativos nos testes de triagem, ainda que na trigésima primeira semana epidemiológica do mesmo ano, Sergipe tenha alcançado o sétimo lugar no racking de casos de dengue da região Nordeste e o terceiro no Levantamento Rápido do Índice de Infestação por Aedes aegypti. O presente trabalho teve como objetivo conhecer da dengue no estado de Sergipe no primeiro semestre de 2016 relacionando-os com alguns parâmetros epidemiológicos. Os 437 pacientes suspeitos de dengue, de janeiro a junho do presente ano, foram cadastrados no LACEN/SE. Após critérios de exclusão, 382 amostras séricas destes pacientes com diagnóstico e resultado negativo para dengue através da metodologia ELISA NS1 Antígeno do kit Platelia foram analisadas pela técnica de RT-PCR em tempo real no sistema multiplex para a confirmação dos casos suspeitos de dengue. A distribuição dos pacientes de acordo com as variáveis epidemiológicas e demográficas revelou que houve predominância dos casos na região centro-sul do estado e da grande Aracaju (53,5%) podendo-se inferir a facilidade no acesso ao atendimento ambulatorial e laboratorial nessa região, o gênero mais acometido foi o feminino (62,8%) e a faixa etária de maior incidência foi 20 a 59 anos (59,3%). A maior frequência foi na zona urbana (84,9%) e os três primeiros dias dos sintomas (63,9%) com maior índice de coletas. A incidência de dengue nos casos que incialmente se apresentaram como suspeita foi de 22,5% (86) distribuídas entre os sorotipos DENV4 82,5% (71), DENV1 9,3% (8) e DENV3 8,1% (7). Em comparação aos dados oficias que informavam que não houve casos de dengue no referido período, este estudo revela a positividade para dengue com co-circulação de três sorotipos distintos sendo o DENV4 o predominante. Ademias, relata a primeira evidência, dos últimos dez anos, de circulação do sorotipo DENV3 no estado. A análise dos dados permitiu visualizar uma subnotificação dos casos triados e, assim, sugerir uma reavaliação dos métodos utilizados como triagem diagnóstica para que se possam tomar medidas de controle para riscos potenciais de formas graves da doença numa população.
Lagarto, SE
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Lohrmann, Florens [Verfasser], and Otto [Akademischer Betreuer] Haller. "Charakterisierung einer C-terminalen Elongation des Influenza-A NS1-Proteins in vitro und in vivo." Freiburg : Universität, 2014. http://d-nb.info/1120020549/34.
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Richard, Audrey. "Oncolytic H-1 parvovirus NS1 protein : identifying and characterizing new transcriptional and posttranslational regulatory elements." Phd thesis, Université du Droit et de la Santé - Lille II, 2011. http://tel.archives-ouvertes.fr/tel-00826936.
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H 1 parvovirus (H-1PV) is a little single stranded DNA virus that preferentially replicates in a lytic manner in transformed cells due to their expression profile that meets the requirements for the activation of H¬ 1 PV life cycle unlike normal cells. This feature is known as oncotropism. H 1PV genome is constituted by two transcriptional units. The first one is driven by the proliferation and transformation dependent P4 promoter and allows the expression of both non structural proteins NS1 and NS2, and the second one controls the expression of both capsid proteins VP1 and VP2 through the activation of P38 promoter. H-1PV life cycle tightly depends on NS1 protein that is involved in crucial events, including viral DNA replication, P38 promoter activation as well as cytotoxicity.NS1 protein is regulated at both transcriptional and post translational levels.My thesis aimed at identifying new determining elements for both of these regulations and characterizing their involvement in both H-1PV life cycle and oncotropism.
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Xisto, Mariana Fonseca. "Expressão heteróloga da proteína não-estrutural 1 (NS1) do vírus dengue-2 em Arabidopsis thaliana." Universidade Federal de Viçosa, 2015. http://www.locus.ufv.br/handle/123456789/6696.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A dengue é a doença mais importante causada por um arbovírus no mundo. O vírus da dengue pertence à família Flaviviridae e possui 4 sorotipos antigenicamente distintos. São vírus de RNA fita simples, polaridade positiva, com o genoma de aproximadamente 11 Kb que é traduzido em uma poliproteína subdividida em três proteínas estruturais e sete proteínas não- estruturais (que estão relacionadas com a replicação viral, expressão das proteínas virais e virulência dos sorotipos). Nos últimos vinte anos têm sido observado um incremento significativo na atividade epidêmica, expansão da distribuição geográfica e transmissão contínua dos diferentes sorotipos em áreas onde a doença não era prevalente. Um dos eventos mais alarmantes tem sido o aumento do número de casos da Febre da Dengue Hemorrágica (FDH) nas Américas. Um ponto ainda limitante dos testes diagnósticos que capturam o anticorpo presente no soro de pacientes infectados consiste na dificuldade de obtenção de baixo custo e produção em grande escala dos antígenos. Diante destas considerações objetivou-se expressar a proteína não-estrutural (NS1) do vírus dengue-2 em plantas transgênicas de Arabidopsis thaliana. O gene da proteína NS1 foi otimizado para a expressão em plantas e clonado no plasmídeo pCAMBIA3301. O vetor de expressão pCAMBIA3301/NS1 foi utilizado para transformação de Arabidopsis thaliana. A terceira geração de plantas transformadas foi selecionada em homozigose dominante. A transformação de Arabidopsis thaliana foi evidenciada pela imunolocalização da proteína NS1 marcada dentro do lúmen do retículo endoplasmático em tecidos foliares. Este sistema será utilizado para obtenção do antígeno NS1 com finalidade de aplicação em testes de diagnóstico da dengue.
Dengue fever is the most important disease caused by an arbovirus in the world. The dengue virus belongs to the Flaviviridae family and has four antigenically distinct serotypes. They are single stranded RNA viruses, positive polarity, with a genome of approximately 11 Kb which is translated into a polyprotein subdivided in three structural proteins and seven non-structural proteins (that are related to viral replication, expression of viral proteins and virulence of serotypes). In the last twenty years, it was observed a significant increase in the epidemic activity, expansion of the geographical distribution and streaming of different serotypes in areas where the disease was not prevalent. One of the most alarming events was the increase in the number of cases of dengue hemorrhagic fever (DHF) in the Americas. A further limiting point of the diagnostic tests that capture antibody present in the serum of infected patients is the difficulty of obtaining low-cost and large-scale production of the antigens. Due to this factor, we aimed to build a system in transgenic Arabidopsis thaliana expressing the nonstructural protein (NS1) of the dengue-2 virus. The gene of the NS1 protein was optimized for expression in plants and cloned into the plasmid pCAMBIA3301. The expression vector pCAMBIA3301/NS1 was used for transformation of Arabidopsis thaliana. The third generation of transformed plants were selected by dominant homozygous. The construction of a transgenic Arabidopsis thaliana system was demonstrated by immunolocalization of the NS1 protein into the lumen of the endoplasmic reticulum in the leaf tissues. This system will be used to obtain the NS1 antigen with application purpose of dengue diagnostic tests.
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Zielecki, Florian [Verfasser]. "Analyse molekularer Pathomechanismen des viralen NS1 Proteins eines Influenza A Virus vom Subtyp H5N1 / Florian Zielecki." Berlin : Freie Universität Berlin, 2010. http://d-nb.info/1024104028/34.
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Li, Shoudong. "Modulations of PACT-PKR Pathway by Cellular Stresses and the NS1 Protein of Influenza A Virus." Connect to text online, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1114788508.
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Chen, Rui. "Functional analysis of interactions between influenza A virus protein NS1 and cellular proteins TRBP and PACT." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25717.
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Seasonal and pandemic Influenza virus infections cause about three to five million cases of severe illness and about 250,000 to 500,000 deaths world-wide annually according to the WHO. Although investigated intensively, Influenza virus pathogenesis is still not very well understood and hard to predict. Influenza A viruses contain a segmented, single-(-) stranded RNA genome encoding at least 10 different proteins and are highly diverse due to hypermutation and reassortment. In previous work, 56 viral genes from six different influenza A virus isolates had been cloned and genome-wide screened for virus-host protein interactions using yeast-two hybrid technology and several human and chicken cDNA libraries, leading to the identification of 127 high-confidence cellular interactors of which 40 have also been identified by RNA interference in other studies. In this thesis, two of the cellular interactors identified which both bound to the viral multifunctional protein NS1, TRBP and PACT, were further investigated with regard to their role in virus life cycle. These two proteins are known to be involved in miRNA silencing and PKR regulation. Both interactions between NS1 and TRBP and NS1 and PACT were confirmed by co-immunoprecipitation, and both TRBP and PACT co-localized with NS1 in a cytosolic compartment. NS1 was also found to be present in the RISC complex in pull-down assays with the RISC core component Ago2. In functional assays, NS1 dose-dependently inhibited RNA silencing. Although no differences in TRBP-binding between NS1 proteins of various different influenza strains could be detected in direct mating Y2H assays, they varied with regard to their inhibitory activity on RNA silencing. TRBP and PACT alone were unable to restore NS1-induced inhibition of RNA silencing activity, however both together restored RNA silencing. Moreover, the siRNA knockdown of PACT abolished the association of NS1 with Ago2, and NS1 competitively inhibited the binding of TRBP and PACT to Ago2. The depletion of either TRBP or PACT led to an inhibition of influenza virus replication. The depletion of TRBP also lifted cellular IFNβ level without infection. However, the knockdown of TRBP but not PACT blocked IFNβ production and increased cell viability post infection. These results indicate that NS1 inhibits the binding of PACT and TRBP to the RISC complex and thereby inhibits miRNA-induced gene silencing. The hypothesis that TRBP supports influenza replication potentially by regulating PKR regulation and IFNβ induction requires further investigation. In conclusion, this study provides evidence for the complexity of virus-host interactions and the dual role of viral proteins in activating both positive and negative regulatory cellular mechanisms.
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Lima, Monique da Rocha Queiroz. "Avaliação de testes de captura de antígeno NS1 para o diagnóstico precoce das infecções por dengue." reponame:Repositório Institucional da FIOCRUZ, 2009. https://www.arca.fiocruz.br/handle/icict/3823.
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Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.
O diagnóstico laboratorial de dengue é muito importante para apoiar os programas Vigilância Epidemiológica considerando-se a dificuldade da confirmação dos casos em bases clínicas apenas, em especial, durante períodos inter-epidêmicos. Atualmente estão disponíveis kits comerciais para o diagnóstico sorológico do dengue, embora o seu custo ainda represente um alto encargo financeiro para países em desenvolvimento. Um diagnóstico rápido pode direcionar as medidas de controle do vetor. A proteína não-estrutural 1 (NS1) do vírus dengue por ser um marcador utilizado durante a fase aguda da doença e tem sido proposto para o diagnóstico da doença. Desta forma, a sensibilidade e especificidade de três kits comerciais para captura de antígeno NS1 disponíveis no mercado foram avaliadas com um painel de 852 amostras obtidos a partir da coleção do Laboratório de Flavivírus no Instituto Oswaldo Cruz, FIOCRUZ, de epidemias ocorridas durante os anos de 1986 a 2008. O desempenho de cada kit foi avaliado individualmente e, a comparação entre os três kits foi baseada na análise de uma sub-população de 450 amostras. Dentre os três kits analisados, o kit NS1 Ag Strip (BioRad Laboratories) foi o mais sensível em confirmar casos de dengue na amostragem testada (89%, 197/220), seguido pelo Platelia NS1 (BioRad Laboratories) (84%, 184/220) .O menos sensível foi o pan -E Early ELISA (PanBio Diagnostics) com 72% (159/220) de sensibilidade. Porém, neste estudo o kit da PanBio foi o mais especifico (100%), enquanto que ambos os kits da BioRad apresentaram 99% de especificidade. Os resultados obtidos demonstraram uma maior sensibilidade de confirmação de casos de infecção primária pelos três kits, porém não houve diferença significativa em relação aos casos de infecção secundária. Os três kits foram mais sensíveis em confirmar casos positivos por isolamento viral do que em casos positivos por RT-PCR. A sensibilidade dos três kits foi maior no período compreendido entre o primeiro ao quinto dia após o inicio dos sintomas. Reações cruzadas foram raramente observadas em vacinados contra o vírus da febre amarela e casos de rubéola. Os resultados obtidos demonstraram que três kits podem ser utilizados para a detecção precoce da infecção viral por dengue.
Dengue virus diagnosis is an important tool to support Epidemiological Surveillance Programs considering the difficulties found in confirm dengue cases based only on the clinical symptoms, especially during inter-epidemic periods. Currently, there are many commercial serological kits for dengue diagnosis, however its costs poses a financial burden for many developing countries. The dengue virus non- structural protein a (NS1) can be used as a marker during the acute phase of the illness and its use has been proposed for the disease diagnosis. Therefore, here we evaluated the sensitivity and specificity of three newly available NS1 antigen capture commercial kits with a panel of 852 samples from the collection of the Flavivirus Laboratory at the Oswaldo Cruz Institute, FIOCRUZ, from epidemics occurred from 1986 to 2008. Each kit was evaluated individually and the comparison among them was based on the analysis of a sub- population of 450 samples. From the three kits analyzed, the NS1Ag Strip (Biorad Laboratories) showed the highest sensitivity (89%, 197/220) in confirming dengue cases, followed by the PlateliaTM NS1 (Biorad Laboratories). The less sensitive one was the pan-E Early ELISA (PanBio Diagnostics) with a sensitivity of 72% (159/220). However, in this study the PanBio kit was the most specific (100%) while the two kits from BioRad showed both 99% of specificity. Primary dengue cases were more frequently confirmed than secondary ones. A higher sensitivity was observed in cases positive by virus isolation, when compared to cases positive by RT-PCR. The highest NS1 antigen detection was from the first to the fifth day after the onset of the symptoms. Cross reactivity were rarely observed in yellow fever vaccinees and rubella cases. The results showed that the three kits can be used in the early diagnosis of dengue infections.
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PURIFICAÇÃO, JUNIOR Antonio Fernando da. "Expressão de proteínas quiméricas contendo epítopos NS1 do vírus da dengue para fins diagnósticos e vacinais." Universidade Federal de Pernambuco, 2017. https://repositorio.ufpe.br/handle/123456789/25092.
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CNPQ
A dengue é uma importante doença de aspecto emergente e um grande desafio para a saúde pública mundial. No mercado, existe apenas uma vacina licenciada que não é totalmente eficaz na prevenção da doença. Também existem dificuldades para se expressar antígenos virais em sistemas procarióticos para uso no diagnóstico sorológico. O presente estudo, por meio da engenharia de proteínas, propõe moléculas alternativas de interesse diagnóstico e/ou vacinal para a dengue. Com o uso de ferramentas computacionais seguindo a estratégia “epitope-scaffolding”, elementos estruturais da NS1 de dengue (epitope) foram transplantados para a TOP7 (scaffold). Os genes sintéticos para as variantes dessa proteína, levando em consideração os 4 sorotipos de dengue, totalizando 4 proteínas recombinantes, foram otimizados, sintetizados comercialmente e clonados em vetor de expressão. As proteínas recombinantes tiveram sua expressão confirmada por ensaios de imunoblot e purificadas por cromatografia de afinidade, apresentando alto rendimento e solubilidade. Foram realizados ELISAs, com soros de pacientes infectados com DENV-3, para avaliação imunológica das proteínas quiméricas expressas. Os sinais detectados para as quimeras sugerem que elas são potencialmente imunogênicas.
Dengue is an important emerging disease and a major public health challenge worldwide. Currently, there is only one licensed vaccine that is not fully effective in preventing the disease. There are also difficulties in expressing viral antigens in prokaryotic systems for use in serological diagnosis. The present study, through the engineering of proteins, proposes alternative molecules to be employed in strategies for diagnostics and/or vaccines for dengue. With the use of computational tools following the epitope-scaffolding approach, structural elements of dengue NS1 (epitopes) were transplanted to the TOP7 protein (scaffold). Synthetic genes for 4 recombinant proteins, taking into account the 4 serotypes of dengue, were optimized, commercially synthesized and cloned into expression vector. The cells of Escherichia coli BL21 were transformed with the respective plasmid constructs. Proteins were confirmed by immunoblot assays and purified by affinity chromatography, exhibiting high yield and solubility. ELISAs, with sera from patients infected by DENV-3, were performed for the immunological evaluation of expressed chimeric proteins. Signals detected for the chimeras suggest that they are potentially immunogenic.
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Stecho, William M. "The effect of adaptive mutations in the influenza A NS1 protein on CPSF30 and PABP1 binding." Thesis, University of Ottawa (Canada), 2009. http://hdl.handle.net/10393/28111.
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The Influenza A NS1 protein is an interferon (IFN) antagonist and a major virulence determinant. To characterize the genetic basis of NS1-mediated virulence, highly pathogenic mouse-adapted Influenza A strains were derived from human A/Hong Kong/1/68 H3N2 by experimental evolution in the mouse lung. Within these strains seven specific NS1 mutations were identified, some of which conferred greater IFN resistance and/or increased viral protein synthesis to the virus. Most of these mutations were shown to affect NS1 CPSF30 binding, which may affect post-transcriptional processing and increase host IFN induction. Some of these mutations were also shown to increase NS1 binding to host translation initiation factor PABP1, supporting a model of increased IFN resistance through direct modulation of host translation.
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Soubies, Sébastien Mathieu. "Caractérisation de facteurs impliqués dans l'adaptation des virus influenza à leurs hôtes : exemple de la protéine NS1." Toulouse 3, 2010. http://www.theses.fr/2010TOU30214.
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Afin de mieux comprendre l'implication de la protéine NS1 dans la pathogénicité des virus influenza aviaires, nous avons reconstitué par génétique inverse la souche A/Turkey/Italy/977/1999 (H7N1). Dans un premier temps, nous avons produit un virus mutant exprimant une NS1 fortement tronquée, nommé H7N1 1-99. Ce virus, bien qu'incapable de se répliquer efficacement dans des fibroblastes embryonnaires de canard (DEF), conserve une capacité réplicative similaire à celle du virus sauvage lors de l'infection de cellules Vero, qui ne peuvent pas produire d'interférons de type I. En outre, le virus H7N1 1-99, à la différence du virus sauvage, a induit une forte production d'interférons de type I lors de l'infection de DEF. Dans un second temps, nous avons étudié les conséquences du polymorphisme C-terminal de la protéine NS1 lors de l'infection de diverses espèces. Pour cela, nous avons produit un virus exprimant une NS1 avec d'un motif C-terminal typiquement aviaire de séquence ESEV (nommé H7N1 ESEV) ainsi qu'un virus mutant dont la NS1 possède le motif RSKV (nommé H7N1 RSKV) préférentiellement associé aux souches humaines. Lors de l'infection de cellules humaines, le motif RSKV a conféré une plus grande capacité réplicative au virus. En revanche, sur cellules murines, le motif ESEV, aviaire, a rendu le virus plus pathogène. Enfin, Le virus H1N1 RSKV s'est répliqué plus efficacement lors de l'infection de canard tout en induisant une plus forte transcription de Mx, un gène induit par les interférons de type I. Ces données montrent que le domaine C-terminal de la protéine NS1 confère une pathogénicité qui dépend des espèces d'hôtes infectésAvian influenza viruses represent a major public health concern. Particularly, the NS1 viral protein, which counteracts the innate immune response, is more and more considered as a virulence factor. How in order to understand how NS1 contributes to avian influenza pathogenicity, we used reverse genetics to reconstitute the avian strain A/Turkey/Italy/977/1999 (H7N1). We first generated a mutant virus expressing a truncated protein of 99 aa, named H7N1 1-99. This virus, whose replication is impaired in duck embryonic fibroblasts (DEF), remains able to replicate to high titres in the interferon deficient cell line Vero. Unlike the wild-type virus, the mutant virus induces a strong interferon production in DEF. These data underline the role of the NS1 protein during the infection of duck cells. In a second step, we evaluated the consequences of C-terminal polymorphisms of NS1 during the infection of several species. We therefore generated a virus whose NS1 protein carries the C-terminal avian sequence ESEV (named H7N1 ESEV) and a mutant virus expressing a NS1 protein with the “human-like” C-terminal sequence RSKV. Viral phenotypes were evaluated in vitro on human, murine or duck cells and in vivo on mice and ducks. In human cells, the H7N1 RSKV virus displayed an increased replicative capacity. Interestingly, the H7N1 ESEV virus was more pathogenic in mouse, with an increased pathogenicity, pulmonary replicationand type I interferon production. In ducks, the H7N1 RSKV virus replicated more efficiently and induced a stronger transcription of Mx, a gene induced by type I interferon. Collectively, these data show that the C-terminal domain of NS1 is a species-depdendent virulence motif
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Silva, Cleide Mara Rosa da. "Renaturação das proteínas não estruturais 1(NS1) dos vírus da zika e da dengue utilizando altas pressões." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/85/85131/tde-26102017-123523/.
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As principais matérias primas necessárias para a preparação de testes diagnósticos são as proteínas dos patógenos que necessariamente apresentem as estruturas nativas. O objetivo do presente estudo foi a obtenção das proteínas não estruturais 1 (NS1) dos vírus da dengue (DENV) e da zika (ZIKV) a partir dos corpúsculos de inclusão (CI) produzidos em bactérias Escherichia coli. Mostramos que a combinação de alta pressão hidrostática (APH) e pH alcalino é eficiente para a solubilização de NS1-CI. A incubação em 2,4 kbar das suspensões de NS1-CI em pH alcalino mostrou-se eficiente para a solubilização da NS1. A presença de Arg promove a dissociação de oligômeros. A aplicação de 2,4 kbar às suspensões de NS1-CI em pH de 10,5 (DENV) e de 11,5 (ZIKV) na presença de Arg e um par redox, seguida de diálise em tampão em pH 8,5, foram as condições escolhidas para o reenovelamento de NS1. Obtivemos ambas NS1 com rendimentos entre 75% e 90% em relação às quantidades totais das proteínas presente nos correspondentes CI de NS1. As NS1 reenoveladas apresentaram reatividade comparável às proteínas obtidas utilizando um protocolo convencional estabelecido, com rendimentos mais de 25 vezes superiores. Foi obtido um processo altamente eficiente para o reenovelamento de NS1 apresentando características biológicas preservadas em relação a reatividade com anticorpos específicos de antígeno, incluindo soro de paciente infectado com zikv e que, portanto, podem ser usados como antígeno para o desenvolvimento de vacinas ou testes de diagnóstico. Além disso, este estudo descreve a criação de um processo inovador, que é a utilização concomitante de APH e pH alcalino, para solubilização e posterior reenovelamento de NS1-CI que podem ser utilizados para outras proteínas relevantes.The main products for the preparation of diagnostic tests are as proteins of the pathogens that necessarily present as the native structures. The objective of the present study was to obtain non-structural proteins 1 (NS1) from dengue virus (DENV) and zika virus (ZIKV) from the inclusion bodies (IBs) produced in Escherichia coli bacteria. We show that it is a combination of high hydrostatic pressure (HHP) and alkaline pH is efficient for a solubilization of NS1-IB. A 2.4 kbar incubation of NS1-IB suspensions at alkaline pH proved to be efficient for NS1 solubilization. The presence of Arg promotes the dissociation of oligomers. The application of 2.4 kbar to the suspensions of NS1-IB at pH 10.5 (DENV) and 11.5 (ZIKV) in the presence of Arg and a redox pair, dialysis in pH 8.5 buffer were as conditions chosen for the refolding of NS1. We obtained both NS1 at yields between 75% and 90% relative to the total amounts of the proteins present in the corresponding NS1 IB. Refolded NS1 showed similar to proteins obtained using an established standard protocol, with yields more than 25 times higher. A highly efficient process for the refolding of NS1 was obtained with preserved biological features regarding reactivity with antigen-specific antibodies, including sera of zikv-infected patients and that can be used as antigen for the development of vaccines or diagnostic tests. In addition, this study describes the creation of an innovative process, which is a concomitant use of HHP and alkaline pH, for solubilization and subsequent refolding of NS1-IB that can be used for other relevant proteins.
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Carneiro, Ana Cláudia Alvarenga. "Estudo dos efeitos da expressão da proteína NS1 de dengue virus na modulação de vias sinalizadoras intracelulares." reponame:Repositório Institucional da UFOP, 2015. http://www.repositorio.ufop.br/handle/123456789/5429.
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Os Dengue virus (DENV) possuem um genoma constituído de RNA senso positivo que expressa proteínas estruturais e não estruturais dentre as quais destaca-se a NS1. Esta desempenha uma atividade na replicação do genoma viral e pode exercer um papel na modulação de vias sinalizadoras celulares. Esta função foi demonstrada por nosso grupo através de em um modelo de células HepG2 expressando NS1 de forma constitutiva em que NS1 altera o perfil de ativação de proteínas da via NF-kB. Dessa forma, este trabalho teve como objetivo avaliar através de ensaios de atividade de luciferase, a atividade transcricional no promotor de IL-6 em células transfectadas de forma estável para a expressão de NS1. Uma vez que altos níveis de IL-6 são detectados no soro de pacientes infectados e que esta citocina pode ser modulada pela via de NF-kB. Os resultados mostraram que a expressão da NS1 aumenta a atividade transcricional no promotor de IL-6 nas células HepG2. Além disso, uma mutação pontual no sítio de ligação de um fator transcricional denominado AP-1 (heterodimero de cFOS e cJUN) no promotor de IL-6, resultou na diminuição desta atividade. Adicionalmente, foi verificado que células expressando NS1 tanto de forma transiente como constitutiva apresentam também uma maior atividade transcricional de AP-1 ativado pela via JNK das MAP cinases. Tais dados sugerem que esta via pode também ser regulada em função da presença de NS1. Outros ensaios de luciferases foram conduzidos para avaliar possíveis alterações nas vias de STAT3 e das MAP cinases MEK/ERK, quando as células foram estimuladas com soro fetal bovino, e avaliar o papel de NS1 na manipulação destas vias. Os resultados obtidos por este trabalho poderão ajudar na compreensão dos mecanismos utilizados pelos DENV na modulação da expressão de genes celulares e auxiliar a condução de pesquisas com foco no desenvolvimento de estratégias terapêuticas para combate ao vírus. _______________________________________________________________________________________________
ABSTRACT: The Dengue virus (DENV) have a genome of RNA positive sense expressing structural and non-structural proteins among which stands out the NS1 which exerts an activity in the viral genome replication and may play a role in the modulation of cell signaling pathways. This function has been demonstrated by our group using HepG2 cells in a model constitutively expressing NS1 in which NS1 alters the protein activation profile of the NF-kB pathway. However, the aim of this work was to evaluate through luciferase activity assays, the expression of pro-inflammatory proteins in HepG2 cells expressing the NS1 protein and also the transcriptional activity in the IL-6 promoter in cells stably transfected for the expression of NS1, since high levels of IL-6 are found in serum of infected patients and that this cytokine may be modulated by the NF-kB pathway. The results showed that the expression of NS1 increases the transcriptional activity of the IL-6 promoter in HepG2 cells. Furthermore, a punctual mutation at the binding site of a transcriptional factor called AP-1 (heterodimer cFos and cJun) in the IL-6 promoter, resulted in decrease of this activity. Additionally, it was verified that cells expressing NS1 both transient and constitutive also exhibit greater transcriptional activity of AP-1 of the JNK pathway activated by MAP kinases. These data suggest that this pathway may also be regulated as a function of the presence of NS1. Further tests were conducted to evaluate possible changes in the STAT3 and MEK/ERK MAP kinase pathway, when the cells were stimulated with fetal bovine serum and also to avaluate the role of NS1 in handling these routes. The results of this study will help to understand the mechanisms used by DENV in modulating the expression of cellular genes and support carrying out researches focused on the development of therapeutic strategies to combat the virus.
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BRASIL, JÚNIOR Aluízio Gonçalves. "Obtenção de bioconjugados anti-NS1 DENV com pontos quânticos fluorescentes como insumos para ensaios diagnósticos da dengue." Universidade Federal de Pernambuco, 2014. https://repositorio.ufpe.br/handle/123456789/24362.
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CAPES
O vírus do dengue (DENV) pertence ao gênero Flavivirus, família Flaviviridae, possuindo RNA genômico de fita simples e apresentando quatro sorotipos antigenicamente relacionados (DENV 1-4). A identificação precoce dos casos de dengue é de vital importância para a tomada de decisões e implementação de medidas de maneira oportuna. No entanto, os métodos de eleição para o diagnóstico das infecções pelos DENV (ensaios imunoenzimáticos de captura de anticorpos das classes IgM e IgG) apesar de possibilitarem a análise rápida e reprodutível, apresentam a limitação da não detecção da enfermidade em sua fase inicial. Tendo por finalidade suplantar essa dificuldade, a obtenção de insumos que possam ser empregados em fluoroimunoensaios de detecção da proteína viral solúvel NS1, apresenta acentuada relevância. Pontos quânticos (PQs) fluorescentes de CdTe foram preparados em água com os estabilizantes, cisteamina (CTM) e ácido mercaptosuccínico (MSA). Os PQs obtidos exibiram elevada fluorescência e estabilidade coloidal, realizando-se a caracterização óptica (emissão/excitação/absorção) e estrutural (DRX/MET). Anticorpos monoclonais anti-NS1 DENV (Mab-anti NS1) específicos para os quatro sorotipos do DENV foram acoplados covalentemente aos PQs, via agentes de acoplamento. Microplacas negras de poliestireno foram previamente sensibilizadas com a proteína viral NS1 nativa, e os bioconjugados obtidos (PQs-Mab anti-NS1) foram utilizados em fluoroimunoensaios de detecção da proteína viral. Os bioconjugados de CdTe/MSA-anti-NS1 DENV para os quatro sorotipos apresentaram resultados promissores nos ensaios de detecção da proteína viral NS1, destacando-se o bioconjugado constituído pelo anticorpo monoclonal anti-NS1 DENV-2. Os bioconjugados foram empregados em ensaios de marcação de células fixadas e não fixadas clone C6/36 infectadas com DENV2, obtendo-se resultados satisfatórios para as células não fixadas.
The dengue virus (DENV) belongs to the Flavivirus gender, Flaviviridae family possessing a single-stranded RNA genome and 4 antigenic related serotypes (DENV1 – 4). The early dengue diagnosis is vital for the efficient medical intervention. The existing diagnostic methods for the DENV infections (immunoenzymatic antibody capture based methods of the IgM and IgG classes) although precise and fast still present limitations related to the diagnosis at early stages of the patology. Aiming the development of new diagnostic products for the immunodetection of the NS1 soluble viral protein is still of great importance. Fluorescent CdTe quantum dots (QDs) were prepared in aqueous medium applying mercaptosuccinic acid (MSA) and cysteamine (CTM) as stabilizing agents and used to produce an immunodiagnostic tool for the DENV detection. The QDs showed high fluorescent yield and great colloidal stability and were characterized by X-ray diffractometry, Electronic transmission microscopy, zeta potential and spectroscopic measurements. Monoclonal anti-NS1 DENV (Mab-anti NS1) antibodies specific for the 4 serotypes were covalently attached to the QDs via coupling agents. Black polystyrene microplates were sensibilized with the viral NS1 native protein and the (QDs-Mab anti-NS1) bioconjugates were applied in immunoassays aiming the detection of the viral protein. The CdTe/MSA-anti-NS1 DENV bioconjugates applied for the four serotypes showed promising results in the detection assays of the viral NS1 protein, being the most efficient the bioconjugate that used the monoclonal anti-NS1 DENV-2. The bioconjugates were used in the staining of fixed and live cells of the clone C6/36 infected with DENV2. The results were satisfactory for the live cells.