Swine influenza viruses (SIV) are a common and an important cause of respiratory disease in pigs. Pigs can serve as mixing vessels for the evolution of reassortment viruses containing both avian and human signatures, which have the potential to cause pandemics. NS1 protein of influenza A viruses is a major antagonist of host defence and it regulates multiple functions during infection by interacting with a variety of host proteins. Therefore, it is important to study swine viruses and NS1-interacting host factors in order to understand the mechanisms by which NS1 regulates virus replication and exerts its host defense functions. Influenza A viruses enter the host through the respiratory tract and infect epithelial cells in the respiratory tract, which form the primary sites of virus replication in the host. Thus, studying SIV infection in primary swine respiratory epithelial cells (SRECs) would resemble conditions similar to natural infection. The objectives of this study were to identify NS1-interacting host factors in the virus-infected SRECs and to understand the physiological role of at least one of the factors in influenza virus infection. The approaches to meet this objective were to generate a recombinant SIV carrying a Strep-tag in the NS1 protein, infect SRECs with the Strep-tag virus, purify NS1-interacting host protein complex from the infected cells by pull-down using strep-tactin resin and then study the physiological role of one of the NS1-interacting partners during influenza infection. Using a reverse-genetics strategy, a recombinant virus carrying the Strep-tag NS1 was successfully rescued and the SRECs were infected with this recombinant virus. The Strep-tag in the NS1 protein facilitated the isolation of an intact NS1-interacting protein complex and the proteins present in the complex were identified by liquid chromatography-tandem mass spectrometry. The identified proteins were grouped to enrich for different functions using bioinformatics. This gave an insight into the different functions that NS1 may regulate during infection and the potential host partners involved in these functions. Among the host proteins identified as potential interaction partners, RNA helicases were particularly of interest to study. Influenza being an RNA virus, RNA helicases could have important functions in the virus life cycle. Among the identified RNA helicases, DDX3 has been shown to regulate IFNβ induction and affect the life cycle of a number of viruses. However, its function in influenza A virus life cycle has not been studied. Hence, this study explored whether DDX3 has any role in the influenza A virus life cycle. Immunoprecipitation studies revealed viral proteins NP and NS1 as direct interaction partners with DDX3. DDX3 is a known component of stress granules (SGs) and influenza A virus lacking the NS1 gene is reported to induce SG formation. Therefore, the role of DDX3 in SG formation, induced by PR8 influenza A virus lacking NS1 (PR8 del NS1) was explored. The results from this study showed that DDX3 co-localized with NP in SGs indicating that DDX3 may interact with NP in the SGs. NS1 protein was found to inhibit virus-induced SGs and DDX3 downregulation impaired virus-induced SG formation. The contribution of the different domains of DDX3 to viral protein interaction and virus-induced SG formation was also studied. While DDX3 helicase domain did not interact with NS1 and NP, it was essential for DDX3 localization in virus induced SGs. Moreover, DDX3 downregulation resulted in the increased replication of PR8 del NS1virus, accompanied by an impairment of SG induction in infected cells. Since DDX3 is reported to regulate IFNβ induction, the role of DDX3 in influenza A virus induced IFNβ induction was also examined. Using small molecule inhibitors and siRNA-mediated gene knockdown, the RIG-I pathway was identified as the major contributor of influenza induced IFNβ induction in newborn porcine tracheal epithelial (NPTr) cells. DDX3 downregulation and overexpression also showed that DDX3 has an inhibitory effect on IFNβ expression induced by both influenza infection and low molecular weight (LMW) poly I:C treatment, which is also a RIG-I ligand. RNA competition assay to identify the mechanism of DDX3-mediated inhibition, showed that RIG-I binding affinity to its ligands LMW poly I:C and influenza viral RNA (vRNA) is much higher than that of DDX3. Furthermore, DDX3 downregulation enhanced titers of the PR8 del NS1 virus, while it did not affect the titers of the wild-type strains of PR8 and SIV/SK viruses. Overall, the results show that DDX3 has an antiviral role and the SG regulatory function of DDX3 has a profound effect on virus replication than the IFNβ regulatory function.

Le virus respiratoire syncytial (RSV) est un virus à ARN de polarité négative. Les études démontrent que toute la population sera infectée par ce virus au moins deux fois avant l’âge de 3 ans. Le RSV peut provoquer plusieurs pathologies respiratoires telles que la bronchiolite aiguë et la pneumonie. Les infections sévères corrèlent avec le développement de l’asthme. Lors d’une infection virale, les particules du RSV sont détectées par le senseur RIG-I qui induit l’activation des facteurs de transcription NF-κB et IRF-3. Respectivement, les facteurs de transcription activeront les réponses inflammatoire et antivirale. Au coeur des pathologies induites par le RSV se trouve une réponse immunitaire mal adaptée. Plus précisément, par l’entremise de NF-κB, le RSV provoque une production exagérée de cytokines et chimiokines qui induisent une réponse inflammatoire démesurée provoquant du dommage tissulaire. Paradoxalement, le RSV est capable d’échapper à la réponse antivirale. Ces deux phénomènes sont contrôlés par l’entremise des protéines non structurales NS1 et NS2. Le mécanisme délimitant le mode d’action de NS1 et NS2 sur la réponse antivirale reste à être déterminé. Avec pour objectif d’élucider comment NS1 et NS2 inhibent la réponse antivirale, nous avons investigué le mécanisme de reconnaissance de l’hôte vis-à-vis de RSV. Nous démontrerons, pour la première fois, que le senseur cytosolique MDA5 est impliqué dans la réponse antivirale contre le RSV. Nous présenterons des résultats préliminaires qui suggèrent que le rôle de MDA5 est non redondant à RIG-I. À l’aide d’ARN interférant dirigé contre RIG-I et de transfection de MDA5, nous démontrerons que MDA5 ne contribue pas à la phosphorylation d’IRF-3, mais plutôt qu’elle régit la stabilité du facteur de transcription. Nous démontrerons aussi que, contrairement à l’hypothèse actuelle sur le fonctionnement de NS1 et NS2, l’inhibition de ces derniers ne provoque pas une augmentation de la cytokine antivirale IFN−β. Cependant, l’expression ectopique de NS1 et NS2 réduit l’activité du promoteur de l’IFN-β et de la protéine cytoplasmic antivirale ISG56 lorsqu’elle est mesurée par essai luciférase. / Respiratory Syncytial Virus (RSV) is a RNA virus with negative polarity. RSV infections are the most common cause of hospitalization among infants. Among populations at risk, infection of RSV can be quite severe. RSV infections can cause bronchiolitis, pneumonia, while severe infections are linked to the development of asthma. Early in the infectious cycle of RSV, the cytosolic sensor RIG-I captures viral particles, and activates the immune response by engaging the transcription factors IRF-3 and NF-κB. At the heart of RSV mediated pathologies is a skewed immune response. More precisely, RSV over stimulates the release of proinflammatory chemokines and cytokines. Intriguingly, while RSV is able to stimulate the production of proinflammatory cytokines and chemokines, RSV under stimulates the antiviral response. The ability of RSV to evade the antiviral response is thought to be mediated by its non-structural proteins: NS1 and NS2. However, the mechanism by which NS1 and NS2 enable RSV to evade the antiviral response remains to be determined. In this memoir we investigated, how RSV is recognized by the innate immune response in airway epithelial cells. With this information we hope to improve our understanding of how NS1 and NS2 allow RSV to circumvent the antiviral response. We show for the first time that cytosolic sensor MDA5 plays a role in the recognition of RSV particles. Using a combination of interfering RNA directed against RIG-I, and transfection of MDA5, we show that MDA5 does not contribute to the phosphorylation of IRF-3. According to the data presented, we suggest that MDA5’s role in the immune response is to prevent the degradation of IRF-3. Contrary to previous research, we show that the inhibition of the nonstructural protein does not increase the production of the antiviral cytokine IFN-β. However, the ectopic expression of NS1 and NS2 does lead to a reduction of the promoter activity of IFN-β and the antiviral protein ISG56 when measured by luciferase assay. This research highlights the importance of MDA5 as a potential therapeutic target in the development of a cure for RSV.

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.

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.

Le virus respiratoire syncytial (RSV) est un virus à ARN de polarité négative. Les études démontrent que toute la population sera infectée par ce virus au moins deux fois avant l’âge de 3 ans. Le RSV peut provoquer plusieurs pathologies respiratoires telles que la bronchiolite aiguë et la pneumonie. Les infections sévères corrèlent avec le développement de l’asthme. Lors d’une infection virale, les particules du RSV sont détectées par le senseur RIG-I qui induit l’activation des facteurs de transcription NF-κB et IRF-3. Respectivement, les facteurs de transcription activeront les réponses inflammatoire et antivirale. Au coeur des pathologies induites par le RSV se trouve une réponse immunitaire mal adaptée. Plus précisément, par l’entremise de NF-κB, le RSV provoque une production exagérée de cytokines et chimiokines qui induisent une réponse inflammatoire démesurée provoquant du dommage tissulaire. Paradoxalement, le RSV est capable d’échapper à la réponse antivirale. Ces deux phénomènes sont contrôlés par l’entremise des protéines non structurales NS1 et NS2. Le mécanisme délimitant le mode d’action de NS1 et NS2 sur la réponse antivirale reste à être déterminé. Avec pour objectif d’élucider comment NS1 et NS2 inhibent la réponse antivirale, nous avons investigué le mécanisme de reconnaissance de l’hôte vis-à-vis de RSV. Nous démontrerons, pour la première fois, que le senseur cytosolique MDA5 est impliqué dans la réponse antivirale contre le RSV. Nous présenterons des résultats préliminaires qui suggèrent que le rôle de MDA5 est non redondant à RIG-I. À l’aide d’ARN interférant dirigé contre RIG-I et de transfection de MDA5, nous démontrerons que MDA5 ne contribue pas à la phosphorylation d’IRF-3, mais plutôt qu’elle régit la stabilité du facteur de transcription. Nous démontrerons aussi que, contrairement à l’hypothèse actuelle sur le fonctionnement de NS1 et NS2, l’inhibition de ces derniers ne provoque pas une augmentation de la cytokine antivirale IFN−β. Cependant, l’expression ectopique de NS1 et NS2 réduit l’activité du promoteur de l’IFN-β et de la protéine cytoplasmic antivirale ISG56 lorsqu’elle est mesurée par essai luciférase. / Respiratory Syncytial Virus (RSV) is a RNA virus with negative polarity. RSV infections are the most common cause of hospitalization among infants. Among populations at risk, infection of RSV can be quite severe. RSV infections can cause bronchiolitis, pneumonia, while severe infections are linked to the development of asthma. Early in the infectious cycle of RSV, the cytosolic sensor RIG-I captures viral particles, and activates the immune response by engaging the transcription factors IRF-3 and NF-κB. At the heart of RSV mediated pathologies is a skewed immune response. More precisely, RSV over stimulates the release of proinflammatory chemokines and cytokines. Intriguingly, while RSV is able to stimulate the production of proinflammatory cytokines and chemokines, RSV under stimulates the antiviral response. The ability of RSV to evade the antiviral response is thought to be mediated by its non-structural proteins: NS1 and NS2. However, the mechanism by which NS1 and NS2 enable RSV to evade the antiviral response remains to be determined. In this memoir we investigated, how RSV is recognized by the innate immune response in airway epithelial cells. With this information we hope to improve our understanding of how NS1 and NS2 allow RSV to circumvent the antiviral response. We show for the first time that cytosolic sensor MDA5 plays a role in the recognition of RSV particles. Using a combination of interfering RNA directed against RIG-I, and transfection of MDA5, we show that MDA5 does not contribute to the phosphorylation of IRF-3. According to the data presented, we suggest that MDA5’s role in the immune response is to prevent the degradation of IRF-3. Contrary to previous research, we show that the inhibition of the nonstructural protein does not increase the production of the antiviral cytokine IFN-β. However, the ectopic expression of NS1 and NS2 does lead to a reduction of the promoter activity of IFN-β and the antiviral protein ISG56 when measured by luciferase assay. This research highlights the importance of MDA5 as a potential therapeutic target in the development of a cure for RSV.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-07-31T19:16:12Z No. of bitstreams: 2 thiago_coimbra_pimenta.pdf: 3361101 bytes, checksum: 026e379b14e8e90fdfd54a1c94b5e25d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-08-14T15:21:20Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) thiago_coimbra_pimenta.pdf: 3361101 bytes, checksum: 026e379b14e8e90fdfd54a1c94b5e25d (MD5) / Made available in DSpace on 2017-08-14T15:21:20Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) thiago_coimbra_pimenta.pdf: 3361101 bytes, checksum: 026e379b14e8e90fdfd54a1c94b5e25d (MD5) Previous issue date: 2017 / A Dengue ? uma doen?a infecciosa viral de transmiss?o vetorial pelo mosquito Aedes aegypti, cujo controle tem se mostrado de solu??o complexa. Al?m disso, o diagn?stico cl?nico ? dif?cil de ser realizado, principalmente na fase aguda da doen?a, em que os sintomas s?o muito similares aos de outras infec??es febris agudas, e por essa raz?o o desenvolvimento de m?todos de detec??o mais efetivos para o seu controle, ganha relev?ncia. M?todos moleculares est?o sendo cada vez mais utilizados para o diagn?stico precoce por serem mais r?pidos e sens?veis. Nesse sentido, o uso de biossensores ganha relev?ncia por serem dispositivos vers?teis, de f?cil constru??o e utiliza??o, sens?veis e seletivos. Combinado com o uso de pol?meros modificadores da superf?cie transdutora, o que favorece a etapa de imobiliza??o das biomol?culas, os biodispositivos assim constru?dos se mostram mais sens?veis devido ? compatibilidade das caracter?sticas do elemento biol?gico e do filme polim?rico. Desta forma, este trabalho visou desenvolver um imunossensor impedim?trico para diagn?stico de Dengue utilizando eletrodos impressos de grafite (EI) funcionalizados com filmes polim?ricos derivados do ?cido 4-aminofenilac?tico (4-AFA). O imunossensor baseou-se na intera??o espec?fica entre o ant?geno da Dengue, a prote?na NS1, e os anticorpos anti-NS1. No estudo de caracteriza??o por voltametria c?clica (VC) da plataforma funcionalizada, observou-se que o filme polim?rico formado apresentou dois picos de oxida??o em +0,17 e +0,35 V em meio de solu??o de ?cido sulf?rico 0,50 M, o que mostra sua adsor??o e eletroatividade na superf?cie do eletrodo. Medidas de espectroscopia de imped?ncia eletroqu?mica (EIE) mostraram maior resist?ncia ? transfer?ncia eletr?nica do EI modificado com o filme polim?rico quando comparado ao EI sem modifica??o, corroborando o estudo anterior. Para o estudo de melhor concentra??o de NS1 para imobiliza??o, verificou-se que 1,00 ng/mL apresentou melhor sinal anal?tico, devido ? diminui??o nas intera??es que ocorre entre os ant?genos. O tempo de imunorrea??o entre ant?geno (Ag) e anticorpo (Ac) tamb?m foi estudado, no qual observou-se que o tempo ideal para que ocorra a imunocomplexa??o foi de 20 minutos. Em seguida, foi realizado estudo de dilui??o dos soros positivos e negativos por EIE, no qual foi poss?vel observar, para o soro positivo, que quanto mais dilu?do o soro maior foi o valor de Rct encontrado. O soro negativo tamb?m apresentou sinal anal?tico, provavelmente devido ? presen?a de anticorpos n?o espec?ficos, por?m, o sinal gerado apresentou valores mais pr?ximos do sinal da NS1, mostrando a seletividade da plataforma proposta. O estudo foi repetido em eletrodo sem modifica??o a fim de verifica a import?ncia do filme polim?rico na constru??o do biossensor. Observou-se que quando a NS1 ? imobilizada ao eletrodo n?o modificado, o Rct aumento muito pouco quando comparado ao eletrodo modificado com o filme derivado do 4-AFA, o que mostra a efici?ncia e sensibilidade da plataforma proposta. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / Dengue is a viral infectious disease transmitted by the Aedes aegypti mosquito, whose control has shown a complex solution. In addition, the clinical diagnosis is difficult to perform, especially in the acute phase of the disease, in which the symptoms are very similar to other acute febrile infections, and for this reason the development of more effective detection methods for its control gains relevance. Molecular methods are being increasingly used for early diagnosis because they are faster and more sensitive. In this sense, the use of biosensors gains relevance because they are versatile devices, of easy construction and use, sensitive and selective. Combined with the use of transducing surface modifying polymers, which favors a stage of immobilization of the biomolecules, the biodevices constructed in this way are more sensitive due to the compatibility of the biological element characteristics and the polymer film. Thus, this work aimed to develop an impedimetric immunosensor to Dengue diagnose using screen-printed electrode (SPE) functionalized with polymer films derived from 4-aminophenylacetic acid (4-APA). The immunosensor was based on the specific interaction between Dengue antigen, NS1 protein, and anti-NS1 antibodies, IgG e IgM. In characterization study by cyclic voltammetry (CV) of the functionalized platform, it has been observed that the polymer film shown two oxidation peaks in +0.17 and +0.35 V in 0,5 M sulfuric acid solution medium, which shows its adsorption and electroactivity at the SPE surface. Electrochemical impedance spectroscopy (EIS) measurements has shown a higher charge transfer resistance to the polymer film modified SPE when compared to the non-modified SPE, corroborating the previous study. For the study of NS1 best concentration to immobilization, it has been verified that 1,00 ng/mL presented better analytical signal, due to the decrease of the interactions that occur between the antigens. Immunoreaction time between antigens (Ag) and antibody (Ab) has also been studied, in which it has been observed that the ideal time for immunocomplexation was 20 minutes. Afterwards, a positive and negative sera dilution study was carried out by EIE, in which it has been possible to observe, for the positive serum, that the more diluted the serum the greater the Rct found. The negative serum also presented analytical signal, probably due to the presence of non-specific antibodies, however, the generated signal presented values closer to the NS1 signal, showing the selectivity of the proposed platform. The study was repeated in electrode without modification in order to verify the importance of the polymeric film in the biosensor construction. It was observed that when NS1 is immobilized to the non-modified electrode, Rct increases very little when compared to the 4-AFA derived film modified electrode, which shows the efficiency and sensitivity of the proposed platform.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-03-29T13:29:04Z No. of bitstreams: 1 monique_rq_lima_ioc_mt_0010_2009.pdf: 1937766 bytes, checksum: d05c52dcbcc952c1a57ff1ff206f06b1 (MD5) / Made available in DSpace on 2012-03-29T13:29:04Z (GMT). No. of bitstreams: 1 monique_rq_lima_ioc_mt_0010_2009.pdf: 1937766 bytes, checksum: d05c52dcbcc952c1a57ff1ff206f06b1 (MD5) Previous issue date: 2009 / 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.

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-04-30T14:39:02Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO - MARINEIDE SOUZA DA SILVA.pdf: 2429345 bytes, checksum: e0dd6988a96097cdd1e4eb856b6b2faf (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-04-30T14:39:25Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO - MARINEIDE SOUZA DA SILVA.pdf: 2429345 bytes, checksum: e0dd6988a96097cdd1e4eb856b6b2faf (MD5) / Made available in DSpace on 2018-04-30T14:39:25Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO - MARINEIDE SOUZA DA SILVA.pdf: 2429345 bytes, checksum: e0dd6988a96097cdd1e4eb856b6b2faf (MD5) Previous issue date: 2017-09-04 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Dengue is currently considered the most important arbovirose. Its main vector is the mosquito Aedes aegypti, present in several regions around the world. The serological diagnosis by means of the NS1 antigen search is the one that has greater applicability, because of easy of execution and the window for detection between the 1st and the 9th day of the onset of symptoms, with a higher frequency until the 5th day. This study aimed to investigate the presence of dengue virus in samples with non-reactive results for the NS1 antigen, assessing if there was a failure in laboratorial diagnosis, or the existence of other arboviruses circulating in the state of Amazonas. Using the RT-qPCR technique, the presence of dengue virus was investigated in 306 serum samples from patients with clinical suspicion of infection. The samples that remained negative were investigated for the presence of the Zika (ZIKV), Chikungunya (CHIKV), Mayaro (MAYV) and Oropouche (OROV) viruses with the same methodology, as well as the presence of IgG and IgM for dengue virus by ELISA. Of the 306 analyzed samples, 17 (5.5%) were positive for DENV, with three sequenced for serotype 4. Thirty-four (10.8%) were positive for ZIKV, one (0.3%) for CHIKV, thirteen 4.2%) for MAYV and nine (2.9%) for OROV. In relation to the NS1 test, all kits evaluated presented 100% agreement in negativity. For the screening of anti-DENV antibodies of the IgG class, of the 306 samples tested 134 (43.8%) had positive results. Regarding the detection of the IgM antibody, different positivities were observed for commercial kits: VIRION (n = 250) 35.6% positive; FOCUS (n = 105) 10.5% positive and PANBIO (N = 80) 20% positive. Our results confirm cases of false negative results for the NS1 tests of three commercial kits, in addition to the circulation of other arboviruses among patients from different municipalities in the state of Amazonas. / A dengue é considerada atualmente a mais importante arbovirose. O seu principal vetor é o mosquito da espécie Aedes aegypti, presente em diversas regiões do mundo. O diagnóstico sorológico por meio da pesquisa do antígeno NS1 é o que tem maior aplicabilidade, pela baixa complexidade na execução e por ser detectado entre o 1º e 9º dia de início dos sintomas, porém com frequência maior até o 5º dia. Esse estudo teve por objetivo principal investigar a presença do vírus dengue em amostras com resultados “Não Reagentes” para o antígeno NS1, avaliando se houve falha no diagnóstico laboratorial, ou a existência de outros arbovírus circulando no estado do Amazonas. Utilizando a técnica de RT-qPCR, pesquisou-se a presença do vírus dengue em 306 amostras de soros de pacientes com suspeita clínica de infecção. As amostras que continuaram negativas foram pesquisadas quanto a presença dos vírus Zika (ZIKV), Chikungunya (CHIKV), Mayaro (MAYV) e Oropouche (OROV), pela mesma metodologia, bem como foi pesquisada a presença de anticorpos das classes IgG e IgM para o vírus dengue por ELISA. Das 306 amostras analisadas 17 (5,5%) foram positivas para DENV, com três sequenciadas para o sorotipo 4. Trinta e quatro (10,8%) foram positivas para o ZIKV, uma (0,3%) para CHIKV, treze (4,2%) para MAYV e nove (2,9%) para OROV. Em relação ao teste NS1 todos os kits avaliados apresentaram 100% de concordância em negatividade. Para a pesquisa de anticorpos anti-dengue da classe IgG, das 306 amostras testadas 134 (43,8%) tiveram resultados positivos. Em relação à detecção do anticorpo IgM foram observadas diferentes positividades para os kits comerciais: VIRION (n=250) 35,6% positivas; FOCUS (n=105) 10,5% positivas e PANBIO (N=80) 20% positivas. Nossos resultados confirmam casos de resultados falso-negativos, para os testes NS1 de três kits comerciais, além da circulação de outros arbovírus entre os pacientes de diferentes municípios do estado do Amazonas.

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.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22810号 / 生博第444号 / 新制||生||59(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 朝長 啓造, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM