No description available.

Neuraminidase (NA) is one of two major antigens on the surface of influenza A viruses. It is comprised of a single N-terminal transmembrane domain (TMD), a stalk domain, and a C-terminal enzymatic head domain that cleaves sialic acid, most notably to release new particles from the host cell surface. NA is only enzymatically active as a homo-tetramer. However, it is not known which properties facilitate the oligomerization of NA during assembly. Our results show that, apart from anchoring the protein to the membrane, the NA TMD also contributes to the assembly process by keeping the stalk in a tetrameric conformation. The ability of the TMD to oligomerize is shown to be dependent on its amphiphilic characteristics that was largely conserved across the nine NA subtypes (N1-N9). Over time the NA TMDs in human H1N1 viruses were found to have become more amphiphilic, which correlated with stronger oligomerization. An old H1N1 virus with a more recent N1 TMD had impaired growth, but readily acquired compensatory mutations in the TMD to restore growth, by reverting the TMD oligomerization strength back to that of the old TMD, demonstrating a biological role of the TMD in folding and assembly. NA and the other viral proteins are spatially and temporally coordinated to achieve optimal viral production. By using a co-transfection analysis, the high AU-content in the NA and HA ER-targeting sequence coding regions (for NA TMD as well as the HA signal sequence) were found to inhibit their expression. The inhibition was alleviated by the early expressed influenza RNA-binding protein NS1, which promoted translation and showed enriched foci at the endoplasmic reticulum (ER). NS1, which expresses early during infection, is therefore likely the regulator of NA and HA to prevent premature expression. These results show that the NA TMD is under substantial selection pressure at both the nucleotide and amino acid level to accommodate its roles in ER-targeting, protein folding, and post-transcriptional regulation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Accepted.</p>

L'objectif principal de cette thèse était de définir un ensemble d'outils méhodologiques pour décrire et étudier la dynamique de la grippe et les paramètres de l'hitsoire naturelle. La cinétique virale (VK) de la grippe est utilisé pour représenter l'infectiosité, qui est la capacité à transmettre l'infection, alors que l'histoire naturele est décrite par la dynamique des symptômes (SD). Nous avons développé un modèle de cinétique virale/dynamique des symptômes (VKSD) basé sur approche de population. Notre modèle prolonge des travaux précédents en incluant la réponse immunitaire innée et en apportant des estimations réalistes des paramètres de l'infection et de la maladie, en prenant en compte la variabilité interindividuelle. Le choix d'un protocole d'étude est crucial pour estimer précisement les paramètres du modèle. Dans ce but nous avons optimisé un ensemble de protocoles afin qu'ils soient économiques et faciles à mettre en place pour aider à la mise en place de futures études. Enfin, nous avons réalisé une étude in silico pour étudier les effets des inhibiteurs de neuraminidases sur l'infection grippale et la maladie ainsi que sur l'émergence de virus résistants. Nous avons exploré l'efficacité de l'oseltamivir pour différentes posologies, chez des sujets sains ou immunodéficients et pour différentes dates de début de traitement. Nous avons identifié les situations pour lesquelles le traitement entraîne un équilibre efficacité/faible émergence de virus résistant. Cette thèse souligne l'importance d'acquérir des connaissance sur la dynamique de l'infection grippale afin de faire face aux épidémies/pandémies et d'en atténuer l'impact

Although seasonal influenza epidemics represent a significant threat to public health, their treatment options remain limited. With deaths from the 1918 influenza pandemic estimated at >50,000,000 worldwide and future pandemics predicted, the need for a potent broad-spectrum influenza therapy is critical. In this thesis, I describe the use of a structurally modified zanamivir, an influenza neuraminidase inhibitor that blocks the release of nascent virus, to deliver attached therapeutic agents specifically to the surfaces of viruses and virus-infected cells, leading to simultaneous inhibition of virus release and immune-mediated destruction of both free virus and virus-infected cells. Chapter 1 describes the major characteristics of the influenza virus, the morbidity and mortality associated with annual infections by current strains of the virus, and the treatments available to reduce the disease burden associated with these infections. Chapter 2 describes the design, synthesis, and evaluation of a zanamivir-related targeting ligand and its conjugation to two orthogonal imaging agents which are then used to characterize the binding specificity and biodistribution of the targeting ligand in influenza virus-infected cells and in infected mice. Chapter 3 describes the development of an influenza virus-targeted immunotherapy, where a zanamivir-targeted hapten is exploited to redirect the immune system to destroy influenza virus and virus-infected cells. When tested in vivo, this immunotherapy is shown to be significantly superior to zanamivir in protecting mice from lethal influenza virus infections. Finally, both a zanamivir-targeted chemotherapy and a CAR-T cell therapy with different mechanisms of cytotoxicity against neuraminidase expressing cells are introduced in Chapter 4.

Sialidose é uma doença neurossomática causada pela deficiência congênita da neuraminidase-1 (Neu1), enzima envolvida na regulação do catabolismo de sialoglicoconjugados nos lisossomos. Com o acúmulo de sialoglicoconjugados, ocorre comprometimento sistêmico e neurológico. Achados histológicos musculares incluem expansão da matriz extracelular (MEC) devido à proliferação anormal de fibroblastos, invasão das fibras musculares por componentes da MEC, fragmentação do citoplasma, formação vacuolar e atrofia das fibras musculares. Entretanto o mecanismo da atrofia muscular na deficiência de Neu1 não está completamente esclarecido, sendo o objetivo desse estudo. Desnervou-se o músculo gastrocnêmio direito de camundongos com deficiência de Neu1 (Neu1 -/-) e de controles Neu1 +/+. Os animais foram eutanasiados 0, 3, 7, 14 e 21 dias pós desnervação. Os músculos desnervados e contralaterais foram submetidos às seguintes análises: 1) histologia geral e medida da área transversa das fibras; 2) autofagia, através da avaliação da presença de vacúolos autofágicos por estudo ultraestrutural e da análise da expressão da proteína LC3; 3) ativação do sistema lisossomal, por reação de fosfatase ácida e análise da expressão proteica de catepsina L e lamp1; 4) deposição de colágeno e infiltração de tecido conjuntivo no tecido muscular; 5) níveis das proteínas Akt e GSK3b; 6) expressão dos atrogenes MuRF1 e Atrogina-1; 7) níveis da proteína MyoD, relacionada à diferenciação muscular; e 8) expressão dos genes Neu1, Neu2, Neu3 e Neu4. Os animais Neu1-/- apresentaram menor peso corporal e muscular compararando-se com animais Neu1 +/+. Houve redução progressiva da área das fibras dos músculos desnervados em relação aos músculos contralaterais. Os animais Neu1-/- apresentaram atrofia muscular basal, com aumento acentuado dos espaços endomisiais e perimisiais. Ocorreu formação de vacúolos autofágicos a partir de 14 dias de desnervação tanto em animais Neu1+/+ quanto em Neu1-/-. Os níveis de expressão proteica de catepsina L e de lamp1 aumentaram a partir de 14 dias de desnervação, mais notadamente em músculos desnervados de camundongos Neu1-/-. A expressão proteica de colágeno III mostrou-se aumentada em animais Neu1-/-, principalmente após desnervação. A expressão proteica da forma fosforilada do Akt (forma ativada) diminuiu após 21 dias de desnervação principalmente em músculos desnervados de animais Neu1+/+. Os níveis de PGSK3 b, forma inativa de GSK3b, diminuíram após a desnervação, em animais Neu1+/+ e animais Neu1-/-. Houve aumento na expressão gênica de Atrogina-1 e MuRF1 após 3 e 7 dias de desnervação, respectivamente; a expressão gênica de Atrogina-1 nos camundongos Neu1-/- teve um aumento atrasado, mostrando diferença significante após 7 dias de desnervação. Não houve diferença significativa entre níveis proteicos de MyoD. A expressão gênica de Neu1 mostrou-se elevada em músculos desnervados de animais Neu1+/+. Conclui-se, portanto, que a Neu1 parece atuar na regulação da massa muscular principalmente controlando o processo de ativação do sistema lisossomal, porém aparentemente sem afetar a autofagia / Sialidosis, a severe neurosomatic disease, results from congenital neuraminidase-1 (Neu1) deficiency. This enzyme regulates the catabolism of sialoglycoconjugates in the lysosomes. Systemic and neurologic manifestations occur due to the sialoglycoconjugates accumulation. In the mouse model for Neu1 deficiency, the muscle histologic findings include extracellular matrix (ECM) expansion, due to abnormal fibroblast proliferation, muscle fibers invasion by ECM components, cytoplasm fragmentation, vacuolar formation and muscle atrophy. Nevertheless the mechanisms of muscle atrophy in Neu1 deficiency are not completely known. This study was designed to investigate Neu1 involvement in muscle atrophy process. Denervation of gastrocnemius muscle was performed by sectioning sciatic nerve from Neu1 deficient mice (Neu1 -/-) and from normal control Neu1 +/+; the animals were euthanized 0, 3, 7, 14 and 21 days after denervation. Denervated and control muscles were collected and submitted to several analysis: 1) histological; 2) autophagic vacuoles formation, performed by ultrastructural analysis and LC3 protein expression; 3) acid phosphatase reaction, lamp1 and cathepsin L protein expression, to analyze lysosomal activation; 4) collagen deposition and fibrous formation; 5) proteins involved with muscle trophism, Akt and GSK3b; 6) MuRF1 and Atrogin-1 gene expression; 7) MyoD protein expression; 8) Neu1, Neu2, Neu3 and Neu4 genes expression. Neu1 -/- mice presented decreased body and muscle weight comparing to Neu1 +/+ animals. Muscle fiber cross-sectional area was reduced in denervated muscles comparing to contralateral muscles. Neu1 -/- mice muscles presented basal atrophy and increase of endomisial and perimisial spaces, which became more evident after denervation. After 14 days of denervation, autophagosome formation was noticed on Neu1 +/+ and Neu1-/- animals. Cathepsin L protein levels were increased after 14 and 21 days of denervation, especially in denervated muscles from Neu1 -/- mice. Lamp1 protein expression was increased in Neu1-/- animals. Type III collagen protein levels were increased in Neu1-/- animals. There were no significant differences between MyoD protein levels. P-Akt, active form of Akt protein levels, decreased after 21 days of denervation, especially in denervated muscles from control group animals, indicating that protein synthesis is decreased. P-GSK3b, inactive form of GSK3b decreased in denervated muscles from Neu1 -/- and Neu1 +/+ animals, which indicates that this protein remained activated during muscle atrophy process. There were significant differences in Atrogin-1 and MuRF1 gene expression levels after 3 and 7 days of denervation. Neu1 -/- animals muscles presented a delayed Atrogin-1 response. Neu1 gene expression was increased in denervated muscles from Neu1 +/+ mice. These findings suggest that Neu1 seems to act in the regulation of muscle mass mainly by controlling the process of lysosomal system activation, but apparently without affecting autophagy

Sialidose é uma doença neurossomática causada pela deficiência congênita da neuraminidase-1 (Neu1), enzima envolvida na regulação do catabolismo de sialoglicoconjugados nos lisossomos. Com o acúmulo de sialoglicoconjugados, ocorre comprometimento sistêmico e neurológico. Achados histológicos musculares incluem expansão da matriz extracelular (MEC) devido à proliferação anormal de fibroblastos, invasão das fibras musculares por componentes da MEC, fragmentação do citoplasma, formação vacuolar e atrofia das fibras musculares. Entretanto o mecanismo da atrofia muscular na deficiência de Neu1 não está completamente esclarecido, sendo o objetivo desse estudo. Desnervou-se o músculo gastrocnêmio direito de camundongos com deficiência de Neu1 (Neu1 -/-) e de controles Neu1 +/+. Os animais foram eutanasiados 0, 3, 7, 14 e 21 dias pós desnervação. Os músculos desnervados e contralaterais foram submetidos às seguintes análises: 1) histologia geral e medida da área transversa das fibras; 2) autofagia, através da avaliação da presença de vacúolos autofágicos por estudo ultraestrutural e da análise da expressão da proteína LC3; 3) ativação do sistema lisossomal, por reação de fosfatase ácida e análise da expressão proteica de catepsina L e lamp1; 4) deposição de colágeno e infiltração de tecido conjuntivo no tecido muscular; 5) níveis das proteínas Akt e GSK3b; 6) expressão dos atrogenes MuRF1 e Atrogina-1; 7) níveis da proteína MyoD, relacionada à diferenciação muscular; e 8) expressão dos genes Neu1, Neu2, Neu3 e Neu4. Os animais Neu1-/- apresentaram menor peso corporal e muscular compararando-se com animais Neu1 +/+. Houve redução progressiva da área das fibras dos músculos desnervados em relação aos músculos contralaterais. Os animais Neu1-/- apresentaram atrofia muscular basal, com aumento acentuado dos espaços endomisiais e perimisiais. Ocorreu formação de vacúolos autofágicos a partir de 14 dias de desnervação tanto em animais Neu1+/+ quanto em Neu1-/-. Os níveis de expressão proteica de catepsina L e de lamp1 aumentaram a partir de 14 dias de desnervação, mais notadamente em músculos desnervados de camundongos Neu1-/-. A expressão proteica de colágeno III mostrou-se aumentada em animais Neu1-/-, principalmente após desnervação. A expressão proteica da forma fosforilada do Akt (forma ativada) diminuiu após 21 dias de desnervação principalmente em músculos desnervados de animais Neu1+/+. Os níveis de PGSK3 b, forma inativa de GSK3b, diminuíram após a desnervação, em animais Neu1+/+ e animais Neu1-/-. Houve aumento na expressão gênica de Atrogina-1 e MuRF1 após 3 e 7 dias de desnervação, respectivamente; a expressão gênica de Atrogina-1 nos camundongos Neu1-/- teve um aumento atrasado, mostrando diferença significante após 7 dias de desnervação. Não houve diferença significativa entre níveis proteicos de MyoD. A expressão gênica de Neu1 mostrou-se elevada em músculos desnervados de animais Neu1+/+. Conclui-se, portanto, que a Neu1 parece atuar na regulação da massa muscular principalmente controlando o processo de ativação do sistema lisossomal, porém aparentemente sem afetar a autofagia / Sialidosis, a severe neurosomatic disease, results from congenital neuraminidase-1 (Neu1) deficiency. This enzyme regulates the catabolism of sialoglycoconjugates in the lysosomes. Systemic and neurologic manifestations occur due to the sialoglycoconjugates accumulation. In the mouse model for Neu1 deficiency, the muscle histologic findings include extracellular matrix (ECM) expansion, due to abnormal fibroblast proliferation, muscle fibers invasion by ECM components, cytoplasm fragmentation, vacuolar formation and muscle atrophy. Nevertheless the mechanisms of muscle atrophy in Neu1 deficiency are not completely known. This study was designed to investigate Neu1 involvement in muscle atrophy process. Denervation of gastrocnemius muscle was performed by sectioning sciatic nerve from Neu1 deficient mice (Neu1 -/-) and from normal control Neu1 +/+; the animals were euthanized 0, 3, 7, 14 and 21 days after denervation. Denervated and control muscles were collected and submitted to several analysis: 1) histological; 2) autophagic vacuoles formation, performed by ultrastructural analysis and LC3 protein expression; 3) acid phosphatase reaction, lamp1 and cathepsin L protein expression, to analyze lysosomal activation; 4) collagen deposition and fibrous formation; 5) proteins involved with muscle trophism, Akt and GSK3b; 6) MuRF1 and Atrogin-1 gene expression; 7) MyoD protein expression; 8) Neu1, Neu2, Neu3 and Neu4 genes expression. Neu1 -/- mice presented decreased body and muscle weight comparing to Neu1 +/+ animals. Muscle fiber cross-sectional area was reduced in denervated muscles comparing to contralateral muscles. Neu1 -/- mice muscles presented basal atrophy and increase of endomisial and perimisial spaces, which became more evident after denervation. After 14 days of denervation, autophagosome formation was noticed on Neu1 +/+ and Neu1-/- animals. Cathepsin L protein levels were increased after 14 and 21 days of denervation, especially in denervated muscles from Neu1 -/- mice. Lamp1 protein expression was increased in Neu1-/- animals. Type III collagen protein levels were increased in Neu1-/- animals. There were no significant differences between MyoD protein levels. P-Akt, active form of Akt protein levels, decreased after 21 days of denervation, especially in denervated muscles from control group animals, indicating that protein synthesis is decreased. P-GSK3b, inactive form of GSK3b decreased in denervated muscles from Neu1 -/- and Neu1 +/+ animals, which indicates that this protein remained activated during muscle atrophy process. There were significant differences in Atrogin-1 and MuRF1 gene expression levels after 3 and 7 days of denervation. Neu1 -/- animals muscles presented a delayed Atrogin-1 response. Neu1 gene expression was increased in denervated muscles from Neu1 +/+ mice. These findings suggest that Neu1 seems to act in the regulation of muscle mass mainly by controlling the process of lysosomal system activation, but apparently without affecting autophagy

No description available.

Protein sequencing has produced overwhelming amount of protein sequences, especially in the last decade. Nevertheless, the majority of the proteins' functional and structural classes are still unknown, and experimental methods currently used to determine these properties are very expensive, laborious and time consuming. Therefore, automated computational methods are urgently required to accurately and reliably predict functional and structural classes of the proteins. Several bioinformatics methods have been developed to determine such properties of the proteins directly from their sequence information. Such methods that involve signal processing methods have recently become popular in the bioinformatics area and been investigated for the analysis of DNA and protein sequences and shown to be useful and generally help better characterise the sequences. However, there are various technical issues that need to be addressed in order to overcome problems associated with the signal processing methods for the analysis of the proteins sequences. Amino acid indices that are used to transform the protein sequences into signals have various applications and can represent diverse features of the protein sequences and amino acids. As the majority of indices have similar features, this project proposes a new set of computationally derived indices that better represent the original group of indices. A study is also carried out that resulted in finding a unique and universal set of best discriminating amino acid indices for the characterisation of allergenic proteins. This analysis extracts features directly from the protein sequences by using Discrete Fourier Transform (DFT) to build a classification model based on Support Vector Machines (SVM) for the allergenic proteins. The proposed predictive model yields a higher and more reliable accuracy than those of the existing methods. A new method is proposed for performing a multiple sequence alignment. For this method, DFT-based method is used to construct a new distance matrix in combination with multiple amino acid indices that were used to encode protein sequences into numerical sequences. Additionally, a new type of substitution matrix is proposed where the physicochemical similarities between any given amino acids is calculated. These similarities were calculated based on the 25 amino acids indices selected, where each one represents a unique biological protein feature. The proposed multiple sequence alignment method yields a better and more reliable alignment than the existing methods. In order to evaluate complex information that is generated as a result of DFT, Complex Informational Spectrum Analysis (CISA) is developed and presented. As the results show, when protein classes present similarities or differences according to the Common Frequency Peak (CFP) in specific amino acid indices, then it is probable that these classes are related to the protein feature that the specific amino acid represents. By using only the absolute spectrum in the analysis of protein sequences using the informational spectrum analysis is proven to be insufficient, as biologically related features can appear individually either in the real or the imaginary spectrum. This is successfully demonstrated over the analysis of influenza neuraminidase protein sequences. Upon identification of a new protein, it is important to single out amino acid responsible for the structural and functional classification of the protein, as well as the amino acids contributing to the protein's specific biological characterisation. In this work, a novel approach is presented to identify and quantify the relationship between individual amino acids and the protein. This is successfully demonstrated over the analysis of influenza neuraminidase protein sequences. Characterisation and identification problem of the Influenza A virus protein sequences is tackled through a Subgroup Discovery (SD) algorithm, which can provide ancillary knowledge to the experts. The main objective of the case study was to derive interpretable knowledge for the influenza A virus problem and to consequently better describe the relationships between subtypes of this virus. Finally, by using DFT-based sequence-driven features a Support Vector Machine (SVM)-based classification model was built and tested, that yields higher predictive accuracy than that of SD. The methods developed and presented in this study yield promising results and can be easily applied to proteomic fields.

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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_PerfilGenotipicoResistencia.pdf: 2327447 bytes, checksum: c13b76b67d930ccdd5d15036d66ff32a (MD5) Previous issue date: 2012 / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / O vírus Influenza é o responsável pela gripe, uma doença que ocasiona milhões de mortes e hospitalizações todos os anos. Nas infecções severas, especialmente em pessoas com risco para complicações, os antivirais tornam-se os principais meios para o manejo clínico, merecendo especial destaque os inibidores da neuraminidase (INAs). De fato, na pandemia de 2009 a Organização Mundial da Saúde (OMS) recomendou o uso do oseltamivir para o tratamento dos doentes. Porém, devido à evolução genética viral, surgiram cepas com mutações no gene codificador da neuraminidase (NA) responsáveis por substituições aminoacídicas que levam à resistência aos fármacos INAs. Assim, a OMS passou a recomendar a vigilância de resistência genotípica para os vírus Influenza. Este trabalho teve como objetivos verificar a ocorrência de mutações no gene codificador da NA dos vírus Influenza A (H1N1) pandêmico que possam estar relacionadas à resistência aos INAs em cepas circulantes na mesorregião metropolitana de Belém no período de maio de 2009 a maio de 2012 e analisar, através da modelagem de proteínas, as substituições aminoacídicas da NA que possam estar influenciando na conformação protéica. Durante o período de estudo, foram recebidas no Laboratório de Vírus Respiratórios 2619 amostras clínicas de pacientes que apresentavam sinais e sintomas de infecção respiratória aguda com até cinco dias de evolução. Para a detecção do genoma viral foi feita a extração do RNA viral, seguida de RT-PCR em tempo real utilizando marcadores específicos para Influenza A H1N1pdm, resultando em 744 (28,4%) positivas. Parte das amostras positivas foram então inoculadas em células MDCK. Para as amostras isoladas em cultura de células, foi feita uma nova extração do RNA viral seguida de uma RT-PCR e semi-nested (PCR) utilizando iniciadores específicos para o gene NA, e posterior análise em sequenciador automático ABI Prism 3130xl (Applied Biosystems). A modelagem molecular da NA foi realizada através dos softwares SWISS-MODEL, MODELLER 9.10, PROCHECK, VERIFY3D e PYMOL. A análise parcial das sequências da neuraminidase nas amostras sequenciadas mostrou que não houve a circulação de cepas de vírus H1N1pdm com a mutação H275Y, a principal envolvida na resistência ao oseltamivir. Porém, em duas amostras foi identificada a substituição D199N que já foi relatada em vários estudos mostrando uma possível associação com o aumento da resistência ao oseltamivir. As amostras de 2012 apresentaram duas substituições (V241I e N369K) que estão relacionadas com um possível papel na compensação dos efeitos negativos causados pela mutação H275Y. A modelagem molecular mostrou que na mutação D199N houve uma alteração na estrutura da proteína NA próxima ao sítio de ligação ao antiviral. A análise filogenética revelou que as amostras de 2012 formaram um cluster isolado, demonstrando uma variação muito mais temporal do que geográfica. Este representa o primeiro estudo de resistência dos vírus Influenza H1N1pdm na mesorregião metropolitana de Belém, representando um importante instrumento para que os profissionais de saúde adotem estratégias mais eficazes no manejo da doença e no desenvolvimento de novos fármacos anti-influenza. / Influenza virus is responsible for the flu, a disease that causes millions of hospitalizations and deaths every year. In severe infections, especially in people at risk with comorbidities for complications, antiviral become the main means for clinical management, mainly the neuraminidase inhibitors (INAs). By fact, in the 2009 pandemic episode the World Health Organization (WHO) recommends the use of Oseltamivir for the treatment of patients. However, due to viral genetic evolution emerged strains with mutations in the gene coding for the neuraminidase (NA) responsible for amino acid substitutions that lead to drug resistance INAs. Thus, WHO began recommending surveillance genotypic resistance to influenza viruses. Our study aimed to verify the occurrence of mutations in the NA encoding gene of Influenza virus A (H1N1) pandemic strains that could be related to INAs resistance in the Belém mesoregion from May 2009 to May 2012 and analyze, through modeling of proteins, NA amino acid substitutions that may be altering the protein conformation. During the study period, were received 2619 clinical samples at the Virus Respiratory Lab in Evandro Chagas Institute in Belém – Brazil, from patients presenting signs and symptoms of acute respiratory infection with up to five days from the start of symptoms. For the detection of the viral genome viral RNA extraction was made followed by real time RT- PCR using specific markers for Influenza A H1N1pdm, resulting in 744 (28.4%) positive samples. A portion of the positive samples were then inoculated in MDCK cells and isolated samples were then submitted to a new viral RNA extraction followed by real time RT-PCR and semi-nested reaction (PCR) using primers specific for the NA gene. Subsequent analysis was done in 3130xl ABI Prism automatic sequencer (Applied Biosystems ). Molecular modeling was performed to the NA gene using SWISS-MODEL software, MODELLER 9.10, Procheck, VERIFY3D and PYMOL. The analysis of partial sequences of neuraminidase showed no circulation of the H1N1 pdm with H275Y mutation, the principal involved in resistance to oseltamivir. However in two samples were identified the D199N substitution that has been reported in several studies showing a possible association with increased resistance to oseltamivir. The samples of 2012 showed two substitutions (V241I and N369K) which are relate to a possible role in offsetting the negative effects caused by the H275Y mutation. Molecular modeling shows that the mutation D199N caused a change in protein structure near the NA-antiviral binding site. Phylogenetic analysis revealed that 2012 samples formed an isolated cluster, showing a much more temporal variation than geographic. This represents the first study of drug resistance of influenza virus in H1N1pdm metropolitan mesoregion of Belém - Brazil an important tool for health professionals to adopt more effective strategies for disease management and the development of new anti-influenza drugs.

Antiviral drug resistance is a major problem in the treatment of viral infections, including influenza and hepatitis C virus (HCV). Influenza neuraminidase (NA) is a viral sialidase on the surface of the influenza virion and a primary antiviral target in influenza. Two subtypes of NA predominate in humans, N1 and N2, but different patterns of drug resistance have emerged in each subtype. To provide a framework for understanding the structural basis of subtype specific drug resistance mutations in NA, we used molecular dynamics simulations to define dynamic substrate envelopes for NA to determine how different patterns of drug resistance have emerged in N1 and N2 NA. Furthermore, we used the substrate envelope to analyze HCV NS3/4A protease inhibitors in clinical development. In addition, influenza hemagglutinin (HA) is a primary target of neutralizing antibodies against influenza. Novel broadly neutralizing antibodies (BnAbs) against the stem region of HA have been described and inhibit several influenza viral subtypes, but antibody neutralization escape mutations have emerged. We identified potential escape mutations in broadly neutralizing antibody F10 that may impact protein dynamics in HA that are critical for function. We also solved crystal structures of antibody fragments that are important for understanding the structural basis of antibody binding for influenza BnAbs. These studies can inform the design of improved therapeutic strategies against viruses by incorporating an understanding of structural elements that are critical for function, such as substrate processing and protein dynamics, into the development of novel therapeutics that are robust against resistance.