Les inhibiteurs de la neuraminidase (INAs) jouent un rôle central dans le contrôle des infections grippales, tant dans le cas des épidémies et des pandémies comme chez les patients immunosuprimés et d'autres patients à risque. Cependant, le développement et la dissémination de la résistance compromettent l'utilité à long terme de cette intervention. En fait, le problème de la résistance aux INAs a été mis en évidence pendant les épidémies de grippe annuelles de 2007-09, avec la dissémination globale d’une variante de la souche A(H1N1) saisonnière résistante à l'oseltamivir. Dans ce cas, les observations préliminaires ont spéculé avec l'existence d'un ensemble de mutations “permissives” qui auraient facilité cette transmission mondiale. Heureusement, l'émergence et la propagation mondiale de la souche pandémique en 2009 a mené au remplacement de la souche saisonnière A/Brisbane/59/2007 (H1N1) résistante à l'oseltamivir, par le virus A(H1N1)pdm09 naturellement sensible aux INA, et, par conséquent, l'oseltamivir a récupéré son utilité clinique. En fait, la plupart des virus A(H1N1)pdm09, A(H3N2) et B circulants à ce jour restent sensibles à l'oseltamivir, avec seulement 1-2% de souches résistantes. Néanmoins, le nombre croissant de souches résistantes récemment détectées en l’absence de traitement fait craindre que ce problème puisse encore augmenter. À cet égard, l'impact de l'émergence et la dissémination de la résistance sur le choix limité des antiviraux actuellement disponibles renforce la nécessité d’une meilleure compréhension des mécanismes sous-jacents à ce phénomène ainsi que de nouvelles approches thérapeutiques. Les différentes études présentées dans le cadre de cette thèse convergent vers l'objectif général de mieux décrire les mécanismes de développement de la résistance aux INAs dans les virus de la grippe. En outre, nous prévoyons que les thérapies combinées pourraient induire une meilleure réponse virologique et immunologique par rapport à la monothérapie antivirale. À la fin, nous nous attendons à ce que notre travail ait un impact sur la gestion des infections grippales en guidant la surveillance mondiale des marqueurs potentiels de résistance, ainsi qu’en proposant des traitements novateurs qui minimisent le développement de souches résistantes. / Neuraminidase inhibitors (NAIs) play a central role in the control of influenza infections, with important implications in the management of outbreaks and pandemics as well as in immunocompromised and other at risk patients, with both prophylactic and therapeutic indications. However, the development and dissemination of antiviral drug resistance represents a major limitation that compromises the long-term usefulness of this intervention. Actually, the problem of resistance to NAIs was highlighted by the worldwide dissemination of the oseltamivir-resistant seasonal A(H1N1) neuraminidase H274Y variant during the 2007-09 annual influenza epidemics. In that case, preliminary observations speculated with the existence of a set of “permissive” mutations that could have facilitated this global transmission. Fortunately, the antigenic shift that enabled the emergence of and global spread of the 2009 pandemic strain meant the replacement of the oseltamivir-resistant seasonal A/Brisbane/59/2007 (H1N1) virus by the naturally NAI-susceptible A(H1N1)pdm09 virus, and, consequently, oseltamivir recovered its clinical utility. In fact, most of the circulating A(H1N1)pdm09, A(H3N2) and B viruses remain susceptible to oseltamivir with only 1-2% of tested strains exhibiting phenotypic or genotypic evidence of resistance. Nevertheless, the growing number of resistant strains recently detected in the absence of therapy raises concern that this problem could increase. In that regard, the impact of the emergence and dissemination of resistance on the limited choice of antivirals currently available underscores a better understanding of the mechanisms underlying this phenomenon as well as the necessity for innovative therapeutic approaches. The different studies presented in this thesis converge to the general objective of better describing the mechanisms underlying the development of resistance to NAIs in influenza viruses. Also, we anticipate that combination therapies will induce better virological and immunological responses compared to antiviral monotherapy. In the end, we expect that our work will have an impact on the management of influenza infections by guiding the global surveillance of potential drug resistance markers, as well as proposing innovative ways to improve the clinical outcome and minimizing the development of drug-resistant strains.

A ce jour, les données génétiques et moléculaires se rapportant aux virus influenza de type A (VIs) présents dans la population porcine au Québec sont relativement rares. Pourtant, ces informations sont essentielles pour la compréhension de de l'évolution des VIs à grande échelle de 2011 à 2015. Afin de remédier à ce manque de données, différents échantillons (pulmonaires, salivaires et nasaux) ont été prélevés à partir de 24 foyers dans lesquelles les animaux présentaient des signes cliniques. Ensuite, les souches virales ont été isolées en culture cellulaire (MDCK) ou sur oeufs embryonnés. Les 8 segments génomiques des VIs de 18 souches virales ont par la suite été séquencés et analysés intégralement. La résistance aux drogues antivirales telles que l’oseltamivir (GS4071) carboxylate, le zanamivir (GS167) et l’amantadine hydrochloride a également été évaluée par des tests d'inhibition de la neuraminidase (INAs) ainsi que par un test de réduction sur plaque. Deux sous-types viraux H3N2 et H1N1 ont été identifiés dans la population porcine au Québec. Douze souches des VIs de sous-type trH3N2 ont été génétiquement liées au Cluster IV, avec au moins 6 profils de réassortiment différents. D'autre part, 6 souches virales ont été trouvées génétiquement liées au virus pandémique A(H1N1)pdm09 avec au moins trois profils de réassortiment génétique différents. Le sous-type trH3N2 des VIs est le plus répandu dans la population porcine au Québec (66,7%). La cartographie d'épitope de la protéine HA de sous-type H3 a présenté la plus forte variabilité avec 21 substitutions d’acides aminés sur 5 sites antigéniques A (5), B (8), C (5), D (1), et E (2). Toutefois, la protéine HA du sous-type H1 avait seulement 5 substitutions d'aa sur les 3 sites antigéniques Sb (1), Ca1 (2) et Ca2 (2). Un isolat H1N1 (1/6 = 16,7%) et 1 autre trH3N2 (1/12 = 8,3%) ont été trouvés comme étant résistants à l'oseltamivir. En revanche, 2 isolats du H1N1 (2/6 = 33,3%) et 2 autres du trH3N2 (2/12 = 16,7%) ont révélé être résistants au zanamivir. Dans l'ensemble, le taux de résistance aux INAs et à l’amantadine était compris entre 33,3% et 100%. La présence des VIs résistants aux drogues antivirales chez les porcs ainsi que l'émergence possible de nouvelles souches virales constituent des préoccupations majeures en la santé publique et animale justifiant ainsi la surveillance continue des VIs dans la population porcine au Québec. / Data about genomic variability of swine influenza A viruses (SIV) in Quebec herds are scarce. Yet, this information is important for understanding virus evolution in Quebec from until 2015. Different clinical samples were obtained from 24 outbreaks of swine flu in which animals were experiencing respiratory disease. Samples including lung tissues, saliva and nasal swabs were collected and virus isolation was attempted in MDCK cells and embryonated eggs. All eight gene segments of the 18 isolated SIV strains were sequenced and analysed. Antiviral drugs resistance against oseltamivir carboxylate (GS4071), zanamivir (GS167) and amantadine hydrochloride was evaluated by neuraminidase inhibition assays (NAIs) and plaque reduction assay. Two subtypes of SIV, H3N2 and H1N1, were identified in Quebec pig herds. Twelve SIV strains were genetically related to trH3N2 Cluster IV and at least 6 different reassortment profiles were identified. On the other hand, 6 Quebec SIV strains were found to be genetically related to the pandemic virus A(H1N1)pdm09 and from which three reassortment profiles were identified. Overall, the trH3N2 was the most prevalent subtype (66.7%) found in Quebec swine herds. The epitope mapping of HA indicated that the H3 subtype was the most variable with a possibility of 21 amino acids (aa) substitutions within the 5 antigenic sites A(5), B(8), C(5), D(1) and E(2). However, the HA protein of the H1 subtype had only 5 aa substitutions within 3 antigenic sites Sb(1), Ca1(2) and Ca2(2). One H1N1 (1/6 = 16.7%) and one trH3N2 (1/12 = 8.3%) were identified as strains resistant against oseltamivir. In contrast, two H1N1 (2/6 = 33.3%) and two trH3N2 (2/12 = 16.7%) strains were found to be resistant against zanamivir. Overall, the SIV resistance against antiviral neuraminidase inhibitor drugs was (33.3%). All strains were resistant against the M2 inhibitor antiviral drug, amantadine. The presence of antiviral drug resistance in Quebec swine herds and the possible emergence of new SIVs strains are public health concerns supporting the surveillance of SIVs.

INTRODUÇÃO:A neuraminidase 1 (chamada a seguir por Neu1) regula o catabolismo de sialoglicoconjugados nos lisossomos. A deficiência congênita da Neu1 é a base da sialidose, doença neurossomática grave associada a deformidades osteoesqueléticas, hipotonia e fraqueza muscular. Camundongos com deficiência de Neu1 desenvolvem uma forma atípica de degeneração muscular caracterizada porproliferação anormal de fibroblastoslevando a invasão nas fibras musculares, expansão da matriz extracelular (MEC), fragmentação do citoplasma, formação vacuolar e atrofia muscular. A ocorrência de atrofia muscular indica que a deficiência da Neu1 podeestar relacionada com o controle da massa muscular, a qual é dependente do equilíbrio entre síntese e degradação proteica.Uma característica principalnaMacroautofagia (denominada a seguir por autofagia) é a principal via de degradação intracelular sendo expressamente essencial para a homeostase celular e remoção de materiais citoplasmáticos. Objetivos: Avaliarseos efeitos da deficiência da Neuraminidase 1 afetama indução de autofagia e a formação de autofagossomos e determinar os efeitos do bloqueio da função lisossomal sobre o fenótipo muscular na deficiência da Neuraminidase1.Metodologia:Camundongos Neu1+/-foram cruzados e os filhotes genotipados, onde camundongos Neu1-/-(nocaute) e Neu1+/+(normal),foram utilizados no estudon total=90, sendo 10 em padronizações, 20 para coleta de fibroblastos e 60 para procedimentos in vivo, os grupos experimentais foram divididos em privação alimentar por 2 dias que, por meio da inibição do mTOR, induz a autofagia;grupo detratamento com Colchicina por 4 dias onde irá impedir a junção autofagossomo/lisossomonão havendo a degradaçãolisossomaleadicionado nos dois últimos dias de privação alimentar e o grupo de tratamento comRapamicina por 7 dias, droga com função de inibir seletivamente o mTOR.Após os tratamentos, os animais foram eutanasiadospara coleta dos músculos gastrocnêmios e tibiais anterior. Os músculos foram analisados apósas coloraçõeshistológicas porHematoxilina & Eosina e Fosfatase Ácida; Imunofluorescência de LC3-I/II; Western Blottingdas proteínas LC3-I/II, Lamp-1 e p62/SQSTM e a análise Ultra-estrutural. Além disso, foi realizada cultura de fibroblastos, os quais foram submetidos à privação de nutrientes e ao tratamento com Rapamicina, seguidos dasmesmas metodologias de análise invivo. Resultados:As análises histológicas através de H&E e Fosfatase Ácida não revelaram alterações consistentes na musculatura esquelética de animais submetidos aos tratamentos, mostrando em animais com deficiência de Neu1 um aumento anormal do espaço endomisial, e aumento da atividade lisossomalintracitoplasmáticos. Na análise ultra-estruturalobservou-se em todos os grupos, a presença de diversas estruturas com aspecto autofágico, de diferentes tamanhos, formas e constituintes. Naanálise da expressão do LC3 através de Western Blottingmostrou importante ativação do LC3-II na privação alimentar (com e sem administração de Colchicina) tanto em animais controles quanto em animais com deficiência de Neu1 e uma importante ativação do LC3-I em Rapamicina em ambos os grupos mostrando assim que houve um aumento da via da autofagia através do bloqueio do mTOR. Já na análise de Imunofluorescência não foi possível observar diferença consistenteentre os grupos.A análise in vitrode Western Blottingmostrou que tal ativação foi similar entre fibroblastos Neu1+/+e Neu1-/-. Conclusão:Os experimentos relacionados com a ativação ou inibição da autofagia, não resultaram em diferenças consideráveis entre músculos normais e músculos com deficiência de Neu1. Desta forma, podemos concluir com estes experimentos que aparentemente a Neu1, apesar de ser uma importante enzima lisossomal, não interfere com o processo de autofagia / Introduction:Neuraminidase 1 (hereinafter Neu1) regulates the catabolism of sialoglycoconjugates in lysosomes. The congenital deficiency of Neu1 is the basis of sialidosis, a severe neurosomatic disease associated with osteo-skeletal deformities, hypotonia, and muscle weakness. Mice with Neu1deficient develop an atypical form of muscle degeneration characterized by abnormal fibroblast proliferation leading to muscle fiber invasion, extracellular matrix expansion (ECM), cytoplasm fragmentation, vacuolar formation, and muscle atrophy. The occurrence of muscle atrophy indicates that deficiencyofNeu1 may be related to the control of muscle mass, which is dependent on the balance between synthesis and protein degradation. A major feature in Macroautophagy (hereinafter referred to as autophagy) is the main pathway of intracellular degradation being expressly essential for cellular homeostasis and removal of cytoplasmic materials. Objectives:To evaluate whether the effects of neuraminidase 1 deficiency affect autophagy induction and autophagosome formation and to determine the effects of lysosomal function block on muscle phenotype in neuraminidase 1 deficiency. Methodos:Mice Neu1+/-were crossbred and the genotyped pups, where mice Neu1-/-(knockout) and Neu1+/+(normal), were used in the study ntotal = 90, 10 for standardization, 20 for fibroblast collection and 60 for in vivo procedures, experimental groups were divided into food deprivation for 2 days that, through mTOR inhibition, induces autophagy; Colchicine treatment group for 4 days whereit will prevent autophagosome/lysosome junction without lysosomal degradation and added in the last two days of food deprivation and Rapamycin treatment group for 7 days, drug with function to selectively inhibit mTOR. After the treatments, the animals were euthanized to collect the anterior gastrocnemius and tibial muscles. The muscles were analyzed after histological staining by Hematoxylin & Eosin and Acid Phosphatase; LC3-I/II immunofluorescence; Western Blottingof LC3-I/II, Lamp-1 and p62/SQSTM proteins and Ultra-structural analysis. In addition, fibroblasts were cultured and subjected to nutrient deprivation and Rapamycin treatment, followed by the same in vivo analysis methodologies. Results:Histological analyzes by H&E and Acid Phosphatase did notreveal consistent changes in skeletal muscle of animals submitted to treatments, showing in animals with Neu1 deficiency an abnormal increase in endomysial space and increased intracytoplasmic lysosomal activity.In ultrastructural analysis, it was observed in all groups, the presence of several structures with autophagic aspect, of different sizes, shapes and constituents. The analysis of LC3 expression by Western Blottingshowed important activation of LC3-II in food deprivation (with and without Colchicine administration) in both control and Neu1 deficient animals and an important activation of LC3-I in Rapamycininboth groups,thus showing an increase in the autophagy pathway through mTOR blockade. In the immunofluorescence analysis, it was not possible to observe consistent difference between the groups. In vitro Western Blottinganalysis showed that such activation was similar between Neu1+/+and Neu1-/-fibroblasts. Conclusion: Experiments related to activation or inhibition of autophagy did not result in considerable differencesbetween normal muscles and Neu1 deficient muscles. Thus, we can conclude from these experiments that apparently Neu1, despite being an important lysosomal enzyme, does not interfere with the autophagy process

L'élastine est la protéine matricielle responsable de l'élasticité des tissus retrouvée dans des tissus soumis à de fortes contraintes mécaniques tels que les poumons, les artères ou la peau. La dégradation de cette protéine lors de processus physiopathologiques produit des peptides bologiquement actifs nommés peptides d'élastine portant le motif GXXPG essentiel à leur activité. Ces peptides régulent diverses fonctions biologiques telles que le chimiotactisme, la synthèse de protéases, la prolifération. Tous ces effets dépendent de la fixation des peptides d'élastine au complexe récepteur de l'élastine. Ce complexe est composé de trois sous-unités : une protéine périphérique de 67 kDa, l'Elastin Binding Protein (EBP), et deux protéines associées à la membrane, la Protective Protein/Cathepsin A (PP/CA) et la Neuraminidase-1 (Neu-1) de 55 et 61 kDa respectivement. L'activité sialidase de Neu-1 est responsable de l'activation de ERK 1/2 après fixation des peptides d'élastine au complexe récepteur de l'élastine.Dans cette étude, nous démontrons que l'EBP et les radeaux lipidiques sont colocalisés à la membrane plasmique. Nous montrons, de plus, que la déstructuration de ces microdomaines aussi bien que leur déplétion en glycolipides bloque la signalisation du récepteur. L'utilisation d'un anticorps monoclonal bloquant dirigé contre le GM3 montre qu'il est essentiel à la signalisation. Après traitement par les peptides d'élastine, le contenu cellulaire en GM3 diminue alors que celui en lactosylcéramide augmente suggérant une conversion du GM3 en lactosylcéramide. L'utilisation de lactose ou de siRNA Neu-1 bloque cette conversion ce qui tend à démontrer que le complexe récepteur de l'élastine est impliqué dans ce mécanisme. Une analyse par cytométrie en flux confirme cette production de lactosylcéramide induite par les peptides d'élastine.L'analyse par spectrométrie de masse mettrait en évidence deux lactosylcéramides (C23:0 et C24:1) potentiellement bioactifs dont la synthèse chimique a été entreprise. La purification des radeaux lipidiques par ultracentrifugation différentielle en gradient de saccharose ainsi que leur identification par Dot-blot couplé à la fluorescence montre un changement de densité de ces microdomaines après stimulation par les peptides d'élastine.L'évaluation biologique in vitro de ces lactosylcéramides montre qu'ils miment les effets des peptides d'élastine sur l'activation de ERK 1/2, la prolifération et la synthèse de MMP-1. Enfin, l'évaluation ex vivo des lactosylcéramides démontre une réduction de la zone de tissu cardiaque nécrosé suggérant un rôle cardioprotecteur de ces molécules. Ce travail propose un mécanisme original de transduction du signal à la membrane plasmique et nous laisse envisager le complexe récepteur de l'élastine, les peptides d'élastine et le lactosylcéramide comme de nouveaux agents thérapeutiques potentiels. / Elastin is the matrix protein responsible for the elasticity of tissues where resilience is required such as lung, arteries or skin. Elastin degradation during physiopathological processes produces biologically active peptides named elastin peptides bearing the GXXPG pattern essential for their activity. These peptides regulate various biological functions such as chemotaxis, proteases synthesis and proliferation. These effects are dependent of elastin peptide binding to the elastin receptor complex (ERC). This complex is composed of three subunits: a peripheral protein of 67 kDa called elastin binding protein (EBP) and two membrane-associated proteins, protective protein/cathepsin A (PP/CA) and neuraminidase-1 (Neu-1) of 55 and 61 kDa, respectively. The sialidase activity of Neu-1 is responsible for ERK 1/2 pathway activation following binding of elastin peptide on the elastin receptor complex.In this study, we demonstrate that EBP and lipid rafts colocalize at the plasma membrane. We also show that the disruption of these microdomains and their depletion in glycolipids block the receptor signaling. The use of a monoclonal anti-GM3 blocking antibody shows that this glycosphingolipid is essential for signaling. Following elastin peptide treatment, cellular GM3 level decreases while the lactosylceramide one increases consistently with a GM3/LacCer conversion. The use of lactose or Neu-1 siRNA blocks this process suggesting that the elastin receptor complex is involved in this mechanism. Flow cytometry analysis confirms this elastin peptide-driven LacCer generation.Mass spectrometry analysis of elastin peptide-stimulated cell membrane extracts identified two potentially bioactive lactosylceramides (C23:0 and C24:1) and their synthesis has been realized. Lipid rafts purification by differencial ultracentrifugation in sucrose gradient shows a variation of the microdomains density as well as their identification by fluorescence linked-Dot-blot following elastin peptide stimulation.In vitro biological evaluation of these lactosylceramides shows that they mimic the elastin peptide effects on ERK 1/2 activation, proliferation and MMP-1 synthesis. Finally, ex vivo lactosylceramides evaluation demonstrates a decrease of cardiac tissue necrosis area suggesting that these molecules could be cardioprotective agents. This work proposes an original mechanism of signal transduction at the plasma membrane and let us foresees the elastin receptor complex, elastin peptides and lactosylceramide as new potential therapeutical targets.

The natural reservoir of influenza A virus (IAV) is wild waterfowl, and all human IAVs have their genetic origins from avian viruses. Neuraminidase inhibitors (NAIs) are currently the best drugs for treatment of human influenza; therefore, the orally available NAI oseltamivir (Tamiflu®) has been stockpiled worldwide as part of pandemic preparedness planning. Re-sistance to NAIs is related to worse clinical outcomes and if a new pandemic influenza virus would be oseltamivir-resistant its public health impact would be substantially worsened. The active metabolite oseltamivir carboxylate (OC) is not removed by sewage treatment and ends up in river water, where OC-concentrations up to 0.86µg/L have been detected. We hypothesize that occasional OC exposure of wild waterfowl carrying IAVs may result in circulation of resistant variants that may potentially evolve to become human-pathogenic. We tested the hypothesis in an in vivo Mallard (Anas platyrhynchos) model in which birds were infected with avian IAVs and exposed to OC. Excreted viruses were analyzed regarding genotypic and phenotypic resistance by neuraminidase (NA) sequencing and a functional NA inhibition assay. Two viruses with NAs of the phylogenetic N2-group, H6N2 and H7N9, acquired the NA substitutions R292K and I222T when host ducks were exposed to 12µg/L and 2.5µg/L of OC, respectively. Drug susceptibilities were at previously described levels for the substitutions. To test persistence of resistance, an OC resistant avian H1N1/H274Y virus (with a group N1 NA-protein) from a previous study, and three resistant H6N2/R292K variants were allowed to replicate in Mallards without drug pressure. Resistance was entirely maintained in the H1N1/H274Y virus, but the H6N2/R292K variants were outcompeted by wild type virus, indicating retained fitness of the resistant H1N1 but not the H6N2 variants. We conclude that OC in the environment may generate resistant IAVs in wild birds. Resistant avian IAVs may become a problem to humans, should the resistance trait become part of a new human pathogenic virus. It implies a need for prudent use of available NAIs, optimized sewage treatment and resistance surveillance of avian IAVs of wild birds.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

The outbreak of pandemic influenza and its ability to spread rapidly makes it a severe threat to public health. Antiviral drugs such as oseltamivir (Roche’s Tamiflu™) and zanamivir (GlaxoSmithKline’s Relenza™) are neuraminidase (NA) inhibitors (NI), which bind more tightly to NA than its natural substrate, sialic acid. However, the virus can acquire resistance to antiviral drugs by developing single point mutations (such as H274Y) in the target protein. Thus in some cases the drugs may not be as effective as expected. The high level of inconsistency exhibited by fluorometric assays and the short half-life of the chemiluminescent assay for monitoring drug resistance lead to the need for a simple, label-free, reliable assay. To address this problem, this work focused on three main objectives: 1) to determine the binding affinities of two common anti-viral drugs (oseltamivir and zanamivir) against the influenza NA wild type and drug resistant mutants using bioinformatics software Schrodinger Suite™ 2010. 2) To develop a reliable label-free, real-time, surface plasmon resonance (SPR) assay to measure the binding affinity between influenza viral coat protein neuraminidase (wild type and mutant) and anti-viral drugs. 3) To develop an SPR inhibition assay to quantitatively compare the interactions of sialic acid, zanamivir and oseltamivir with the viral coat protein neuraminidase (wild type and mutant). The entire docking process was carried out using Schrödinger Suite™ 2010. The 2009 pandemic H1N1 neuraminidase (PDB: 3NSS) was used throughout the docking studies as the wild type structure. Five mutants (H274Y, N294S, H274N, A346N and I222V) and three ligands (sialic acid, oseltamivir and zanamivir) were built using the maestro module. The grid-based ligand docking with energetics (GLIDE) module and induced fit docking (IFD) module were used for docking studies. The binding affinities, Gibbs free energy change (∆G) and molecular mechanics-generalized born energy/ solvent accessible area (MM-GB/SA) values for wild-type NA interactions show that both the antiviral drugs studied interact strongly with the wild-type protein. The ∆G values for all antiviral interactions with mutant NA forms were reduced in magnitude, thereby indicating that they are less favourable than interactions with the wild-type protein. A similar trend was observed with MM-GB/SA results. Amongst all of the computed values, MM-GB/SA was the closest to the experimental data. In several cases, the interactions between the anti-viral drugs and NA mutants were markedly less favourable than those between sialic acid and the same mutants, indicating that these mutations could confer anti-viral resistance. Influenza NA wild-type and H274Y mutant were expressed in baculovirus expression system (BVES) in insect cells. The expressed proteins were partially purified using the standard purification techniques of anion exchange and size exclusion chromatography (SEC). A fluorometric activity assay was performed on the recombinant proteins. Both the wild type and the mutant showed similar level of activities. In addition, the recombinant NAs were used in an inhibition assay. Oseltamivir was found to be sensitive to wild type protein (IC50 = 0.59 nM) and resistant to the H274Y mutant protein (IC50 = 349.43 nM). On the other hand, zanamivir was sensitive to both wild type (IC50 = 0.26 nM) and the H274Y mutant (IC50 = 0.44 nM). This indicated that zanamivir was a more potent inhibitor than oseltamivir. These findings were in good agreement with the literature. An SPR assay for accurate monitoring of influenza antiviral drug resistance was developed. A spacer molecule (1, 6- hexanediamine) was site-specifically tethered to the inert 7-hydroxyl group of zanamivir. The tethered zanamivir was immobilized onto an SPR GLC chip to obtain a final immobilization response of 431 response units (RU). The reference subtracted binding responses obtained for NA wild-type and H274Y mutant were analysed using the ProteOn Manager™ Software tools. The SPR curves were fitted to a simple Langmuir 1:1 model with drift to obtain association rate constant (ka) and dissociation rate constants (kd). The relative binding values obtained from literature and the current SPR assay (1.9 and 1.7 respectively) suggested that the current SPR assay yielded similar results to the existing labelled enzymatic assay. In addition, an SPR inhibition assay was developed. The calculated IC50-spr values were compared and it was observed that oseltamivir was sensitive to wild type protein (IC50-spr = 7.7 nM) and resistant to the H274Y mutant protein (IC50-spr = 256 nM). On the other hand, zanamivir was sensitive to both wild type (IC50-spr = 2.16 nM) and the H274Y mutant (IC50-spr = 2.4 nM). Sialic acid was also found to be sensitive to both wild type (IC50-spr = 5.5 nM) and H274Y mutant (IC50-spr = 3.25 nM). In the cases studied, the viral proteins remained sensitive to sialic acid, consistent with retention of virulence of these mutant strains. It was concluded that zanamivir is a more potent inhibitor than oseltamivir for treating the H274Y mutant. Comparison of the SPR inhibition results with the docking results revealed a similar trend. The wild-type NA and H27Y mutant retained binding affinity for sialic acid and zanamivir. Oseltamivir showed a significant decrease in binding affinity for the H274Y mutant compared with the wild-type. This was because of the disruption of the salt bridge formation within NA that was vital for oseltamivir activity. To my knowledge, this is the first SPR biosensor assay developed to monitor influenza antiviral drug resistance. There is a tremendous scope to extend this study to more mutants and new antiviral drugs. This could pave the way for a reliable SPR biosensor assay to replace low consistency labelled enzymatic assays.

No description available.

La transmission des virus grippaux de type A s’effectue via l’eau, l’air ou les surfaces. Elle implique donc toujours une étape dans l’environnement, durant laquelle les virus sont inactivés plus ou moins rapidement en fonction du sous-type ou de la souche virale analysés. Cependant, à ce jour, les facteurs moléculaires déterminant la stabilité des particules virales en dehors de l’hôte restent largement méconnus. Dans le but d’identifier ces déterminants, nous avons généré différentes combinaisons de réassortiments entre deux virus grippaux de sous-types H1N1 possédant un phénotype de stabilité différent. Les stabilités respectives de ces virus réassortants ont été évaluées dans un environnement-modèle, puis comparées entre elles. Pour cela, nous avons utilisé un système d’analyse en temps réel des cultures cellulaires, permettant de calculer, pour chacun des virus testés, une pente d’inactivation moyenne et, in fine, de mesurer l’influence respective de chacun des segments viraux sur le phénotype de stabilité des virus. D’après nos résultats, le phénotype de stabilité des virus grippaux est majoritairement déterminé par l’hémagglutinine (HA) et la neuraminidase (NA), qui sont les principales glycoprotéines de surface de ces virus. De plus, nous avons identifié des changements d’acides aminés dans la HA et dans la NA, qui ont pour effet une diminution ou une augmentation de la stabilité des particules virales dans l’environnement. Nous avons également montré qu’un virus avec un gène de la HA codons-optimisés, et donc porteur de mutations synonymes, suffit pour augmenter significativement la stabilité des particules virales dans l’environnement. La stabilité de la HA à pH acide, le taux d’expression de la HA dans les cellules infectées, et le nombre de sites de fixation aux ions calcium dans la NA sont modifiés par les mutations décrites dans cette étude, et sont donc des facteurs de stabilité des particules virales. De plus, une analyse en microscopie a permis de montrer que les virus inactivés dans l’environnement peuvent fixer leurs récepteurs cellulaires, mais sont incompétents pour induire l’étape de fusion dans l’endosome nécessaire à l’entrée des virus dans la cellule. Ces deux étapes du cycle viral sont dépendantes de la HA. Dans l’ensemble, nos résultats montrent l’importance de la HA et de la NA des virus grippaux dans la détermination du phénotype de stabilité des virus grippaux dans l’environnement. Par conséquent, la diversité connue des HA et NA dans la nature laisse supposer des variations fréquentes du phénotype de stabilité de ces virus. Leur étude pourrait permettre de mieux décrire l’écologie et l’épidémiologie de ces virus. L’analyse des données épidémiologiques et climatiques des épidémies de grippe saisonnière, sur 5 ans et dans 13 pays, a ainsi révélé une différence de distribution des virus H1N1 et H3N2, en fonction de la température hebdomadaire dans ces pays. La comparaison de la stabilité de ces virus sur des surfaces, à 4 °C et à 20 °C, suggère que la distribution des sous-types viraux au début des épidémies est en partie régulée par leur stabilité en fonction de la température / The transmission of Influenza A viruses (IAV), either airborne in mammals or oro-faecal in aquatic birds, submits viral particle to a wide range of environmental conditions. These environmental conditions modulate IAV survival outside the host, which is also dependent on the viral subtype or strains. To date, the molecular drivers of IAV environmental persistence remain to be identified. In order to identify IAV molecular drivers of the environmental persistence, we generated different reassortant viruses between two H1N1 viruses that do not have the same stability outside the host. To this purpose, we performed survival kinetic and compared the inactivation slope of generated reassortant viruses in our controlledenvironment, using a real time cell analysis system. Our results demonstrate that the hemagglutinin (HA) and the neuraminidase (NA) are the main viral segments driving IAV environmental persistence. In addition, mutations driving viral stability in the environment were identified in the HA and NA amino-acid sequences. We also demonstrated that synonymous mutations introduced in the HA, using a codon-optimization strategy, drive the environmental persistence of IAV. The HA stability at low pH, HA surface expression levels in infected cells and the number of calcium binding sites of the NA were alternately changed by the mutations described in our study, indicating that these are stability determinants of IAV survival outside the host. Then, the sequential events of viral entry were analysed with fluorescence microscopy assays, showing that viral particles being exposed for a long period in saline water at 35°C are still able to bind their cellular receptor whereas the HA-mediated fusion within the endosome is not possible anymore. These two steps of the viral cycle are mainly mediated by the HA protein. Altogether, these result highlight the importance of the HA and the NA proteins, driving the environmental persistence of IAV. Given the known diversity of these two proteins in nature, this arouses interest in studying IAV environmental persistence at a more global scale. Such study could improve our knowledge on IAV ecology and epidemiology. Epidemiologic and climatic data analyse of human seasonal influenza viruses during 5 years and from 13 countries revealed that H1N1 virus and H3N2 virus distribution differs according to the mean weekly temperature in these countries. We then compared the H1N1 virus and H3N2 virus persistence on stainless steel surface at 4 °C and 20 °C, and the preliminary results suggest that IAV seasonal subtypes distribution might be partly regulated by their stability according to the temperature

The proper orchestration of voltage-gated ion channel gating is vital to maintaining normal heart rhythms throughout an animal's lifespan. Voltage-gated sodium channels, Nav, are responsible for the initiation of the cardiac action potential, which leads to cardiac systole. Comparison of neonatal ventricular and atrial myocyte Nav gating with adult indicated that the neonatal ventricular Nav gated following a ~10 mV greater depolarization than did atrial or adult ventricular Nav. In this study I questioned whether development- and/or chamber-dependent changes in Nav-associated functional sialic acids could account for these differences. When desialylated with neuraminidase, all gating characteristics for the lower voltage activated atrial and adult ventricular Nav shifted significantly to more depolarized potentials. However, desialylation of the higher voltage activated neonatal ventricular Nav had no effect on channel gating. Furthermore, channels were stripped of their N-glycosylation via PNGase-F in an attempt to separate the potential effects of the remaining glycosylation structure on Nav gating. Following treatment, neonatal ventricular Nav gating remained unchanged while atrial and adult ventricular Nav gating again shifted to depolarized potentials nearly identical to those of the neonatal ventricular channel. Immunoblot analyses indicated that atrial and adult ventricular Nav α subunits are more heavily sialylated than the neonatal ventricular a subunit, with approximately 15 more sialic acid residues. The data indicate that differential sialylation of myocyte Nav α subunits is responsible for much of the developmental and chamber-specific remodeling of Nav gating observed here. In addition, the Nav1.5 α subunit can associate with β subunits, also believed to be sialylated. The potential for functional β1 trans sialic acids to further modulate Nav1.5 gating was tested via co-transfection of β1 with the Nav1.5 α subunit into the Pro5/Lec2 mammalian expression system. Co-transfection revealed that the additional b1 trans sialic acids caused a hyperpolarizing shift in all tested gating parameters. When transfected into neonatal ventricular myocytes, β1 expression revealed no effect, implying that β1 expression alone is not responsible. Together, the myocyte and expression system studies describe a novel mechanism by which Nav gating, and subsequently cardiac excitability, are modulated by the regulated change in channel-associated functional sialic acids.