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.

University of Macau / Institute of Chinese Medical Sciences

The lithiation of 3-sulfolene followed by subsequent treatment with an alkyl halide electrophile has been previously established as a method to produce 2-substituted-3-sulfolenes. Tandem reactivity with bis-alkyl halides has been observed to afford relatively simple bicyclic products. We hypothesized that it may be possible to access more complex bicyclic systems through use of bis-vinyl ketones as the electrophilic component. Herein, we present the outcome and mechanistic insights for the reaction between a variety of 3-sulfolene and substituted-3-sulfolene anions with bis-vinyl ketones to afford a variety of stereochemically complex fused, bridged and spiro bicyclic archetypes. The potential of these bicyclic-sulfone frameworks to act as molecular scaffolds for the generation of conformationally-restricted enzyme inhibitors is explored. Potent monocyclic small molecules that inhibit influenza’s neuraminidase enzyme have been developed as commercially successful antivirals. Similarly potent inhibitors against prokaryotic or eukaryotic neuraminidases have yet to be described. Selective inhibitors of these latter neuraminidase isozymes may provide useful treatments for bacterial infections (such as cholera and pneumonia) as well as a variety of cancers and metabolic disorders. A conformationally-restricted scaffold may prove ideal for designing selective (and potent) inhibitors against these underexplored enzymes. As a proof of principle, one of our rigid bicyclic-sulfone archetypes is elaborated to a drug-like scaffold that is shown to inhibit viral, bacterial and human neuraminidase enzymes. / Graduate / mgbrant@uvic.ca

This study designed and computational docked a group of ligands intended to find potent inhibitors for Neuraminidase 4 which would have strong interactions with 8 conserved amino acids in the active site. Several trials of ligands were designed based on derivatives of neuraminic acid and evaluated as inhibitors of influenza neuraminidase. Optimized geometries of those ligands were determined using HF/B3LYP/6-311++G** techniques. Binding energies of the ligands bound to the N4 subtype of the neuraminidase protein were determined using AutoDock 4.0. Currently used inhibitors for influenza viruses will also be analyzed in the exactly same way. Comparing the binding information of those candidates and current ligands can provide a useful data about the potential of these species as antiviral drugs. / Department of Chemistry

Erysipelothrix rhusiopathiae, a Gram-positive bacillus, has long been an important pathogen in veterinary medicine as well as a cause of serious disease in humans. Infections caused by this organism have economic impact on animal industries, causing erysipelas in swine and morbidities in other farmed animals. Human infections are commonly erysipeloid (skin cellulitis) and occasionally septicaemia or endocarditis. Little is known of the diagnosis, epidemiology and pathogenesis of such infections in Western Australia. The aims of this thesis were to establish new diagnostic techniques for the detection and recovery of E. rhusiopathiae, to describe the epidemiology of Erysipelothrix infection in Western Australia in humans and animals, and to characterize virulence-associated characteristics, especially focusing on the neuraminidase produced by the organism. A protocol using 48 h Brain Heart Infusion enrichment followed by subculture to selective agar containing antibiotics achieved the highest recovery rate of 37% in a seafood survey. Twentyone isolates of Erysipelothrix spp., of which 19 were identified as E. rhusiopathiae, were obtained. Two published PCR assays for differentiating E. rhusiopathiae and other Erysipelothrix species were evaluated and the best PCR detection rate achieved was 67% following selective enrichment. The PCR method was 50% more sensitive than the culture method. Epidemiological surveys using the above methods showed that E. rhusiopathiae infection is present in farmed animals in Western Australia. The PCR positive frequencies (3.3-3.7%) and isolate recovery rate (2.8-3.3%) in samples from pig and sheep abattoirs and carcass washings indicate a potential threat to the economy of the farmed animal industry as well as a public health concern with the occurrence of E. rhusiopathiae in meat for consumption. Positive PCR results (1.1%) from human skin swabs of patients with cellulitis and wounds may suggest the existence of Erysipelothrix colonization in the general population. Genetic relatedness of 92 isolates of Erysipelothrix species from various sources was analyzed and a total of 64 distinct PFGE patterns identified. Isolates were further classified into 20 clonal groups based on pattern similarities, and most E. rhusiopathiae were clustered into six groups. A few patterns of other Erysipelothrix species were clustered into separate groups from E. rhusiopathiae but shared greater than 70% similarity with E. rhusiopathiae. The genetic relatedness of colonial variants was well demonstrated using this method. PFGE typing promises to be a useful tool for epidemiological and taxonomic studies of Erysipelothrix. Several virulence-associated factors were characterized in 86 isolates of Erysipelothrix spp. A rapid and sensitive peanut lectin hemagglutination assay for neuraminidase was developed and the influence of media, incubation conditions and pH on the production of the enzyme was investigated. All 61 isolates of E. rhusiopathiae produced neuraminidase in cooked meat broth with titres between 1:10 and 1:320, with no significant difference in titre among isolates from different sources. The enzyme activity was not detected in non-pathogenic Erysipelothrix spp. Capsule was produced by 78.7% of isolates of E. rhusiopathiae but not by other species, while both hyaluronidase and haemolysin were produced by non-pathogenic Erysipelothrix spp. It was concluded that neuraminidase and capsule are most likely to be virulence factors of E. rhusiopathiae. The gene encoding neuraminidase was cloned from the type strain E. rhusiopathiae ATCC 19414. The cloned fragment was a functional partial nanH gene with a mol% G+C of 39.7. The predicted amino acid sequence displayed homology with many microbial neuraminidases and contained conserved sequences found in most bacterial neuraminidases. Southern hybridization experiments demonstrated that the gene was present as a single copy on the bacterial genomic DNA. A neuraminidasenegative mutant vector was constructed by insertional inactivation using a tetM cassette. This has provided starting material for developing a neuraminidase-deficient E. rhusiopathiae mutant, which will permit the study of the role of neuraminidase in pathogenesis. Based on the cloned sequence, a sensitive neuraminidase-specific nested PCR technique was designed and optimized. The specificity was tested in 61 isolates of E. rhusiopathiae, 25 Erysipelothrix species, and 62 other species of neuraminidaseproducing and non-producing bacteria. All isolates of E. rhusiopathiae were PCR positive and all other bacteria were negative; thus this PCR is a highly specific method suitable for application in clinical investigations of Erysipelothrix infection. In conclusion, the present study has contributed new knowledge of the biology of Erysipelothrix spp. and current occurrence of Erysipelothrix infections in Western Australia, as well as to the understanding of pathogenesis of E. rhusiopathiae. Development of several new cultural and molecular approaches in combination with other established techniques will facilitate future studies of the epidemiology, taxonomy and pathogenesis of this bacterial species.

Influenza viruses cause respiratory illnesses which can vary in severity depending on the strain of the virus, as well as the age and health condition of the host. Influenza remains a major threat to public health due to its nature prone to suffer mutations. As a result, vaccines have to be reformulated annually and new strains may cause sporadic global pandemics. Furthermore, the recent emergence of resistant strains of the virus against the current standard of care (oseltamivir and zanamivir) underlines the need of novel anti-influenza therapeutics. The aim of this dissertation work is to contribute to the discovery of new anti-influenza inhibitors either by rational drug-design and optimization of oseltamivir structure, or by developing screening assays suitable for the discovery of novel inhibitors of the enzymes neuraminidase or RNA-polymerase. Scheme 1. Overview of the strategy used for the development of new anti-influenza therapeutics. The dashed arrows indicate the inhibitors that were converted into probes and their corresponding target enzymes Two main modification points were explored for the improvement of oseltamivir properties (Scheme 1); modifications at carbon C-3 aimed to overcome oseltamivir resistance caused by common mutations like H274Y, meanwhile modifications at carbon C-5...

Influenza viruses cause respiratory illnesses which can vary in severity depending on the strain of the virus, as well as the age and health condition of the host. Influenza remains a major threat to public health due to its nature prone to suffer mutations. As a result, vaccines have to be reformulated annually and new strains may cause sporadic global pandemics. Furthermore, the recent emergence of resistant strains of the virus against the current standard of care (oseltamivir and zanamivir) underlines the need of novel anti-influenza therapeutics. The aim of this dissertation work is to contribute to the discovery of new anti-influenza inhibitors either by rational drug-design and optimization of oseltamivir structure, or by developing screening assays suitable for the discovery of novel inhibitors of the enzymes neuraminidase or RNA-polymerase. Scheme 1. Overview of the strategy used for the development of new anti-influenza therapeutics. The dashed arrows indicate the inhibitors that were converted into probes and their corresponding target enzymes Two main modification points were explored for the improvement of oseltamivir properties (Scheme 1); modifications at carbon C-3 aimed to overcome oseltamivir resistance caused by common mutations like H274Y, meanwhile modifications at carbon C-5...

No description available.

Influenza is an infectious disease caused by the influenza virus. This virus causes a severe viral infection that spreads easily from person to person in yearly pandemics. Vaccination is the most effective way to prevent the infection, however, due to the high rate in mutations of the virus, the vaccine needs to be often reformulated. Another option to combat influenza is based on administration of antiviral drugs. Clinical studies of isolated influenza strains ("avian flu" H5N1, 2004; "swine flu" H1N1, 2009) revealed resistance towards known influenza neuraminidase inhibitors (zanamivir, oseltamivir). The resistance is caused by structural changes close to the enzymatic site. This calls for the development of new neuraminidase inhibitors as well for development of inhibitors targeting different influenza enzymes. This Thesis is focused on design and synthesis of new inhibitors of influenza neuraminidase and RNA-dependent RNA polymerase, namely PA subunit and the assembly of PA-PB1 heterodimer enzymes (Scheme 1). Influenza neuraminidase inhibitors were prepared by C-5 derivatization of oseltamivir followed by subsequent extension of its structure with binders of 150-cavity. Binding potencies of new oseltamivir derivatives against two influenza strains were determined. The next part contributed to...