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Influenza A is involved in hundreds of thousands of deaths globally every year resulting from viral infection-related complications. Previous efforts to subdue the virus by preventing proper function of wild-type (WT) neuraminidase (N), and M2 proteins using oseltamivir and amantadine (AMT) or rimantadine (RMT), respectively, exhibited success initially. Over time, these drugs began exhibiting mixed success as the virus developed drug resistance. M2 is a proton channel responsible for the acidification of the viral interior which facilitates release of the viral RNA into the host. M2 has a His37-tetrad that is the selective filter for protons. This protein has been demonstrated to be a feasible target for organic compounds. However, due to a mutation from serine to asparagine at residue 31 of M2, which is found in the majority of influenza strains circulating in humans, AMT and RMT block is insufficient. From simulations, it is unclear whether the insensitivity results from weak binding or incomplete block. The question of how the S31N mutation caused MT and RMT insensitivity in M2 is addressed here by analyzing the binding kinetics of AMT and RMT using the two-electrode voltage clamp electrophysiology method. The dissociation rate constant (k2) is dramatically increased compared to WT for both AMT and RMT, by 1500-fold and 17000-fold respectively. Testing of AMT at 10 mM demonstrates complete block, albeit weak, of the S31N M2 channel. At 10 mM, RMT does not reach complete block even though the binding site is saturated. When RMT is in the bound state, it is not blocking all the current, and is binding without block. These results motivated the development of novel M2 blockers using copper complexes focusing on the His37 complex in M2. I hypothesized that copper complexes would bind with the imidazole of a histidine in the His37 complex and prevent proton conductance. The His37 complex is highly conserved in the M2 channel and, therefore, would be important target for influenza therapeutics. By derivatizing the amines of known M2 blockers, AMT and cyclooctyalmine, to form the iminodiacetate or iminodiacetamide, we have synthesized Cu(II) containing complexes and characterized them by NMR, IR, MS, UV–vis, and inductively coupled plasma mass spectroscopy (ICP-MS). The copper complexes, but not the copper-free ligands, demonstrated H37-specific blocking of M2 channel currents and low micromolar anti-viral efficacies in both Amt-sensitive and Amt-resistant IAV strains with, for the best case, nearly 10-fold less cytotoxicity than CuCl2. Isothermal titration calorimetry was used to obtain enthalpies that showed the copper complexes bind to one imidazole and curve fitting to the electrophysiology data provided rate constants for binding in the M2 channel. Computational chemistry was used to obtain binding geometries and energies of the copper complexes to the His37-tetrad. The results show that the copper complexes do bind with the His37 complex and prevent proton conductance and influenza infection.

Human influenza is a contagious respiratory disease resulting in substantial morbidity and mortality worldwide. With the recent cases of avian influenza infections in humans and the heightened concern for an influenza pandemic arising from these infections, it is essential to understand host responses that would confer protective immunity to influenza. The cell-mediated immune responses to influenza virus play an important role during influenza infection. To analyze the specificity and diversity of memory T-cell responses, we performed a genome-wide screening of T cell epitopes to influenza A virus in healthy adult donors. We identified a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in interferon-(IFN-γ) enzyme-linked immunosorbent spot assays (ELISPOT) using peripheral blood mononuclear cells (PBMCs) from four healthy adult donors. We found that among 11 influenza viral proteins, hemagglutinin (HA) and matrix protein 1 (M1) had more T-cell epitopes than other viral proteins. The donors were not previously exposed to H5N1 subtype, but we detected H5 HA T cell responses in two of the four donors. To confirm that HA is a major target of T cell responses we also analyzed H1 and H3 HA-specific T-cell responses using PBMC of additional 30 adult donors. Fifteen out of thirty donors gave a positive response to H3 HA peptides, whereas five of thirty donors gave a positive response to H1 HA peptides. Because we detected T cell responses to the H5 HA peptides in donors without prior exposure to H5N1 subtype, we asked if cross-reactive T cells to H5 HA peptides can be attributed to a prior exposure to H2N2 subtype, the closest HA to the H5 based on their phylogeny. We compared younger donors who have no prior exposure to H2N2 subtype and older donors who were likely to be exposed to H2N2 subtype, and both groups responded H2N2 peptides at similar level, suggesting that memory T cells cross-reactive to H5 HA peptides can be generated by prior exposure to the H1N1 and H3N2 subtypes, and the exposure to H2N2 subtype is not necessary. We subsequently identified a CD4+ T cell epitope that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of HA of influenza A and the HA of the influenza B virus. A CD4+ T cell line specific to this epitope recognizes target cells infected with various influenza A viruses including seasonal H1N1 and H3N2, a reassortant H2N1, the 2009 pandemic H1N1, H5N1 and influenza B virus in cytotoxicity assays and intracellular cytokine staining assays. Individuals who have the HLA-DRB1*09 allele have ex vivo IFN-γ responses to this epitope peptide in ELISPOT. Although natural infection or standard vaccination may not induce strong T and B cell responses to this very conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4+ T cells which are cross-reactive to both influenza A and B viruses.

Molecular dynamics simulation was used to study ion permeation through different membrane proteins embedded in a lipid bilayer (DMPC) with different saline solutions. The potential of mean force (PMF) for ion transport was obtained by umbrella sampling simulations. A revised MacKerell force field for tryptophan residues was studied using gramicidin A (gA) channel as a test model. The revised force field contribution to the Na+ PMF was consonant with the prediction from the experimental results, but in stark contrast to the prediction of the CHARMM force field, version 22, for the tryptophan side-chain. A new grid-based correction map algorithm by MacKerell group, called CMAP, was introduced into the CHARMM force field, version 31. The CMAP algorithm focused on optimizing phi, psi dihedral parameters for the peptide backbone. The CMAP corrections reduced the excessive translocation barrier. Decomposition demonstrated the reduction in the translocation barrier was due to effects on the K+ PMFH2O rather than on K+ PMFgA. The presence of negatively charged sulfonate group at the entrance and exit of the gA channel affected the depth and the location of the highly occupied sites. The negatively charged sulfonate group produced a strong attraction for the cations in the bulk towards the channel mouth. In the M2 transmembrane domain channel (M2-TMD), three M2-TMD structures were studied, differing only in whether the selectivity-filter (four His37 side-chains) was uncharged, +2 charged, or +3 charged. M2-TMD structural properties were compared with the structural properties of other models extracted from NMR and X-ray studies. The spontaneous cation and anion entry into the charged selectivity-filter was different from that into a neutral selectivity-filter. Cl- ions had a lower free-energy barrier in the selectivity-filter than either Na+ or NH4+ ions through the M2-TMD channel. NH4+ ions had a lower free-energy barrier in the selectivity-filter than Na+ ions. Based on accessible rotamer conformations, a revised conductance mechanism was proposed. In this conductance mechanism, the His37 side-chain functioned as an acceptor and donor group, whereas the Trp41 side-chain functioned as a carrying group.

Les interactions entre le circovirus porcin de génotype 2b (PCV2b), le virus du syndrome reproducteur et respiratoire porcin (VSRRP) et le virus de l’influenza porcine (VIP) dans le complexe respiratoire porcin (CRP) sont souvent associées à une augmentation des signes cliniques respiratoires chez les animaux. L’objectif général de cette étude était de caractériser les effets et les mécanismes moléculaires impliqués dans la pathogenèse de la co-infection de PCV2b avec le VSRRP ou le VIP dans des modèles cellulaires des voies respiratoires du porc. La réplication virale, la viabilité cellulaire, l’expression de l’ARNm des cytokines et la modulation de l’expression des gènes cellulaires induite par l’infection virale ont été évalués dans les cellules co-infectées versus les cellules infectées par un seul virus. Les résultats obtenus ont permis de confirmer que la réplication du PCV2b augmente en présence du VSRRP dans les cellules épithéliales des voies respiratoires porcines (NPTr) génétiquement modifiées pour exprimer le récepteur CD163 (NPTr-CD163), tandis que celle du VSRRP est diminuée. Il a été mis en évidence que le PCV2b provoque une diminution ou une augmentation de la réplication du VIP dans les cellules NPTr et les macrophages alvéolaires porcins (iPAM 3D4/21), respectivement. Cependant, la réplication du PCV2b n’est pas affectée en présence du VIP. L’expression des ARNm des cytokines varie différemment selon le modèle de co-infection analysé et le type cellulaire infecté. L’expression des interférons de type I (α/β) a été significativement réduite dans les cellules NPTr-CD163, co- infectées par le PCV2b et le VSRRP (PCV2b/VSRRP) par rapport aux mêmes cellules infectées uniquement par le PCV2b. Cependant, la co-infection du PCV2b et du VIP (PCV2b/VIP) a causé une augmentation synergique de l’expression des IFNs de type I et de type II dans les cellules NPTr, tandis que dans les cellules iPAM 3D4/21, le PCV2b a affecté la réponse des IFNs induite par le VIP. À la suite des analyses transcriptomiques, plusieurs gènes différentiellement exprimés ont été identifiés dans les cellules co-infectées ou infectées par un seul virus. Dans les cellules co-infectées PCV2b/VSRRP, le niveau d’expression de l’ARNm et de la protéine du gène cellulaire codant pour la phosphatase 1 à double spécificité (DUSP1) ont été significativement augmentés. La réduction de l’expression de DUSP1, à l’aide des petits ARN interférents (ou siRNA pour small interfering RNA) dans les cellules co-infectées a significativement réduit la réplication du PCV2b, suggérant un rôle de DUSP1 dans la pathogenèse de la co-infection PCV2b/VSRRP. / Interactions of porcine circovirus genotype 2b (PCV2b), porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SwIV) in the porcine respiratory complex (PRC) are often associated with increased respiratory clinical signs in animals. The general objective of this study was to characterize the effects and molecular mechanisms involved in the pathogenesis of PCV2b co-infection with PRRSV or SwIV using cellular models of the porcine respiratory tract. Viral replication, cell viability, cytokine mRNA expression and modulation of cell gene expression induced by viral infection were evaluated in co-infected cells versus single infected cells. The results obtained confirmed that PCV2b replication increases in the presence of PRRSV in porcine respiratory epithelial cells (NPTr) genetically modified to express the CD163 receptor (NPTr-CD163), whereas PRRSV is decreased. It was demonstrated that PCV2b causes a decrease or an increase in SwIV replication in NPTr cells and porcine alveolar macrophages (iPAM 3D4/21), respectively. However, PCV2b replication was not affected in the presence of SwIV. The impact of co-infections on cytokine mRNA expression varied according to the co- infection model and the type of infected cell. Type I IFNs (α/β) expression was significantly reduced in PCV2b/PRRSV co-infected NPTr-CD163 cells compared to PCV2b single-infected cells. However, PCV2b/SwIV co- infection caused a synergistic increase in type I IFNs expression in NPT cells, while in iPAM 3D4/21 cells, PCV2b affected SwIV-induced IFN response. As result of transcriptomic analyses, differentially expressed genes were identified in co-infected and single infected cells. It was observed that in PCV2b/PRRSV co- infected cells, the mRNA and protein expression levels of the cellular gene coding for the dual specificity protein phosphatase 1 (DUSP1) were significantly increased. Knockdown of DUSP1 expression in co- infected cells, using specific siRNA, significantly reduced PCV2b replication, suggesting a role for DUSP1 in the pathogenesis of PCV2b/PRRSV co-infection.

Chong, Ka Chun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 125-141). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Les travaux de recherche effectués dans le cadre de cette thèse ont été guidés par le manque d’information et une compréhension limitée des mécanismes épidémiologiques à l’origine de l’émergence et de la diffusion de la grippe aviaire hautement pathogène, souche H5N1 en Chine du Sud, aussi reconnue comme l’épicentre potentiel de l’émergence des virus influenza aviaires à caractères pandémiques. <p>Dans ce cadre, des données spatio-temporelles relatives aux foyers de la maladie ainsi que des données de surveillance virologiques (isolement du virus effectué dans le cadre du système de surveillance nationale) ont été collectées sur une période de quatre ans et analysées afin d’éxplorer les facteurs de risque relatifs à l’émergence et persistence de la maladie dans certaine zones de production du sud de la Chine. Les analyses ainsi effectuées ont permis d’identifier, à travers l’utilisation de méthodes statistiques robustes ayant fait leur preuve dans le domaine de la santé ou de l’écologie (la régression logistique classique et les arbres de regression logistique), des facteurs de risque liés à certains types de production de volailles (canards élevés en plein air, zones riches en eau et par extension associées à la riziculture) ou des facteurs associés à l’activité humaine. A travers une représentation cartographique des facteurs ainsi identifiés, des cartes de risque ont été produites permettant ainsi de visualiser d’une part les zones à haut risque de persistence de l’infection virale et d’autre part les zones vulnérables à l’apparition de foyers de la maladie, donnant aux autorités nationales la possibilité de mieux cibler leurs politiques de surveillance et de contrôle. <p>Dans un second temps, notre étude s’est portée sur les marchés à volailles traditionnels du sud de la Chine qui représentent un risque permanent de persistence, d’évolution et de diffusion des virus influenza aviaires, ainsi qu’un risque important en matière de santé publique. La dynamique de ces marchés et les liens qui les unissent ont été étudiés à travers des outils d’analyse empruntés à la sociologie tels que l’Analyse des Réseaux Sociaux (Social Network Analysis). Grace à cette approche, l’importance de l’hygiène de ces marchés et notamment du nettoyage et de la désinfection des cages dans la persistence du virus a été mise en évidence. Enfin, des enquêtes effectuées auprès des vendeurs de volailles ont permis d’identifier l’origine et la destination des animaux vendus et de reconstruire des réseaux plus ou moins intriqués de liens commerciaux qui unissent ces marchés entre eux dans trois provinces du sud de la Chine. L’analyse de ces réseaux et de leurs configurations ont permis d’identifier des marchés à plus haut risque de persistence de l’infection du fait de leur position centrale au sein de ces réseaux. De même qu’il est indispensable de cibler la surveillance et le contrôle de la maladie dans des zones écologiquement favorables à la persistence des virus influenza aviaires, cette étude révèle l’importance de certaines pratiques hygiéniques et commerciales dans la persistence de la maladie et la nécessité de cibler la surveillance et le contrôle au niveau de certains de ces marchés situés au centre d’un réseau dense et connecté, pour pouvoir in fine mieux contrôler la maladie au niveau national.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished