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Lai, Chun-cheong, and 黎振昌. "STD-NMR as a novel method to study influenza virus-receptor interactions." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47849745.
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Influenza infections continue to be a global health concern that causing both
seasonal epidemics and unpredictable pandemics. Hemagglutinin (HA) and
Neuraminidase (NA) are the two major surface glycoproteins of influenza viruses,
which are important for their host cell sialic acid (Sia) receptor binding and
cleaving activities. Although numerous methods have been developed to study the
HA and NA interactions with sialic acid, x-ray crystallography remained the only
method to provide detailed information at atomic resolution.
The aim of this study is to develop and evaluate a novel strategy for the
investigation of influenza virus-receptor interactions, which is able to provide
information about an interaction down to atomic resolution. Influenza virus-like
particles (VLPs) containing HA and NA separately were developed and it was
reported here for the first time that sole expression of NA in mammalian cell led
to VLP formation. Characterization of these VLPs demonstrated that they are
non-infectious, but morphologically and biochemically mimic the native viruses.
Therefore the VLPs can be regarded as an ideal research model to study the
HA-Sia interaction without the interference of NA, or vice versa. Saturation
transfer difference (STD) NMR spectroscopy is a state-of-the-art technology to
determine how a binding-ligand interacts with its target protein. Modification of
STD-NMR methodology was performed to adapt the technique to influenza VLP
system. HA-Sia interaction was investigated in great detail and group epitope
mapping of the interacting ligands was performed by analyzing the STD-NMR
spectra. The data obtained are in a good agreement with the well established
crystallography technique, reflecting the reliability of the STD-NMR technology.
Regarding the NA-Sia interaction, my data demonstrated that
substrate-hydrolysis specificity of NA is dependent on the binding of NA to those
ligands. In addition, using competition experiments with NA inhibitor, a
secondary sialic acid binding site was detected. It is the first direct experimental
evidence that confirms avian, seasonal human and human pandemic swine-origin
influenza virus N1 neuraminidases exhibit a distinct secondary binding site.
In conclusion, here I presented a novel interdisciplinary strategy using VLP
and NMR technology to study the interaction of influenza virus with its receptor.
This method is unique in its ability to provide detailed information on the HA and
NA interactions with sialic acid leading to group epitope mapping of the binding
ligands, which will help us not only to understand the virus tropism but also to
define new therapeutic targets.published_or_final_version
Microbiology
Doctoral
Doctor of Philosophy
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MACCHI, ELEONORA. "NMR as a tool for structural characterization of carbohydrates and glycan-protein interactions." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/69274.
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Il virus dell’influenza A è un virus a RNA formato da 8 geni, tre dei quali - emagglutinina (HA), neuramminidasi (NA) e polimerasi (PB) –, risultano particolarmente critici nell’infezione e nella trasmissione uomo-uomo. L’infezione inizia con il legame dell’HA del virus ai recettori glicanici presenti sulle cellule dell’ospite; questa interazione è altamente specifica, ed è governata dal tipo di legame tra l’acido sialico e il galattosio all’interno del recettore.
I recettori umani glicanici, siti di riconoscimento per i virus human-adapted, sono espressi principalmente nel tratto superiore dell’epitelio respiratorio umano e presentano un legame α2→6 tra l’acido sialico e il galattosio nell’estremità non riducente. I virus aviari invece, riconoscono recettori glicanici che presentano un legame α2→3 tra acido neuraminico e il galattosio.
Studi precedenti dell’interazione tra HA e trisaccaridi hanno dimostrato che sia la conformazione dei glicani, che il diverso tipo di legame tra acido sialico e galattosio sono fattori chiave per la regolazione dell’interazione. Partendo dalle sopracitate considerazioni questo lavoro di ricerca si è occupato di studiare la dinamica e la conformazione in soluzione di due pentasaccaridi, usati come modelli per il recettore aviario (LSTa, Neu5Ac-α(2→3)-Gal-β(1→3)-GlcNAc-β(1→3)-Gal-β(1→4)-Glc) e umano (LSTc, Neu5Ac-α(2→6)-Gal-β(1→4)-GlcNAc-β(1→3)-Gal-β(1→4)-Glc), utilizzando tecniche NMR (Nuclear Magnetic Resonance) e simulazioni di dinamica molecolare (MD). I nostri studi dimostrano che in soluzione i due recettori presentano diverse conformazioni, dinamiche e topologie. Queste peculiarità uniche comportano caratteristiche molecolari diverse per il riconoscimento di HA, dimostrando quindi la specificità dell’interazione tra recettore e emagglutinina.
La relazione tra la specificità dell’HA verso i recettori e la trasmissibilità del virus è stata precedentemente dimostrata usando il prototipo del virus SC18 (H1N1) A/South Carolina/1/1918. Combinando tecniche di Risonanza Magnetica Nucleare e di dinamica molecolare, abbiamo dimostrato come, durante l’interazione, il sito di binding dell’emagglutinina imponga differenti vincoli conformazionali al recettore. Il virus pandemico SC18, che presenta un’efficacia di trasmissione negli uomini molto alta, a confronto con il singolo (NY18, Asp225 → Gly) e doppio (AV18, Asp190 → Glu e Asp225 → Gly) mutante, impone maggiori vincoli alla conformazione del recettore umano, proprietà correlata all’affinità dell’interazione recettore-HA, misurata tramite saggi biochimici.
Questa relazione tra affinità e vincoli conformazionali imposti al recettore è stata osservata anche per il virus aviario-adattato AV18, il quale impone vincoli conformazionali maggiori al recettore aviario in confronto a quelli imposti a quest’ultimo da NY18. In particolare, è interessante osservare come emagglutinine differenti impongano vincoli conformazionali diversi a seconda che leghino recettori umani o aviari. In ultimo, abbiamo esteso il nostro studio a un virus meno pandemico, H7N9, e due suoi mutanti, i quali sono in grado di legare sia il recettore umano che aviario, allo scopo di capire come avviene l’interazione tramite l’utilizzo di tecniche NMR e di dinamica molecolare.
In questo studio descriviamo le basi strutturali dell’interazione tra l’emagglutinina di nuovi virus e i recettori umani e aviari, combinando l’approccio sperimentale a tecniche computazionali. Questa metodologia potrà essere usata come strumento utile per la sorveglianza di nuovi virus pandemici.
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Liao, Shu-Yu Ph D. Massachusetts Institute of Technology. "Structure and dynamics of full-length M2 protein of influenza A virus from solid-state NMR." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/113974.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017.Cataloged from PDF version of thesis.
Includes bibliographical references.
Solid-state nuclear magnetic resonance (SSNMR) has been frequently used to elucidate the structure and dynamics of membrane proteins and fibrils that are difficult to characterize by Xray crystallography or solution NMR. This thesis focuses on the structure determination and the proton conduction mechanism of the full-length matrix protein 2 (M2) of influenza A virus. The M2 membrane protein can be separated into three domains: an N-terminal ectodomain (1-2 1), an cc-helical transmembrane domain (TM) (22-46) connected to an amphipathic helix (AH) and a Cterminal cytoplasmic tail (63-97). The TM domain of M2 is responsible for proton conduction ant the ectodomain has been the target for vaccine development. The cytoplasmic tail has been implicated in M2 interaction with other viral proteins from mutagenesis studies. Given the importance of both N- and C-termini, it is essential to determine the structure and the dynamics of M2FL. Furthermore, we are interested in how the cytoplasmic tail affects proton conduction and the interaction of the anti-viral drug amantadine with M2 in the presence of the C-terminus. Using uniformly ¹³C, ¹⁵N-labeled M2FL, our water-selected 2D ¹³C-¹³C correlation experiment indicated that N- and C- termini are on the surface of the lipid bilayer moreover combining with chemical shift prediction, we determined that these two domains are mostly disordered. Deleting the ectodomain of M2FL (M2(21-97)) proved that a small [beta]-strand is located at the N-terminus only in the DMPC-bound state. The M2 conformation is found to be cholesterol-dependent since [beta]-strand is not found in cholesterol-rich membranes. M2(21-97) shows cationic histidine at higher pH, in contrast to M2TM, indicating that the cytoplasmic tail shifts the His37 pKa equilibria. Quantification of the ¹⁵N intensities revealed two pKa's as opposed to of four in M2TM suggesting cooperative proton binding. A possible explanation is that the large number of positively charged residues in the cytoplasmic tail facilitates proton conduction. The cytoplasmic tail was also found to restore drug-binding as amantadine no longer binds to M2(21-61) a in virus-mimetic membrane. These results have extended our understanding of the influence of the cytoplasmic domain on the structure and proton conduction of M2.
by Shu-Yu Liao.
Ph. D.
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Hornick, Emma E. L. "Contributions of NLRS to pathogenic and protective immune responses during influenza virus infection." Diss., University of Iowa, 2018. https://ir.uiowa.edu/etd/6139.
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Pattern recognition receptors, including members of the NBD and LLR-containing (NLR) family, are key sensors of infection and injury. Early sensing of pathogen invasion and subsequent activation of pro-inflammatory signaling cascades is essential for controlling infection. However, signaling pathways activated upon pathogen recognition can also contribute to inflammation-mediated tissue damage. The studies detailed in Chapters 3 and 4 are primarily concerned with the roles of two NLR family members, Nlrp12 and Nlrc4, during influenza A virus (IAV) infection. While IAV itself is cytopathic, the immune response is responsible for a great deal of the tissue damage during infection in some contexts. NLR family members are involved in both pathogen sensing and modulation of pro-inflammatory signaling, thus they are perfectly situated to shift the balance between pathogen clearance and immunopathology.
Nlrp12 has been implicated in regulation of pro-inflammatory signaling through NFκB family members. In Chapter 3 we report that during IAV infection, we find no differences in those pathways, and instead we describe a novel role for Nlrp12 in regulating transcript stability. Previous work has shown that one of the key differences between lethal and sublethal IAV infections is the early and exaggerated recruitment of neutrophils. Previous studies in our laboratory had established a role for Nlrp12 in CXCL1-mediated neutrophil recruitment during respiratory bacterial infections. We therefore hypothesized that Nlrp12-/- mice would be protected from pathogenic neutrophil recruitment during lethal IAV infection due to decreased CXCL1 production. In Chapter 3 we show that indeed, Nlrp12-/- mice have improved survival, decreased pulmonary microvascular permeability, and decreased necrosis and hemorrhage in their airways compared to WT mice. Nlrp12-/- mice also have fewer neutrophils in their lungs, due to decreased production of CXCL1 by neutrophils, DCs and macrophages. Our data showing decreased Cxcl1 transcript stability in R848-treated Nlrp12-/- BMDCs strongly suggest that the reduction in CXCL1 production by DCs in the Nlrp12-/- lungs is a result of decreased Cxcl1 transcript stability.
Nlrc4 is a best known as a member of the Nlrc4 inflammasome, which is activated upon sensing of Gram-negative bacterial pathogens. However, a recent study from our laboratory showed an inflammasome-independent role for Nlrc4 in supporting critical anti-tumor T cell responses. Given that T cells are also critical for successful resolution of IAV infection, we hypothesized that during IAV infection, Nlrc4-/- mice would have compromised IAV-specific T cell responses and therefore poorer survival. Indeed, our studies in Chapter 4 show that in IAV-infected Nlrc4-/- mice, the pulmonary IAV-specific CD4 T cell response is significantly diminished and mortality is significantly increased compared to WT mice. During IAV infection, the blunted CD4 T cell response is a result of increased death of the CD4 T cells, perhaps due to increased expression of FasL on CD11c+ cells in the Nlrc4-/- lung environment.
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Williams, Jonathan K., Alexander A. Shcherbakov, Jun Wang, and Mei Hong. "Protonation equilibria and pore-opening structure of the dual-histidine influenza B virus M2 transmembrane proton channel from solid-state NMR." AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2017. http://hdl.handle.net/10150/626055.
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The influenza A and B viruses are the primary cause of seasonal flu epidemics. Common to both viruses is the M2 protein, a homotetrameric transmembrane proton channel that acidifies the virion after endocytosis. Although influenza A M2 (AM2) and B M2 (BM2) are functional analogs, they have little sequence homology, except for a conserved HXXXW motif, which is responsible for proton selectivity and channel gating. Importantly, BM2contains a second titratable histidine, His-27, in the tetrameric transmembrane domain that forms a reverse WXXXH motif with the gating tryptophan. To understand how His-27 affects the proton conduction property of BM2, we have used solid-state NMR to characterize the pH-dependent structure and dynamics of His-27. In cholesterol-containing lipid membranes mimicking the virus envelope, N-15 NMR spectra show that the His-27 tetrad protonates with higher pKa values than His-19, indicating that the solvent-accessible His-27 facilitates proton conduction of the channel by increasing the proton dissociation rates of His-19. AM2is inhibited by the amantadine class of antiviral drugs, whereas BM2 has no known inhibitors. Wemeasured the N-terminal interhelical separation of the BM2 channel using fluorinated Phe-5. The interhelical F-19-F-19 distances show a bimodal distribution of a short distance of 7 angstrom and a long distance of 15-20 angstrom, indicating that the phenylene rings do not block small-molecule entry into the channel pore. These results give insights into the lack of amantadine inhibition of BM2 and reveal structural diversities in this family of viral proton channels.
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Harter, Cordula. "Zum Mechanismus der Interaktion der Ektodomäne von Influenza Virus Hämagglutinin mit Liposomen /." Zürich, 1988. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=8739.
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Delaforge, Elise. "Dynamique structurale et fonctionnelle du domaine C-terminal de la protéine PB2 du virus de la grippe A." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV037/document.
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La capacité du virus de la grippe aviaire à traverser la barrière des espèces et à devenir fortement pathogène chez les mammifères est un problème majeur de santé publique. Chez les oiseaux, la réplication a lieu dans l'intestin, à 4C, tandis que chez les humains elle a lieu dans l'appareil respiratoire, plus froid, à 33C. Il a été montré que l'adaptation à la température du virus de la grippe a lieu par de nombreuses mutations de la polymérase virale, notamment dans le domaine 627-NLS situé en C-terminal de la protéine PB2. Ce domaine est impliqué dans l'adaptation à l'hôte et interagit avec la protéine de l'hôte, importine alpha, étant donc indispensable pour l'entrée de la polymérase virale dans le noyau de la cellule [Tarendeau et al., 2008]. Les structures cristallographiques du 627-NLS et du complexe importine alpha/NLS existent. Cependant, lors de la superposition de ces structures via leur domaine NLS commun, un important choc stérique entre le domaine 627 et l'importine alpha devient évident. Ceci indique qu'une autre conformation du 627-NLS est requise pour l'interaction avec l'importine alpha [Boivin and Hart, 2011]. Dans cette étude, nous avons examiné les bases moléculaires de l'adaptation inter-espèces du virus à travers l'étude de la structure et de la dynamique du 627-NLS aviaire et humain. Nous avons identifié deux conformations du 627-NLS en échange lent (10-100 s-1), correspondant apparemment à une conformation ouverte et une conformation fermée des deux domaines. Nous proposons que la conformation ouverte du 627-NLS est la seule conformation compatible avec l'interaction avec l'importine alpha, et que l'équilibre entre conformation ouverte et fermée pourrait jouer le rôle de thermostat moléculaire, contrôlant l'efficacité de la réplication virale chez différents hôtes. La cinétique et la dynamique de ce comportement conformationnel important ainsi que de l'interaction entre le 627-NLS et l'importine alpha ont été caractérisées par résonance magnétique nucléaire (déplacements chimique, augmentation paramagnétique de la relaxation, relaxation de spin, transfert de saturation par l'échange chimique), combinée à la diffusion des rayons X et des neutrons aux petits angles ainsi qu'au transfert d'énergie par résonance de type Förster. Aussi, nous avons déterminé les affinités d'une série de mutants évolutifs du 627-NLS pour l'importine alpha et du 627-NLS aviaire ou humain pour différents isoformes de l'importine alpha, montrant que les affinités observées sont cohérentes avec les préférences d'interactions vues in vivoThe ability of avian influenza viruses to cross the species barrier and become dangerously pathogenic to mammalian hosts represents a major threat for human health. In birds the viral replication is carried out in the intestine at 40°C, while in humans it occurs in the cooler respiratory tract at 33°C. It has been shown that temperature adaption of the influenza virus occurs through numerous mutations in the viral polymerase, in particular in the C-terminal domain 627-NLS of the PB2 protein. This domain has already been shown to participate in host adaptation and is involved in importin alpha binding and therefore is required for entry of the viral polymerase into the nucleus [Tarendeau et al., 2008]. Crystallographic structures are available for 627-NLS and the complex importin alpha/NLS, however, a steric clash between importin alpha and the 627 domain becomes apparent when superimposing the NLS domain of the two structures, indicating that another conformation of 627-NLS is required for binding to importin alpha [Boivin and Hart, 2011]. Here we investigate the molecular basis of inter-species adaptation by studying the structure and dynamics of human and avian 627-NLS. We have identified two conformations of 627-NLS in slow exchange (10-100 s-1), corresponding to an apparently open and closed conformation of the two domains. We show that the equilibrium between closed and open conformations is strongly temperature dependent. We propose that the open conformation of 627-NLS is the only conformation compatible with binding to importin alpha and that the equilibrium between closed and open conformations may play a role as a molecular thermostat, controlling the efficiency of viral replication in the different species. The kinetics and domain dynamics of this important conformational behaviour and of the interaction between 627-NLS and importin alpha have been characterized using nuclear magnetic resonance chemical shifts, paramagnetic relaxation enhancement, spin relaxation and chemical exchange saturation transfer, in combination with X-ray and neutron small angle scattering and Förster resonance energy transfer. Also, we have determined the affinities of various evolutionnary mutants of 627-NLS to importin alpha and of avian and human 627-NLS to different isoforms of importin alpha, showing that the observed affinities are coherent with the preferred interactions seen in vivo
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Höfer, Chris Tina. "Influenza virus assembly." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2015. http://dx.doi.org/10.18452/17251.
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Influenza A Viren besitzen ein segmentiertes, einzelsträngiges RNA-Genom, welches in Form viraler Ribonukleoprotein (vRNP)-Komplexe verpackt ist. Während das virale Genom im Zellkern repliziert wird, finden Assemblierung und Knospung reifer Viruspartikel an der apikalen Plasmamembran statt. Für die Virusbildung müssen die einzelnen viralen Komponenten hierher gebracht werden. Während intrinsische apikale Signale der viralen Transmembranproteine bekannt sind, sind der zielgerichtete Transport und der Einbau des viralen Genoms in neuentstehende Virionen noch wenig verstanden. In dieser Arbeit wurden potentielle Mechanismen des vRNP-Transportes untersucht, wie die Fähigkeit der vRNPs mit Lipidmembranen zu assoziieren und die intrinsische subzellulären Lokalisation des viralen Nukleoproteins (NP), eines Hauptbestandteils der vRNPs. Es konnte gezeigt werden, dass vRNPs nicht mit Lipidmembranen assoziieren, was mittels Flotation aufgereinigter vRNPs mit Liposomen unterschiedlicher Zusammensetzung untersucht wurde. Die Ergebnisse deuten jedoch darauf hin, dass das virale M1 in der Lage ist, Bindung von vRNPs an negativ-geladene Lipidmembranen zu vermitteln. Subzelluläre Lokalisation von NP wurde des Weiteren durch Expression fluoreszierender NP-Fusionsproteine und Fluoreszenzphotoaktivierung untersucht. Es konnte gezeigt werden, dass NP allein nicht mit zytoplasmatischen Strukturen assoziiert, stattdessen aber umfangreiche Interaktionen im Zellkern eingeht und mit hoher Affinität mit bestimmten Kerndomänen assoziiert, und zwar den Nukleoli sowie kleinen Kerndomänen, welche häufig in der Nähe von Cajal-Körperchen und PML-Körperchen zu finden waren. Schließlich wurde ein experimenteller Ansatz etabliert, welcher erlaubt, den Transport vRNP-ähnlicher Komplexe mittels Fluoreszenzdetektion aufzuzeichnen und Einzelpartikelverfolgungsanalysen durchzuführen. Unterschiedliche Phasen des vRNP-Transportes konnten beobachtet werden und ein 3-Phasen-Transportmodell wird skizziert.Influenza A viruses have a segmented single-stranded RNA genome, which is packed in form of viral ribonucleoprotein (vRNP) complexes. While the viral genome is replicated and transcribed in the host cell nucleus, assembly and budding of mature virus particles take place at the apical plasma membrane. Efficient virus formation requires delivery of all viral components to this site. While intrinsic apical targeting signals of the viral transmembrane proteins have been identified, it still remains poorly understood how the viral genome is transported and targeted into progeny virus particles. In this study, potential targeting mechanisms were investigated like the ability of vRNPs to associate with lipid membranes and the intrinsic ability of the viral nucleoprotein (NP) – which is the major protein component of vRNPs – for subcellular targeting. It could be shown that vRNPs are not able to associate with model membranes in vitro, which was demonstrated by flotation of purified vRNPs with liposomes of different lipid compositions. Results indicated, however, that the matrix protein M1 can mediate binding of vRNPs to negatively charged lipid bilayers. Intrinsic subcellular targeting of NP was further investigated by expression of fluorescent NP fusion protein and fluorescence photoactivation, revealing that NP by itself does not target cytoplasmic structures. It was found to interact extensively with the nuclear compartment instead and to target specific nuclear domains with high affinity, in particular nucleoli and small interchromatin domains that frequently localized in close proximity to Cajal bodies and PML bodies. An experimental approach was finally established that allowed monitoring the transport of vRNP-like complexes in living infected cells by fluorescence detection. It was possible to perform single particle tracking and to describe different stages of vRNP transport between the nucleus and the plasma membrane. A model of three-stage transport is suggested.
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Green, P. C. "Serological and immunocytochemical studies on influenza virus and influenza virus infected cells." Thesis, University of Manchester, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356114.
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Morgan, David John. "Defective interfering influenza virus reverses the immunopathological effects of standard influenza virus in mice." Thesis, University of Bristol, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.332491.
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Seekings, Amanda Hanna. "Emergence of H7 highly pathogenic avian influenza virus from low pathogenicity avian influenza virus." Thesis, Imperial College London, 2017. http://hdl.handle.net/10044/1/52910.
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Outbreaks of highly pathogenic avian influenza virus (HPAIV) may result in the infection of millions of poultry, causing devastating disease and up to 100% mortality. Avian influenza outbreaks and laboratory experiments have shown that HPAIV can emerge from low pathogenicity avian influenza virus (LPAIV) precursors. The multi-basic cleavage site (MBCS) in the haemagglutinin protein is described as the main pathogenic determinant of HPAIV infection in poultry. Identifying a precursor LPAIV is important for understanding the molecular changes involved in the emergence of HPAIV. In 2008, H7N7 HPAIV was confirmed in Oxfordshire, UK. The presence of a LPAIV precursor with a rare di-basic cleavage site (DBCS) was identified. The DBCS contains an additional basic amino acid compared to common circulating LPAIVs that harbour a single basic amino acid at the cleavage site (SBCS). Using reverse genetics, isogenic viruses based on A/chicken/England/11406/2008 H7N7 HPAIV, from the outbreak, were rescued with the MBCS replaced with either a DBCS (H7N7DB) as seen in the putative LPAIV precursor or a SBCS representative of common H7 LPAIVs (H7N7SB). Intravenous pathogenicity index testing of the recombinant viruses confirmed that only the MBCS conferred the highly pathogenic phenotype. Following passage in ovo, H7N7DB showed evidence of spontaneous evolution to a HPAIV genotype and phenotype as demonstrated by the acquisition of a MBCS, and by influenza virus-specific immunohistochemistry staining in embryo vascular endothelial cells. In contrast, deep sequencing of tissues from embryos in which H7N7SB was serially passaged up to three times showed retention of the LPAIV genotype. Thus, in chicken embryos, an H7N7 virus containing a DBCS displays an unstable nature allowing for rapid evolution to HPAIV. In ovo passage presents a novel approach to assess the likelihood of a LPAIV to evolve into HPAIV, and allows a laboratory-based dissection of molecular mechanisms behind the emergence of HPAIV.
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Mittelholzer, Camilla Maria. "Influenza virus - protection and adaptation /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-656-5/.
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Jia, Nan. "Glycobiology studies of influenza virus." Thesis, Imperial College London, 2015. http://hdl.handle.net/10044/1/50787.
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Glycans represent a class of macromolecules that exhibit vital biological roles in living organisms. They are not only essential for maintaining the normal functionalities of a cell, but are also involved in many pathogenic processes. The influenza A virus binds to glycan receptors that are expressed on the surface of respiratory epithelial cells of human airway and thereby initiates infection. Deciphering the structural features of glycans and comprehending their functional implications are thus crucial to expand our understandings of the disease. To validate the alternative models that are used in the studies of influenza, we generated the glycomic profiles from in vivo and in vitro experimental systems by mass spectrometry. A combination of MALDI-TOF MS, MALDI-TOF-TOF MS/MS, GC-EI-MS and enzymatic digestion experiments were utilised to characterise the structure of glycans. The ferret has been used as an experimental animal to investigate the transmission and replication of influenza viruses. To verify the validity of this model, we carried out glycomic characterisation of ferret respiratory tissues to complement the data that was generated from human airway tissues. The mass spectrometric analysis indicates that the respiratory glycosylation of ferret highly resembles that of human, although distinctive expression of glycans displaying the Sda epitope are detected exclusively in ferret. Nonetheless, in comparison to other lab animals such as mouse and swine, ferret remains a better alternative model for studying the pathogenicity of influenza viruses. In the second project, we generated the glycomic profiles from human and ferret respiratory epithelial cells that were cultured under experimental conditions. Glycosylation patterns between these two in vitro systems are largely comparable, except the presence of the Sda epitope in ferret cells. However, when compared to their corresponding in vivo tissues, diminished structural repertoires especially the high-mass structures were observed.
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Wallensten, Anders. "Influenza A virus in wild birds." Doctoral thesis, Linköping : Linköping University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-7643.
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Poon, Leo L. M. "The polyadenylation of influenza virus mRNA." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312548.
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Bishop, K. J. "Study of influenza A virus ribonucleoproteins." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596669.
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Two expression systems were utilised in an attempt to produce suitable quantities of the influenza polymerase proteins for protein purification. Using the methylotrophic yeast Pichia pastoris, both PB1 and PB2 were expressed at around 0.1-0.4 mg/l. However, it did not prove possible to purify the polypeptides which appeared to be aggregated. The P proteins and NP were also expressed in mammalian cells using recombinant Semliki Forest viruses. However, soluble protein did not accumulate at levels sufficient for large scale purification studies. A transient "reverse genetics" assay whereby the expression of a synthetic model viral RNA segment is driven by influenza P proteins expressed by recombinant vaccinia viruses and NP from a T7 driven plasmid, was used for the analysis of mutant influenza proteins in the processes of viral transcription and replication. Recombinant vaccinia viruses expressing mutant forms of PB1 were generated and their ability to support viral replication determined. One mutant which was shown not to bind PB2 retained reduced but signifciant replication activity. In contrast, mutants that were unable to bind PA were defective in viral RNA synthesis. Of the mutants that were defective in viral transcription, one acted as a trans-dominant inhibitor of wild type protein function. Mutants that were unable to bind RNA were unable to support viral transcription, whilst those with reduced RNA binding capacity were similarly impaired in transcription. The mutants that were unable to bind RNA were also shown to be transdominant inhibitors of replication, indicating that they were able to compete with wild type NP for essential components of the transcription machinery. Mutations in the N-terminal region of NP previously described as the karyopherin α binding site were also analysed for their ability to support viral transcription. Neither mutation of a residue described as essential for karyopherin α binding nor deletion of the karyopherin α binding site affected viral RNA synthesis, though these mutants localised in the cell differently to wild type NP.
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Duhaut, Susan. "The assembly of influenza virus ribonucleoproteins." Thesis, University of Reading, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306344.
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Read, Eliot Keith Curtis. "Investigating influenza A virus RNA trafficking." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609127.
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Tan, E.-Pien. "Screening for influenza virus resistance genes." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608229.
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Kudryavtseva, Katerine. "Genome packaging in influenza A virus." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648592.
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Mahallawi, Waleed. "Natural immunity to influenza virus in humans following 2009 pandemic H1N1 influenza." Thesis, University of Liverpool, 2013. http://livrepository.liverpool.ac.uk/13137/.
Full textAbstract:
Influenza is a highly contagious and acute respiratory infection caused by influenza virus in the mucosa of the respiratory tract. Both seasonal and pandemic influenza continue to cause substantial morbidity and mortality in humans. The 2009 pandemic H1N1 (pH1N1) influenza and the potential of a highly pathogenic avian H5N1 (aH5N1) pandemic highlighted the need for effective preventative strategies. Understanding the development of natural immunity following the pH1N1 pandemic may provide important information on host protective immunity in humans, which could inform future more effective vaccination strategies against influenza. In this thesis, naturally developed mucosal immunity to 2009 pH1N1 virus was studied in children and adults using cells derived from human nasal-associated lymphoid tissue (NALT). Firstly, the frequency of HA-specific memory B cells in human NALT to pH1N1 virus and their ability to produce cross-reactive antibodies were studied. Patients who had serological evidence of previous exposure to pH1N1 virus developed large numbers of IgG memory B cells in NALT that produce crossreactive neutralizing antibodies against a number of influenza subtypes upon pH1N1 virus antigen stimulation. The presence of such memory B cells in human NALT appears to have primed the host for cross-reactive mucosal memory response against other H1N1 and the highly pathogenic aH5N1 virus strains. These findings may have important implications in future vaccination strategies against influenza. Secondly, serum specific anti-pH1N1 HA IgG antibodies were analysed using ELISA. HA-specific antibody levels to pH1N1 in adults were significantly higher than that of children. The results may suggest that adults had been exposed to more cross-reactive influenza viruses than children, and developed more cross-reactive memory responses against some influenza viruses than in children. Significantly higher HA-specific IgG antibody titres to pH1N1 HA (measured using ELISA) were found in subjects who had HAI titres≥40 than in those with HAI antibody titre<40. This suggests that following the 2009 pH1N1 pandemics, large numbers of people developed anti-pH1N1 HA antibodies to both the circular head and the stalk regions of HA which may have broader protective immunity. Thirdly, HA-specific memory CD4+ T cell response to pH1N1 virus was shown in tonsillar cells from children and adults. This suggests that following the 2009 pandemic H1N1 influenza, humans developed memory T cell response to the pH1N1 HA protein antigen at the mucosal level in the nasopharynx. There appeared to be an age-associated increase in this memory response. Finally, mucosal antibody responses in NALT to HAs of a number of influenza A viruses were investigated following in vitro stimulation of adenotonsillar cells with LAIV vaccine which contains a 2009 pandemic H1N1 virus, a seasonal H3N2 and a B influenza strain. Significant antibody responses of all 3 isotypes (IgG, IgA and IgM) to the HA of pandemic H1N1 virus were observed in tonsillar cells following LAIV stimulation. It suggests that the in vitro model of human NALT using adenotonsillar cell culture could be used to study the LAIV-induced immune responses which may predict the immunogenicity and efficacy of candidate LAIV vaccines in humans.
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Lee, Hung-chiu. "Synthetic RNA interference against influenza A virus." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B35537814.
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Pavan, Carmen M. "Influenza B virus : segment 7 gene expression." Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=55673.
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Smith, D. B. "The production of influenza virus spliced mRNAs." Thesis, University of Cambridge, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355039.
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Jung, Tanis E. "Mutational analysis of the influenza virus polymerase." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426398.
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Lee, Hung-chiu, and 李洪釗. "Synthetic RNA interference against influenza A virus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B35537814.
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Lau, Lee-hang Lincoln, and 劉力恆. "Influenza virus shedding and transmission in households." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hdl.handle.net/10722/196093.
Full textAbstract:
Background The dynamics of influenza virus transmission are not yet fully understood, hindering the appropriate development and implementation of control and mitigation strategies. One major uncertainty relates to the profile of infectiousness over time in humans infected by influenza virus, and how variation in infectiousness might contribute to the risk of transmission in households.
Methods In 2008 and 2009, two large community-based studies were conducted to study the household transmission of influenza viruses in Hong Kong. I analyzed data on viral shedding and disease in participants, and used statistical models to examine how viral shedding patterns and other factors might affect the risk of influenza virus transmission in households, both within individuals over time, and between individuals with different patterns in viral shedding.
Results The patterns of viral shedding relative to the time of illness (acute respiratory illness; ARI) onset in naturally acquired infections were found to be largely comparable to the patterns observed in experimental infections. Viral shedding detected by RT-PCR peaks around the day of ARI onset after which levels of shedding declined over around 7 days, and viral shedding tended to be greater in children than adults. The patterns of viral shedding in cases of seasonal A subtypes were similar, although the trends of shedding in cases of seasonal B differed with some indication of a plateau in shedding for up to 5 days after illness onset. The risk of household influenza transmission was significantly associated with log10 viral shedding, though not with influenza related signs and symptoms such as cough.
Conclusions The patterns of viral shedding observed in naturally-acquired influenza A virus infections correlated with the pattern of infectiousness over time after onset of illness. The majority of infectiousness was estimated to occur within 2-3 days of illness onset, with implications for isolation strategies. The heterogeneous nature of individual viral shedding suggests the possibility of substantial variation in infectiousness, particularly among children.published_or_final_version
Community Medicine
Doctoral
Doctor of Philosophy
28
Harvey, Ruth. "Studies of influenza A virus H5 haemagglutinin." Thesis, University of Reading, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270832.
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Tibbles, K. W. "Studies on the influenza virus RNA polymerase." Thesis, University College London (University of London), 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380686.
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Blok, Vivian Carol. "Studies of the influenza virus RNA polymerase." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292959.
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Doty, Daniel S. M. Massachusetts Institute of Technology. "CD8⁺ T Cell Response to Influenza Virus." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/86280.
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Thesis: S.M., Massachusetts Institute of Technology, Department of Biology, 2005.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 10-12).
The flu is an extremely prevalent and potentially devastating disease, especially dangerous to the very young, the elderly, and to people with compromised immune systems. Influenza has a characteristic course of infection, and is often effectively dispatched by the immune system. The cell-mediated lysis of infected cells is a particularly important step in clearing the infection. Antigen specific CD8+ T lymphocytes are selected and activated in the mediastinal lymph node, proliferate and gain effector function, then migrate to the lungs, where they selectively destroy infected cells. The CD8+ effector population pool undergoes a phase of contraction, when most effector cells die. Those that survive become memory T cells, protecting the body from subsequent influenza infections. The molecular and cellular interactions that comprise the CD8+ cytotoxic T cell response to influenza virus are of particular interest because of their implications for the prevention, treatment, and alleviation of the flu.
by Daniel Doty.
S.M.
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Pappworth, Isabel Yseult. "Studies in influenza A virus induced apoptosis." Thesis, University of Birmingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434078.
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Influenza viruses induce apoptosis in tissue culture cells in vitro. In a study to examine the role of individual genes in apoptosis a twelve plasmid based reverse genetic system was used to produce a series of viruses, these being: rA/Victoria and rA/WSN, recombinant forms of the parent viruses A/Victoria/75 (H3N2) and A/WSN/33 (H1N1), rA/Vic/WSNNP and rA/Vic/WSNM, recombinant A/Victoria based viruses with the A/WSN nucleoprotein (NP) and matrix (M) gene respectively. These viruses were examined for their ability to induce apoptosis (determined morphologically), cell lysis (determined by lactate dehydrogenase release) and infection (determined by antibody labelling of nucleoprotein [NP]). Virus replication assays were also performed. The rA/WSN virus behaved differently to rA/Victoria and rA/Vic/WSNNP. A difference in the proportions of morphological apoptosis and cell lysis induced at 24 hours p.i. was observed. When these two measurements were taken over time it was seen that the rA/Victoria and rA/Vic/WSNNP infected cells progressed through apoptosis at a faster rate with more cells lysed at 24 hours post infection than cells infected with the rA/WSN virus. Other viruses that were thought to be reassortants of A/Victoria/75 and A/WSN/33 were found not to be, and were instead clone 7a (a reassortant of A/England/939/69 and A/Puerto Rico/8/34 parents) used in the laboratory. Clone 7a infected cells behaved similarly to rA/WSN infected cells in that progression through apoptosis was slower than for rANictoria and rANic/WSNNP infected cells. Clone 7a replicated to a higher titre and over a more prolonged period suggesting that the purpose of the delay may be to enhance replication. However, inhibition of apoptosis by the pan-caspase inhibitor (z-VAD-fmk) did not affect replication of clone 7a, rA/Victoria or rA/Vic/WSNNP.
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Lu, Lu. "Transmission dynamics of Avian Influenza A virus." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/10481.
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Influenza A virus (AIV) has an extremely high rate of mutation. Frequent exchanges of gene segments between different AIV (reassortment) have been responsible for major pandemics in recent human history. The presence of a wild bird reservoir maintains the threat of incursion of AIV into domestic birds, humans and other animals. In this thesis, I addressed unanswered questions of how diverse AIV subtypes (classified according to antigenicity of the two surface proteins, haemagglutinin and neuraminidase) evolve and interact among different bird populations in different parts of the world, using Bayesian phylogenetic methods with large datasets of full genome sequences. Firstly, I explored the reassortment patterns of AIV internal segments among different subtypes by quantifying evolutionary parameters including reassortment rate, evolutionary rate and selective constraint in time-resolved Bayesian tree phylogenies. A major conclusion was that reassortment rate is negatively associated with selective constraint and that infection of wild rather than domestic birds was associated with a higher reassortment rate. Secondly, I described the spatial transmission pattern of AIV in China. Clustering of related viruses in particular geographic areas and economic zones was identified from the viral phylogeographic diffusion networks. The results indicated that Central China and the Pearl River Delta are two main sources of viral out flow; while the East Coast, especially the Yangtze River delta, is the major recipient area. Simultaneously, by applying a general linear model, the predictors that have the strongest impact on viral spatial diffusion were identified, including economic (agricultural) activity, climate, and ecology. Thirdly, I determined the genetic and phylogeographic origin of a recent H7N3 highly pathogenic avian influenza outbreak in Mexico. Location, subtype, avian host species and pathogenicity were modelled as discrete traits and jointly analysed using all eight viral gene segments. The results indicated that the outbreak AIV is a novel reassortant carried by wild waterfowl from different migration flyways in North America during the time period studied. Importantly, I concluded that Mexico, and Central America in general, might be a potential hotspot for AIV reassortment events, a possibility which to date has not attracted widespread attention. Overall, the work carried out in this thesis described the evolutionary dynamics of AIV from which important conclusions regarding its epidemiological impact in both Eurasia and North America can be drawn.
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Sieben, Christian. "Host cell invasion by influenza A virus." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16743.
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Influenzaviren müssen in die Wirtszelle aufgenommen werden, um dort ihr Genom freizusetzen und ihre Replikation mit Hilfe des Reproduktionsapparats der Zelle einzuleiten. Der komplexe Replikationszyklus der Influenza A Viren ist noch nicht vollständig verstanden. Er beginnt mit der Bindung des viralen Hämagglutinins (HA) an Sialinsäure (SA) auf der Zelloberfläche der Wirtszelle. In dieser Arbeit wurde die Virusbindung an Zellen mit unterschiedlicher Rezeptorkomposition verglichen. Dabei konnte gezeigt werden, dass für die zelluläre Spezifität die Präsentation des Rezeptors innerhalb der Plasmamembran der Zelle eine größere Rolle spielt als die Struktur des Rezeptorglykans selbst. Des Weiteren deuten die Beobachtung sehr kleiner Kräfte und ein stufenweises Öffnen von Bindungen auf eine multivalente Interaktion hin. Multivalenz wird oft in biologischen Bindungsprozessen beobachtet und kann Bindungskräfte enorm verstärken. Basierend auf diesen Ergebnissen wurden inhibitorische Nanopartikel entwickelt, die die natürliche Zelloberfläche als hochaffine Bindungsalternative imitieren. Verschiedenartige Nanopartikel wurden evaluiert und konnten die Virusaktivität um mehr als 80 % hemmen. Nach der Bindung wird das Virus durch Endozytose in die Zelle aufgenommen. Durch spezifische Virusmarkierung und gleichzeitiger Expression von zellulären Markerproteinen wurde der Transport einzelner Viren in lebenden Zellen verfolgt. Dabei konnte gezeigt werden, dass das Virus sowohl durch frühe, als auch durch späte Endosomen wandern muss, um sein Genom erfolgreich in das Zytoplasma zu entlassen. Außerdem verzögert das Virus die endosomale Ansäuerung um eine optimale Aufenthaltsdauer im Endosom und die lokalisierte Fusion in der Nähe des Zellkerns zu gewährleisten. Pharmakologisches Eingreifen in diese Prozesse konnte zudem weitere kritische Faktoren identifizieren, die die Effizienz der Virusinfektion stark beeinflussen.Influenza virus must enter a host cell to deliver its genome, use the cells reproductive machinery and eventually initiate its replication. The replication cycle of influenza A virus is very complex and still not fully understood. It generally starts with binding of the viral protein hemagglutinin (HA) to its cellular receptor sialic acid (SA). In this work, virus-cell attachment forces were investigated at the single molecule level using intact virus binding to living cells, a set-up that closely mimics the in vivo situation. Cells of different surface SA composition were compared. It could be shown that the unique presentation of the ligand within the cells plasma membrane, rather than the structure of the receptor-glycan itself, strongly affects cellular specificity. The low binding forces as well as the observation of stepwise unbinding events suggest a multivalent interaction type. Based on this finding, inhibitory nanoparticles mimicking the cell surface were constructed. Different particles were evaluated and shown to efficiently inhibit virus infection by ≥ 80 %. Since many molecular details of multivalent interactions remain poorly understood parameters such as ligand spacing and presentation were varied and revealed that the density of ligands as well as the interacting surface plays critical roles for virus inhibition. Upon attachment, the virus enters the cell by endocytosis. Virus trafficking was followed at the single-virus level in living cells. The kinetics of virus transport were visualized using fluorescent marker proteins in combination with specific virus labeling. It was found that the virus needs to progress through early and late endosomal compartments in order to efficiently uncoat and release its genome. Further, the virus delays the endosomal acidification to ensure optimal residence time and fusion in the region close to the host cell nucleus. Drug treatment furthermore unraveled critical factors influencing viral infection efficiency.
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Killian, Mary Lea. "Identification and characterization of H2N3 avian influenza virus from backyard poultry and comparison to novel H2N3 swine influenza virus." [Ames, Iowa : Iowa State University], 2009.
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36
Jagger, Brett William. "The influenza A polymerase in viral pathogenesis." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610897.
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Cheng, Ka-yeung, and 鄭家揚. "Diagnosis and surveillance of human influenza virus infection." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48079819.
Full textAbstract:
Background: Early and accurate diagnosis of influenza helps start correct treatment and
prevention strategies at individual level. Ongoing systematic collection, analysis and
dissemination of the surveillance data from aggregated diagnostic results and other early
indicators help gather the foremost disease information for all subsequent control and
mitigation strategies in the community. Disease information from surveillance results then
feed back to medical practitioners for improving diagnosis. By improving this loop of
disease information transfer in terms of accuracy and timeliness, interventions for disease
control can be applied efficiently and effectively.
Methods: Several new influenza diagnosis and surveillance methods were explored and
evaluated by comparing with laboratory reference test results. Logistic regression models
were applied to synthesize a refined clinical guideline for human influenza infections. The performance of QuickVue rapid diagnostic test was evaluated in a community setting.
Weekly positive rates from the above two diagnostic methods, together with three other
different syndromic surveillance systems, including data from school absenteeism, active
telephone survey and internet based survey were evaluated according to the US CDC
public health surveillance systems guideline in terms of their utility, correlations and
aberration detection performance. Different combinations of surveillance data streams and
aberration detection algorithms were evaluated to delineate the optimal use of multi-stream
influenza surveillance data. A framework of efficient surveillance data dissemination was
synthesized by incorporating the merits of the online national surveillance websites and the
principles of efficient data presentation and dashboard design.
Results: A refined clinical diagnostic rule for influenza infection using fever, cough runny
nose and clinic visit during high influenza activity months as predictors was scored the
highest amount all other current clinical definitions. Time series weekly positive rate from
this rule showed better correlation with reference community influenza activity than many
other current clinical influenza definitions. The QuickVue rapid diagnostic test has an
overall diagnostic sensitivity of 68% and specificity 96%, with an analytic sensitivity
threshold of 105 to106 viral copies per ml. Weekly aggregated QuickVue and school
absenteeism surveillance data was found to be highly correlated with hospital laboratory
and community sentinel surveillance data, but the telephone and internet survey was only
moderately correlated. Multiple univariate methods performed slightly better than
multivariate methods for aberration detections in general. More sophisticated outbreak
detection algorithms did not result in significant improvement of outbreak detectionpublished_or_final_version
Community Medicine
Doctoral
Doctor of Philosophy
38
Renfrey, Sian. "Solubilization and reconstitution of influenza haemagglutinin." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260184.
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Lam, Tsan-yuk Tommy. "Molecular evolution and epidemiology of influenza A virus." Click to view the E-thesis via HKUTO View the Table of Contents & Abstract, 2010. http://sunzi.lib.hku.hk/hkuto/record/B44137084.
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Sun, Jian. "Computer-aided drug design for influenza A virus." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B44205156.
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Kitikoon, Pravina. "Strategy to improve swine influenza virus (SIV) vaccination." [Ames, Iowa : Iowa State University], 2007.
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42
Hayhurst, Andrew. "Studies on the influenza A virus M2 protein." Thesis, Imperial College London, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250090.
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Vater, Sandra. "Studies on influenza A virus PB1-F2 protein." Thesis, University of St Andrews, 2011. http://hdl.handle.net/10023/2075.
Full textAbstract:
The influenza A virus genome codes for up to 12 proteins. Segment 2 encodes three proteins, the polymerase subunit PB1, a small protein PB1-F2 and an N-terminally truncated version of PB1 called N40. Different functions have been reported for PB1-F2 such as induction of apoptosis, regulation of the viral polymerase activity, enhancement of secondary bacterial infections and modulation of the innate immune system. So far, no function has been ascribed to N40. To study PB1-F2 in more detail, its coding sequence was deleted from its original position and inserted downstream of the PB1 (segment 2), NA (segment 6) or M (segment 7) open reading frames (ORF) employing different strategies, including the use of an overlapping Stop-Start cassette, a duplicated promoter sequence and the self-cleaving 2A peptide derived from foot-and-mouth disease virus. Viruses with bicistronic segments were rescued and tested for their ability to express PB1-F2. Whereas no expression of PB1-F2 was detected from bicistronic segments 2 and 7, expression of PB1-F2 from segment 6 was observed in high levels. However, the phenotype of all these viruses was similar to that of viruses lacking PB1-F2 which made mutational analysis of PB1-F2 not worthwhile. Previously, the function of PB1-F2 was mainly studied using a virus deficient in PB1-F2 production but showing increased N40 expression. In the present study, recombinant WSN viruses lacking either PB1-F2 or N40, or both proteins were engineered and the effects of these mutations on the viral life cycle were examined. Viruses deficient for PB1-F2 that overexpressed N40 showed the most attenuated phenotype, whereas the loss of PB1-F2 alone did not obviously affect virus replication. Reduced viral polymerase activity was observed for viruses lacking N40, however attenuation in vivo was only seen in combination with the loss of PB1-F2. Neither the loss of PB1-F2 nor N40 alone had a great impact, but changes in the expression level of both proteins were disadvantageous for the virus. Increased levels of N40 shifted the polymerase activity towards replication, suggesting a new function for N40. Thus, it was shown that the segment 2 gene products and their expression level influence viral replication and pathogenicity, and a careful design of mutant recombinant viruses is vital for determining the experimental outcome.
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Wasson, Peter Stewart. "Development of novel virus vectors for influenza vaccination." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/6492.
Full textAbstract:
The influenza virus, a member of the Orthomyxoviridae family, causes regular, large-scale morbidity and mortality in birds and humans and significant human suffering and economic loss. The primary aim of this study was to develop a novel influenza vaccine. Vaccines are an essential tool for the control of influenza because they increase resistance to infection, prevent illness and death and help to limit virus transmission to other birds and mammals, including humans. By reducing the environmental contamination of influenza virus in global poultry stocks, the risk of a new pandemic virus being generated by the human-avian link is diminished. Marek’s Disease is a common lymphoproliferative disease of poultry that is readily controlled worldwide using the live attenuated vaccine, CVI988. The Marek’s Disease Virus (MDV) CVI988 viral genome, available as a Bacterial Artificial Chromosome (BAC), forms viable infectious viral particles when transfected into Chicken Embryo Fibroblast (CEF) cells. Using BAC mutagenesis, two non-essential genes in the MDV CVI988 BAC (UL41 and US10), were identified and replaced by the low pathogenic influenza haemagglutinin 10 (H10) gene. These live recombinant MDV-H10 vectors will allow simultaneous vaccination against both pathogens. In addition, the non-essential genes were also replaced with GFP creating MDV-GFP constructs. Both genes were expressed initially using a CMV promoter, although this disrupted the MDV CVI988 BAC; a second promoter, PGK-1, proved more successful. A third MDV gene (UL50) was deleted, but severe attenuation prevented the incorporation of H10 into this open reading frame. Future work to test the MDV-HA constructs in vivo will be carried out in collaboration with the Istituto Zooprofilattico Sperimentale delle Venezie in Italy. In addition, development of MDV constructs containing multiple HA genes (H10 and H5) linked by the 2A polyprotein can be developed with the goal of establishing heterosubtypic immunity.
45
Campbell, Gillian Mhairi. "Influenza virus infection in a compromised immune system." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/6521.
Full textAbstract:
Severe influenza virus infection, including human infection with highly pathogenic H5N1 viruses is characterised by massive pulmonary inflammation, immunopathology and excessive cytokine production, a process in which macrophages may play a vital role. The aim of this project was to investigate the hypothesis that inhibition of inflammatory responses from infected macrophages, using either alternatively activated bone marrow derived macrophages (BMDMf), or IFNg receptor deficient (IFNgR-/-) mice may ameliorate the devastating immunopathology and inflammation routinely observed in highly pathogenic influenza virus infections. Infection of alternatively activated BMDMf resulted in enhanced positivity for viral proteins, compared with classically activated, inflammatory BMDMf. However, neither subset propagated the infection indicating that while infection is abortive in both classical and alternatively activated BMDMf, the latter may prove more efficient at removing infectious virus from the site of infection due to enhanced infectivity. However, influenza virus was capable of driving expression of proinflammatory mediators such as iNOS and TNFa from classical and alternatively activated BMDMf even in the absence of IFNg signalling. IFNgR-/- BMDMf demonstrated a reduced inflammatory response to infection compared to Sv129 counterparts, suggesting a potentially impaired inflammatory response in vivo. This was investigated by infection of IFNgR-/- mice, which resulted in ameliorated disease, lower viral titres and mild immunopathology, demonstrating that inhibition of IFNg signalling limits the severity of disease. Additionally, mRNA expression for key inflammatory mediators was reduced, demonstrating that inhibition of the overwhelming inflammatory response to influenza virus infection is beneficial to the host, resulting in protection from immunopathology and improved prognosis, without impairing viral clearance.
46
Sun, Jian, and 孙健. "Computer-aided drug design for influenza A virus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B44205156.
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Lam, Tsan-yuk Tommy, and 林讚育. "Molecular evolution and epidemiology of influenza A virus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44137084.
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Leung, Yin-hung Connie, and 梁彥虹. "Ecology, epidemiology and immunology of avian influenza virus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46329626.
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Ng, Chi-ko, and 伍智高. "Antigenicity and oseltamivir resistance of influenza A virus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hub.hku.hk/bib/B50434536.
Full textAbstract:
Although several risk factors for severe influenza infection have been identified in previous studies, many patients having multiple risk factors only developed mild symptoms while many healthy young patients developed severe complications when infected with A(H1N1)pdm09. Thus, there are still undiscovered factors that affect the progression and severity of influenza.
The early innate immune response may be critical in determining the disease progression. Non-neutralizing antibodies existed in the early stage of infection may contribute to the outcome of the disease. In this study, the association of disease severity with the titre and avidity of non-neutralizing antibodies in early stage of influenza infection was investigated. It has been shown that the titre of non-neutralizing antibody was higher in more severe patients in the early stage of infection. Higher antibody avidity was also found to be associated with more severe disease independently. These findings tend to support the view that antigenic drift leads to an excessive production of pro-inflammatory non-neutralizing antibodies in the patients and associated with severe outcome.
Since patients with more severe disease tend to have a delayed clearance of the virus and allow more transmission, the antigenically shifted or drifted influenza virus may gradually become predominant in human population. This idea suggested that the predominance of influenza virus with NA-H275Y mutation in 2007-2008 was contributed by the co-existing, fitness restoring secondary adaptive mutation in HA. NA-H275Y was identified in previous studies to be the mutation encoding for the influenza virus to resist against oseltamivir but would also change the property of NA as a result of compromised viral fitness. Therefore, influenza virus carrying NA-H275Y is unlikely to emerge and spread in human population. However, NA-H275Y mutated strains of influenza virus emerged and spread globally in the influenza season of 2007-2008 and quickly become the predominant strain in 2008. Previous study found NA-R222Q and NA-V234M were the mutations responsible for restoring the viral fitness in oseltamivir resistant clinical isolates. Still, this cannot fully explain the predominance of the resistant strains over the susceptible strains. Therefore secondary adaptive mutation in HA was believed to be present and cause antigenic change to the resistant strains of influenza. In this study, mutual information analysis and HA structural analysis were conducted to screen out HA-A189T and HA-Y94H to be the candidates co-exist with NA-H275Y and possibly critical for antigenic changes. This study further suggested that HA-Y94H mutation leads to a change of antigenic property of the virus by examining the antigenicity and growth kinetics of the recombinant viruses carrying the selected HA mutations. HA-94 may be critical for determining both the receptor binding property and antigenic property of the virus. Review on the evolution of seasonal influenza viruses from 2005 to 2008 suggested that the emergence of HA-Y94H mutation may enhance the presence of NA-H275Y and helps the viruses carrying NA-H275Y to spread and dominate over the oseltamivir susceptible strains during 2007 and 2008.published_or_final_version
Microbiology
Master
Master of Philosophy
50
Otterstrom, Jason John. "Visualizing Influenza Virus Membrane Fusion: Inhibition and Kinetics." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11251.
Full textAbstract:
The influenza virus hemagglutinin (HA) surface protein is a primary antigenic target for neutralization of viral infection. HA also mediates membrane fusion between the virus and a cell, which is the first critical step during infection. Traditional techniques to study infection neutralization by antibodies or the membrane fusion process rely on ensemble measurements, confounding the precise mechanism of infection neutralization and obscuring transient conformational intermediates. This dissertation describes advances made in a fluorescence microscopy-based single-particle fusion assay to overcome the limitations of ensemble measurements in these types of studies. Virus particles are labeled to visualize lipid mixing between a virus and a target membrane formed upon a glass or polymer support. Optionally, the viral lumen can be labeled to visualize the subsequent release of viral contents.