<p>Despite the advanced knowledge of the mechanisms of influenza infection and improved vaccines, Influenza A Virus still causes a life-threatening respiratory disease, especially during pandemics. Past investigations have proposed a synergism between Influenza A virus and a simultaneous or subsequent bacterial superinfection as the predominant cause of death. The recent development of animal models to study these heterologous infections has shed light onto the diverse mechanisms by which Influenza A Virus may increase the susceptibility to contract a secondary bacterial infection. These studies suggested an important role for the innate immune system in mediating such disease. We developed a model of heterologous infection combining Influenza A Virus and <em>Bordetella parapertussis</em> that demonstrated a critical role for MIP-2 to drive pulmonary neutrophilia in the pathology associated with bacterial superinfection of influenza. However, the origin of this increased MIP-2 production and the mechanisms underlying the immunopathology remained to be elucidated. The present studies proposed IL-1β overproduction as the upstream cause of the increased MIP-2 production observed in heterologous infection. This exaggerated IL-1β production was likely related to the increased bacterial burden observed in heterologously infected mice. This study also demonstrated that reduction in IL-1β production by blockade of the inflammasome seemed to provide an improvement in the clinical symptoms and the immunopathology of the disease. Thus, interventions to attenuate the exacerbated bacterial burden and the inflammatory responses derived from the subsequent IL-1β overproduction should be further investigate as possible therapeutic approaches to treat bacterial superinfections.</p> / Master of Science (MSc)

Gold nanoclusters possess both theoretical and practical importance in the development of ultrasensitive biosensors based on surface-enhanced Raman spectroscopy (SERS). Manipulation of gold nanoclusters in a predictable and reproducible manner for the application of refined biochemical analysis still remains challenging. In this study, high-purity gold nanoclusters are isolated via a simple method based on density gradient centrifugation. Three distinct bands including monomers, small aggregates (2-4 nanospheres), and large aggregates (>5 nanospheres) can be separated via density gradient centrifugation. The isolated gold nanoclusters greatly enhance the Raman intensity of the trapped dye molecules such that single nanocluster detection is feasible. To develop a gold nanoparticle-based biosensor for influenza virus, effort was also made to modify recognition moieties such as aptamers to gold nanoparticles via distinct approaches. The increase of hydraulic diameter and the shift of optical absorbance spectrum indicate the success of surface modification to gold nanoparticles. / Master of Science

Physik gibt es überall dort, wo Materie: Maßnahmen wie Energie, Masse, Temperatur, Geschwindigkeit, Größe und Steifigkeit sind alle Beispiele der physikalischen Eigenschaften. Solche Mengen sind wichtige Charakterisierungen für biologische Organismen: Sie verändern die ganze Zeit während des gesamten Lebenszyklus. Für eine Bio-Mechaniker, Steifigkeit ist eine wichtige Maßnahme zur biologischen Design zu verstehen. Weil biologische Bausteine so klein wie 1 nm (Protein / DNA / Lipid) sein können, sind spezielle Techniken erforderlich, um ihre Steifigkeit zu studieren. Beide Rasterkraftmikroskopie (AFM) und optischen Pinzetten können verwendet werden, um aktiv zu verformen die Objekte an pN-nN Kräfte und messen die Verformung auf Nanometer Längenskalen werden. In dieser Arbeit AFM wird angewandt, um die Mechanik von Influenza-Viren, Liposomen und lebenden Zellen zu studieren. Das Genom von Viren von einer Proteinhülle und in einigen Fällen eine zusätzliche Lipidhülle verpackt. Dieser Verbund Shell hat widersprüchliche Rollen: er hat das virale Genom zu schützen, aber es sollte auch ermöglichen Auspacken während der viralen Infektion in das Genom zu lösen. Influenza-Virus ist das weichste Virus jemals gefunden, aber zur gleichen Zeit eine sehr hartnäckige Virus verursacht jährliche Pandemien. Ein besseres Verständnis der mechanischen Eigenschaften des Influenza-Virus kann uns helfen zu verstehen, warum das Virus so erfolgreich ist. Die mechanischen Eigenschaften von Influenza-Viren wurden durch AFM gemessen und mit den Liposomen der viralen Lipid hergestellt. Wir haben gefunden, dass die Influenzavirus-Mechanik durch seine Lipidhülle (~ 70%) werden dominiert. In Kapitel 2 haben wir gezeigt, dass anstelle der Verwendung einer starren Proteinkapsid die Lipidhülle ausreicht, um das Influenza virale Genom zu schützen. In Kapitel 3 haben wir weitere blickte in die Funktion des M1 Proteinhülle während der viralen Infektion. Ein Zwischenprodukt Auspacken Schritt wurde durch Messen der in fluenzavirale Steifigkeit bei pH 7, 6, 5,5 und 5, Bedingungen, die die Ansäuerung Umgebungen auf der viralen Infektion nachahmen Stoffwechselweg entdeckt. Der Zwischenschritt wurde weiterhin als wesentlich erwiesen für eine erfolgreiche Infektion. Wir schlagen vor, dass das Influenza-Virus hat sich zu eng synchronisiert die verschiedenen Schritte ihrer Auspacken mit pH-

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥ 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Ecient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and ecient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

Das Prinzip der Multivalenz ist in der Natur allgegenwärtig, welches auch von Influenza-Viren genutzt wird, um über ihre Oberflächenproteine an epitheliale Wirtszellen zu binden. Diese Interaktion bietet einen interessanten Ansatzpunkt für multivalente Inhibitoren, wenn es gelingt, die Bedingungen für eine effiziente Wechselwirkung mit dem Virus zu entschlüsseln. Hierzu wurde in dieser Arbeit eine Charakterisierung des Hämagglutinin-Trimers (HA) auf viralen Partikeln mittels Kohlenhydrat-Nukleinsäuregerüsten und Kohlenhydrat-Polyethylenglykol (PEG)-Gerüsten vorgenommen. Distanz-Affinitäts-Beziehungen für die Interaktion des trimeren HA mit den bivalenten Präsentationen des Sialyl-LacNAc zeigten, dass bivalente PEG-Konjugate nicht in der Lage sind, eine bivalente Verstärkung der Wechselwirkungen mit der löslichen HA-Ektodomäne oder mit HA auf der viralen Oberfläche herbeizuführen, wobei die räumliche Rasterung mit PNA∙DNA-Gerüsten eine bimodale Distanz-Affinitäts-Beziehung ergab. Ein Affinitätsmaximum in einem Abstand von 52 - 59 Å wurde einer simultanen Bindung an zwei kanonische Bindungsstellen eines HA-Trimers zugeordnet, wobei ein zweites Affinitätsmaximum bei 26 Å auf die Existenz einer sekundären Bindungsstelle hindeutet. In dieser Arbeit wurde erstmals die multivalente Präsentation von Glykoliganden auf langen repetitiven DNA-Templaten demonstriert. Es wurden Nukleinsäure-Komplexe erhalten die eine vollständige Inhibierung der Virus-induzierten Hämagglutination bei einer Konzentration von 10^(-9) M des Templats erzielten, was einer 10^7-fachen Verstärkung bezogen auf den monovalenten Zucker entspricht. Neben einer hochpotenten Inhibition offenbarten distanzoptimierte bivalente und multivalente Binder auf Nukleinsäuregerüsten auch subtypspezifische Inhibition. / The principle of multivalency is omnipresent in nature, which is also used by influenza viruses to bind to epithelial host cells via their surface proteins. This interaction offers an interesting starting point for multivalent inhibitors if the conditions for an efficient interaction with the virus can be deciphered. For this purpose, the hemagglutinin trimer (HA) on viral particles was characterized using carbohydrate-nucleic acid scaffolds and carbohydrate-polyethylene glycol (PEG) scaffolds. Distance-affinity relationships for the interaction of the trimeric HA with the bivalent presentations of the sialyl-LacNAc showed that bivalent PEG conjugates are not capable of a bivalent enhancement of the interactions with the soluble HA ectodomain or with HA on the viral surface, whereby the spatial screening with PNA∙DNA scaffolds resulted in a bimodal distance-affinity relationship. An affinity maximum at a distance of 52 - 59 Å was assigned to simultaneous binding to two canonical binding sites of an HA trimer, with a second affinity maximum at 26 Å indicating the existence of a secondary binding site. In this work the multivalent presentation of carbohydrate ligands on long repetitive DNA templates was demonstrated for the first time. Nucleic acid complexes were obtained which achieved a full inhibition of the virus-induced hemagglutination at a concentration of 10^(-9) M of the template, which corresponds to a 10^7-fold increase in relation to the monovalent sugar. In addition to a highly potent inhibition, distance-optimized bivalent and multivalent binders on nucleic acid structures also revealed subtype-specific inhibition.

No description available.

The Interferon (IFN) family of cytokines are produced in direct response to virus infection and they constitute the first line of defence against virus infection by inducing hundreds of interferon stimulated genes (ISGs) which act in concert to establish the so-called “antiviral state”. Influenza A viruses and parainfluenza virus type 5 (PIV5) are both small negative strand RNA viruses that must circumvent their hosts’ interferon (IFN) response for replication. However, the ways in which these viruses interact with the IFN system are very different. Although PIV5 replication is initially severely impaired in cells in a pre-existing IFN-induced antiviral state, it manages to overcome the antiviral state by targeting an essential component of type I IFN signalling, STAT1, for degradation. Thus the cells cannot maintain the antiviral state indefinitely without continuous signalling. Consequently, the virus resumes its normal replication pattern after 24-48 hours post-infection. In clear contrast, influenza virus fails to establish its replication in the majority of infected cells (90-95%) with a pre-existing IFN-induced antiviral state, although a few cells are still able to produce viral antigens. To further investigate how influenza virus interacts with cells in a pre-existing IFN-induced antiviral state, I have used in situ hybridization to follow the fate of input and progeny genomes in cells that have, or have not, been treated with IFN prior to infection. Here I show for the first time that IFN pre-treatment blocks the nuclear import of influenza A virus genome, which prevents the establishment of virus replication, but this can be overcome by increasing multiplicities of infection. Of those IFN-induced antiviral molecules, human MxA is an essential component of the IFN-induced antiviral state in blocking influenza virus genome import, as this block can be abolished by lentivirus-mediated knockdown of MxA. I also show that in cells constitutively expressing MxA the viral genome still manages to be transported into the nucleus, indicating that MxA might require an unidentified IFN-induced factor to block nuclear import of the influenza virus genome. These results reveal that IFN exerts its action at an early stage of virus infection by inducing MxA to interfere with the transport of viral genome into the nucleus, which is the factory for viral RNA production.

The influenza A virus non-structural (NS1) protein is multifunctional, and during virus-infection NS1 interacts with several factors in order to manipulate host-cell processes. This study reports that NS1 binds directly to p85β, a regulatory subunit of phosphoinositide 3-kinase (PI3K), but not to the related p85α. Expression of NS1 was sufficient to activate PI3K and cause the phosphorylation of a downstream mediator of PI3K signalling, Akt. However, in virus-infected MDCK cells, the kinetics of Akt phosphorylation did not correlate with NS1 expression, and suggested that negative regulation of this signalling pathway occurs subsequent to ~8h post-infection. Mapping studies showed that the NS1:p85β interaction is primarily mediated by the NS1 C-terminal domain and the p85β inter-SH2 (Src homology 2) domain. Additionally, the highly conserved tyrosine at residue 89 (Y89) of NS1 was found to be important for binding and activating PI3K in a phosphorylation-independent manner. The inter-SH2 domain of p85β is a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. As NS1 does not displace p110 from the inter-SH2 domain, a model is proposed whereby NS1 forms an active heterotrimeric complex with PI3K, and disrupts the ability of p85β to control p110 function. Biological studies revealed that a mutant influenza A virus (Udorn/72) expressing NS1 with phenylalanine substituted for tyrosine-89 (Y89F) exhibited a small-plaque phenotype, and grew more slowly in MDCK cells than wild-type virus. Unexpectedly, another mutant influenza A virus strain (WSN/33) expressing NS1-Y89F was not attenuated in MDCK cells, yet appeared to be less pathogenic than wild-type in vivo. Overall, these data indicate a role for NS1-mediated PI3K activation in efficient influenza A virus replication. The potential application of this work to the design of novel anti-influenza drugs and vaccine production is discussed.

A range of simulations have been conducted to investigate the behaviour of a diverse set of complex biological membrane systems. The processes of interest have required simulations over extended time and length scales, but without sacrifice of molecular detail. For this reason, the primary technique used has been coarse-grained molecular dynamics (CG MD) simulations, in which small groups of atoms are combined into lower-resolution CG particles. The increased computational efficiency of this technique has allowed simulations with time scales of microseconds, and length scales of hundreds of nm. The membrane-permeabilizing action of the antimicrobial peptide maculatin 1.1 was investigated. This short α-helical peptide is thought to kill bacteria by permeabilizing the plasma membrane, but the exact mechanism has not been confirmed. Multiscale (CG and atomistic) simulations show that maculatin can insert into membranes to form disordered, water-permeable aggregates, while CG simulations of large numbers of peptides resulted in substantial deformation of lipid vesicles. The simulations imply that both pore-forming and lytic mechanisms are available to maculatin 1.1, and that the predominance of either depends on conditions such as peptide concentration and membrane composition. A generalized study of membrane protein aggregation was conducted via CG simulations of lipid bilayers containing multiple copies of model transmembrane proteins: either α-helical bundles or β-barrels. By varying the lipid tail length and the membrane type (planar bilayer or spherical vesicle), the simulations display protein aggregation ranging from negligible to extensive; they show how this biologically important process is modulated by hydrophobic mismatch, membrane curvature, and the structural class or orientation of the protein. The association of influenza hemagglutinin (HA) with putative lipid rafts was investigated by simulating aggregates of HA in a domain-forming membrane. The CG MD study addressed an important limitation of model membrane experiments by investigating the influence of high local protein concentration on membrane phase behaviour. The simulations showed attenuated diffusion of unsaturated lipids within HA aggregates, leading to spontaneous accumulation of raft-type lipids (saturated lipids and cholesterol). A CG model of the entire influenza viral envelope was constructed in realistic dimensions, comprising the three types of viral envelope protein (HA, neuraminidase and M2) inserted into a large lipid vesicle. The study represents one of the largest near-atomistic simulations of a biological membrane to date. It shows how the high concentration of proteins found in the viral envelope can attenuate formation of lipid domains, which may help to explain why lipid rafts do not form on large scales in vivo.