No description available.

Severe acute respiratory syndrome (SARS) was defined as an invasive respiratory disease in 2002, which originally came from China and rapidly spread all over the globe. Acute pneumonia and lower respiratory tract involvement most affected the middle aged individuals and elderly with a mortality rate of 11%. While SARS Corona virus (SARS-CoV) has maintained its potential capacity to reemerge, clinical study of the immune system of SARS patients, as well as controlled studies may lead to application of new treatment strategies in future. Throughout this work, I have focused on early immune responses to SARS-CoV in humans and in ferrets. CXCL0 has been associated with alterations in the clinical course of several infectious diseases, including SARS and influenza. Here I have cloned ferret CXCL10 gene and have expressed its recombinant protein. I demonstrate that the CXCL10 plasma level in SARS patients is associated with the severity of disease. I also show that endogenous ferret CXCL10 exhibits similar mRNA expression patterns in the lungs of deceased SARS patients and ferrets experimentally infected with SARS-CoV. Type I interferons (IFNs) are indispensable parts of the innate immunity during early stages of infection. A clear distinction between genes upregulated by direct virus-cell interactions and genes upregulated by secondary IFN production has not been made yet. Here, I have investigated differential gene regulation in ferrets upon subcutaneous administration of IFN-a2b and during SARS-CoV infection. In vivo experiments revealed that IFN-a2b causes upregulation of abundant IFN response genes (IRGs), chemokine receptors, and other genes that participate in phagocytosis and leukocyte migration. SARS-CoV infection of ferrets leads to upregulation of varieties of IRGs and a broad range of genes involved in cell migration and inflammation. This work allowed dissection of several molecular signatures present during SARS-CoV infection, which are part of a robust IFN antiviral response. Since localization of CD8+ Tcells may contribute to tissue injury, I have characterized ferret CD8 gene and have generated reagents that can be used in future studies with the aim of evaluating CD8+ T cells localization in the ferret lung during infection with SARS-CoV.

Aus dem Patientengut einer Fachklinik für Klein- und Heimtiere wurden 11 nicht vorbehandelte Katzen zwischen einem und zwölf Jahren mit mittel- bis hochgradiger Gingivo-Stomatitis ausgewählt. Diese wurden für zwölf Wochen (84 Tage) stationär aufgenommen und nach einem standardisierten Therapiekonzept behandelt: Am ersten Tag erfolgte nach dentalem Röntgen eine umfassende Zahnsanierung. An den Tagen 0, 14, 28, 42 und 84 wurde Interferon (Virbagen Omega® des Herstellers Virbac S.A.®, Carros Cedex, Frankreich) unter Sedation lokal, d.h. submukosal mit 1 ME/kg KGW injiziert. An den Tagen 56, 58, 60 und 62 erfolgte die Interferongabe systemisch. Begleittherapien wurde nach Bedarf eingesetzt, jedoch ohne die Verwendung von Glukokortikoiden und Hormonpräparaten. Verfüttert wurde ausschließlich Futter des Herstellers Royal Canin®, Köln, in den ersten 14 Tagen das Feuchtfutter Royal Canin convalescence support®, ab Tag 15 Royal Canin intestinal® Feucht- und Trockenfutter. An allen Behandlungstagen wurden zur qualitativen Virusbestimmung Tupferproben der am stärksten entzündeten Bezirke entnommen, die Maulhöhle nach einem festen System abfotografiert und die Veränderungen in Formblättern (Stärke der Faucites, Gingivitis, Buccostomatitis, Größe der Fläche und Art der Veränderung) und Grafik-charts festgehalten. Am ersten und letzten Tag wurden außerdem Biopsien zur quantitativen Bestimmung der Viruslast entnommen. Die Entwicklungen in folgenden Bereichen wurden anhand fixer Kriterien 14-tägig festgehalten: Allgemeinzustand, Schmerzen bei der Maulöffnung, Halitosis/zäher Speichel, Größe der Mandibularlymphknoten, Appetit, Schmerzen bei Futter-aufnahme oder Gähnen, Hypersalivation, Aktivität, Putztrieb und Zugänglichkeit. Die klinischen Verbesserungen waren bei allen Tieren schon nach 14 Tagen augenfällig. Der Hauptvorstellungsgrund der Besitzer, Appetitlosigkeit und Schmerzen bei der Futteraufnahme waren einer fast ungestörten Futteraufnahme gewichen, diese konnte in den folgenden Wochen kaum noch optimiert werden. Die entzündlichen Ulzerationen und Proliferationen der Maulhöhle halbierten sich innerhalb der ersten 14 Tage, nach 84 Tagen war der Heilungsprozess bei acht der elf Katzen abgeschlossen. Die persistierenden Proliferationen der restlichen Katzen waren allerdings nicht entzündlich und beeinflussten die Futteraufnahme nicht. Allgemeinzustand, Aktivität, Putztrieb und Zugänglichkeit stiegen bei zehn von elf Katzen bis zum 42. Tag etwa linear auf artspezifisches Normalniveau an und blieben hier konstant. Hypersalivation und Schwellung der Mandibularlymphknoten legte sich, so vorhanden, bei allen Tieren bis auf zwei innerhalb von 28 Tagen, bei diesen beiden war bis zum 84. Tag nur eine geringgradige Verbesserung zu beobachten. Nach der systemischen Vier-Tages-Therapie wurde ein erneutes Aufflackern der Gingivo-Stomatitis etwa auf das Niveau des 56. Tages beobachtet, allerdings ohne Folgen für die Verhaltensparameter. Eine Reduktion der Viruslast konnte trotz der eindrucksvollen Verbesserungen im klinischen Bereich in keinem Fall festgestellt werden. Die FIV/FeLV-positiven Katzen sprachen langfristig wesentlich schlechter auf das Therapiekonzept an als die übrigen Probanden. Daher gilt es bei diesen Tieren vor Interferoneinsatz kritisch zwischen den nicht unerheblichen Kosten und der zweifelhaften Prognose abzuwägen. Generell kann das Therapiekonzept Zahnsanierung – Interferon – Begleittherapie nach erfolgtem FIV/FeLV-Test bei der felinen Gingivo-Stomatitis als klinisch erfolgreich betrachtet werden.

Our innate immunity is our first line of defence against pathogens. We require this immunity to control the numerous viral infections we are challenged with during our lives. However, little is known about the exact molecular mechanisms of our innate immunity, particularly components that have specific antiviral potential. One potent mediator of this antiviral activity is the interferon system. Activation of the interferon system leads to the production of several interferon-induced proteins, which inhibit the multiplication of viruses by distinct mechanisms. A key example of one of these mediators is the human MxA protein. Human MxA has the capacity to inhibit many different viruses from diverse families. In many cases it is proposed that MxA interferes with key viral components, such as incoming or newly formed nucleocapsids. It is speculated that MxA traps and missorts these viral components so they are no longer available for virus production and virus dissemination is inhibited. West Nile virus belongs to Flaviviridae family of viruses and was involved in the outbreak of virus-associated encephalitis in New York City in 1999. In this thesis I show that West Nile virus is insensitive to antiviral activity of MxA and describe how West Nile virus has developed a replication strategy that avoids MxA recognition and activation. I show that virus-induced changes of cytoplasmic membranes provide a protective microenvironment for viral replication and the viral components essential for viral replication. This hypothesis was proven by preventing the formation of these membrane structures with the fungal chemical Brefeldin A. Under these conditions I observed that stable expression of MxA could partially restrict West Nile virus RNA replication. Subsequently, I showed that the assembly mechanism of West Nile virus prevents interaction between the MxA protein and the viral capsid proteins. This was achieved by the use of a trans-packaging cell line whereby the West Nile virus structural proteins are expressed stably in trans instead of in cis from the polyprotein. When this cell line was transfected with a West Nile virus replicon expressing the human MxA protein distinct co-localisation and redistribution of the MxA with West Nile virus capsid proteins into large tubular structures within the cytoplasm of transfected cells was observed. Strikingly, these tubular aggregates are visually analogous to structures observed during infection of MxA expressing cells infected with members of the Bunyaviridae, particularly La Crosse virus. Moreover, retargeting MxA to specific sites of the endoplasmic reticulum in cells transfected with the West Nile virus infectious clone resulted in co-localisation between MxA and the West Nile virus capsid proteins and significantly inhibited the production of infectious particles. These results suggest that the sequestering of viral capsids within cytoplasmic inclusions maybe a conserved mechanism for antiviral activity of the MxA protein across the viruses families and highlight the innate ability of such molecules to recognise key molecular patterns within pathogens. Finally, I sought to exploit the antiviral potential of MxA as a therapeutic agent against infection with Influenza A viruses; viruses that have a very high sensitivity for the antiviral activity of MxA. By expressing MxA from the West Nile virus replicon, infection with the highly pathogenic Influenza virus H5N1 strain could be inhibited in vitro. Furthermore, in vivo studies in Mx-negative mice indicated that intranasal inoculation with MxA expressed from the West Nile virus replicon can protect these mice against an otherwise lethal infection with a low pathogenic Influenza A virus. Taken all together, in this thesis I provide evidence that strongly supports the existence of an evolutionary working mechanism of a significant mediator of our immune system, the antiviral MxA protein. Furthermore, I show how an important human pathogen, such as West Nile virus has evolved a replication strategy to evade this antiviral protein. These results will open new pathways for the development of a new type of antiviral therapies that utilize the potent antiviral activity of the MxA protein.

Our innate immunity is our first line of defence against pathogens. We require this immunity to control the numerous viral infections we are challenged with during our lives. However, little is known about the exact molecular mechanisms of our innate immunity, particularly components that have specific antiviral potential. One potent mediator of this antiviral activity is the interferon system. Activation of the interferon system leads to the production of several interferon-induced proteins, which inhibit the multiplication of viruses by distinct mechanisms. A key example of one of these mediators is the human MxA protein. Human MxA has the capacity to inhibit many different viruses from diverse families. In many cases it is proposed that MxA interferes with key viral components, such as incoming or newly formed nucleocapsids. It is speculated that MxA traps and missorts these viral components so they are no longer available for virus production and virus dissemination is inhibited. West Nile virus belongs to Flaviviridae family of viruses and was involved in the outbreak of virus-associated encephalitis in New York City in 1999. In this thesis I show that West Nile virus is insensitive to antiviral activity of MxA and describe how West Nile virus has developed a replication strategy that avoids MxA recognition and activation. I show that virus-induced changes of cytoplasmic membranes provide a protective microenvironment for viral replication and the viral components essential for viral replication. This hypothesis was proven by preventing the formation of these membrane structures with the fungal chemical Brefeldin A. Under these conditions I observed that stable expression of MxA could partially restrict West Nile virus RNA replication. Subsequently, I showed that the assembly mechanism of West Nile virus prevents interaction between the MxA protein and the viral capsid proteins. This was achieved by the use of a trans-packaging cell line whereby the West Nile virus structural proteins are expressed stably in trans instead of in cis from the polyprotein. When this cell line was transfected with a West Nile virus replicon expressing the human MxA protein distinct co-localisation and redistribution of the MxA with West Nile virus capsid proteins into large tubular structures within the cytoplasm of transfected cells was observed. Strikingly, these tubular aggregates are visually analogous to structures observed during infection of MxA expressing cells infected with members of the Bunyaviridae, particularly La Crosse virus. Moreover, retargeting MxA to specific sites of the endoplasmic reticulum in cells transfected with the West Nile virus infectious clone resulted in co-localisation between MxA and the West Nile virus capsid proteins and significantly inhibited the production of infectious particles. These results suggest that the sequestering of viral capsids within cytoplasmic inclusions maybe a conserved mechanism for antiviral activity of the MxA protein across the viruses families and highlight the innate ability of such molecules to recognise key molecular patterns within pathogens. Finally, I sought to exploit the antiviral potential of MxA as a therapeutic agent against infection with Influenza A viruses; viruses that have a very high sensitivity for the antiviral activity of MxA. By expressing MxA from the West Nile virus replicon, infection with the highly pathogenic Influenza virus H5N1 strain could be inhibited in vitro. Furthermore, in vivo studies in Mx-negative mice indicated that intranasal inoculation with MxA expressed from the West Nile virus replicon can protect these mice against an otherwise lethal infection with a low pathogenic Influenza A virus. Taken all together, in this thesis I provide evidence that strongly supports the existence of an evolutionary working mechanism of a significant mediator of our immune system, the antiviral MxA protein. Furthermore, I show how an important human pathogen, such as West Nile virus has evolved a replication strategy to evade this antiviral protein. These results will open new pathways for the development of a new type of antiviral therapies that utilize the potent antiviral activity of the MxA protein.

Zugl.: Giessen, Univ., Diss., 2005

University, Diss., 2006--Giessen.

Universiẗat, Diss., 2005--Giessen.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 117-142).