Les techniques de biologie moléculaire ont pris au cours des 20 dernières années une place importante dans le diagnostic direct des pathogènes viraux. Notre travail a porté sur la mise en place et le développement d’une plate-forme de biologie moléculaire, au sein du laboratoire de virologie de l’hôpital de la Timone, pour répondre aux demandes et contraintes du diagnostic en milieu hospitalier. L’organisation de cette plate-forme a nécessité plusieurs étapes : la prévention des risques de contamination, l’aliquotage et le stockage des réactifs, l’automatisation des techniques d’extraction des acides nucléiques, la mise au point de témoins positifs synthétiques et de témoins internes et l’optimisation des protocoles de PCR. Cette approche optimisée du diagnostic moléculaire des infections virales a été appliqué notamment à la détection de la grippe pandémique A/H1N1v dans les laboratoires de routine hospitalière et d’urgence « Point Of Care ». La mise en place de cette plate-forme a fait progresser de manière considérable le diagnostic moléculaire du laboratoire. Elle nous permet actuellement de détecter un grand nombre de pathogènes (>80) et de réaliser des tests dans un format à haut débit (≈40 000 tests/an). Au total, cette plateforme est au coeur de la capacité du laboratoire pour réagir de manière rapide aux évènements d'émergence en mettant en place rapidement des procédures diagnostiques standardisées. Ces techniques ont été transférées à de nombreux autres laboratoires de virologie partenaires nationaux et internationaux. Nous envisageons maintenant son utilisation dans une approche syndromique avec notamment, le développement du diagnostic des virus respiratoires. / Molecular biology techniques have taken an important role in the direct diagnosis of viral pathogens over the last 20 years. Our work focused on establishing and developing a platform for molecular diagnosis in the laboratory of Virology (Timone Hospital) to meet the demands and constraints of diagnosis in hospitals. The organization of this platform required several steps: prevention of contamination risks, aliquoting and storage of reagents, automation techniques of nucleic acid extraction, development of synthetic positive controls and internal controls and optimization of PCR protocols. This optimized approach of the molecular diagnosis of viral infections has particularly been applied to the detection of pandemic influenza A/H1N1v in hospital laboratories for routine and emergency "Point Of Care." The implementation of this platform has significantly improved molecular diagnosis in our laboratory. It currently allows us to detect a large number of pathogens (> 80) and perform tests in a high-throughput (≈ 40,000 tests per year). In total, this platform is at the heart of the laboratory capacity to react quickly to emerging events by rapidly implementing standardized procedures. These techniques have been transferred to many other partners’ laboratories nationally and internationally. We are now considering its use in a syndromic approach including the development of the diagnosis of respiratory viruses.

Rokshana Parvin Molecular epidemiology and biological properties of avian influenza viruses of subtype H5N1 and H9N2 Institute of Virology Submitted in November 2014 Pages 106, Figures 7, Table 1, References 339, Publications 4 Keywords: Avian Influenza Virus, H5N1, H9N2, Reassortment, Mutation, Replication and Growth kinetics Introduction Avian influenza viruses (AIVs) are the major cause of significant disease outbreaks with high morbidity and mortality worldwide in domestic birds resulting in great economic losses. Especially the subtypes of highly pathogenic avian influenza viruses (HPAIV) H5N1 and low pathogenic avian influenza viruses (LPAIV) H9N2 became the most prevalent AIVs in poultry causing regular disease outbreaks in many countries of Asia, the Middle East and Europe and are still ongoing events. Therefore, continues monitoring, surveillance and characterization of the circulating viruses are of high priority. Objectives The current study was designed for three main objectives; i) Molecular epidemiology of the HPAIV H5N1 in migratory birds in Bangladesh, ii) Molecular characterization of the AIV subtype H9N2 and iii) Biological properties of the AIV subtype H9N2. Materials and methods In first the part of the investigations, two HPAIV H5N1 strains were confirmed from 205 pools of fecal surveillance samples in Bangladesh. The two isolated H5N1 viruses were characterized by genome amplification and sequence analysis of the all eight genome segments. In the second part of the investigations, a confirmed AIV H9N2 from a retrospective analysis derived from a poultry farm in Bangladesh was characterized. Furthermore, three AI-H9N2 viruses were isolated and characterized from a commercial broiler and broiler breeder flock with clinical respiratory manifestations in Egypt. Full length genome amplification, cloning, sequencing and comprehensive phylogenetic analyses were performed for all eight genome segments. In the final part of the study, four selected Eurasian lineage H9N2 viruses - three G1 sub-lineages H9N2 and one European wild bird H9N2 virus - were propagated in embryonated chicken eggs (ECE) and Madin-Darby canine kidney epithelial cell culture systems. The ECE-grown and cell culture-grown viruses were monitored for replication kinetics based on tissue culture infectious dose (TCID50), hemagglutination assay (HA) and quantitative real time RT-PCR (qRT-PCR). The cellular morphology after infections was analyzed by immunofluorescence assay and cellular ELISA was performed to screen the sensitivity of the viruses to amantadine. Results The two newly isolated HPAIV H5N1 strains from migratory birds belonged to clade 2.3.2.1 and clustered together with other recently isolated viruses in Bangladesh derived from ducks, chickens, quails and crow. The amino acid sequences were also genetically similar although, some unique amino acid substitutions were observed. These substitutions were not related to the known conserved region of the molecular determinants of the virus. The phylogenetic analyses of the isolated AIV H9N2 from Bangladesh and Egypt revealed their close relationship with their respective contemporary isolates and maintained ancestor relation with A/Quail/HK/G1/1997 confirming that all studied H9N2 belonged to G1 sub-lineage. All six internal gene segments of the Bangladeshi AIV H9N2 showed high sequence homology with the HPAIV subtype H7N3 from Pakistan. In addition, also the PB1 internal gene showed high nucleotide homologies with a recently circulating HPAI-H5N1 virus from Bangladesh. Thus, the Bangladeshi AIV H9N2 is genetically a unique strain which shares internal gene segments with different HPAI viruses and takes part in reassortment events. On the other hand, the internal gene segments of the Egyptian H9N2 viruses were similar to the other members of the G1 sub-lineage with no evidence of reassortment events. In this virus rather point mutations within their respective gene segments are observed. With regard to the biological characterization, the three G1-H9N2 viruses produced comparatively higher titer than the Eurasian wild type-AIV H9N2. Overall, the ECE-grown viruses yielded higher titers than cell culture-grown viruses. Following a single passage in cell culture, individual nucleotide substitutions were noticed in HA, NA and NS gene sequences but none of them are related to the conserved region that can alter virus pathogenesis or virulence. All of the studied H9N2 viruses were sensitive to amantadine. Conclusion The present study demonstrated for the first time the presence of HPAI H5N1 in the wild migratory bird population in Bangladesh and determine as one of the major cause to introduce the new clade of HPAIV H5N1 into the Bangladeshi poultry flocks. The Bangladeshi AIV H9N2 strain has exhibited two independent reassortment events with HPAIV of subtype H7N3 and H5N1.The Egyptian AIV H9N2 strains were limited to regular point mutations which is very common for AIVs. The G1-H9N2 viruses showed a higher replication profile when compared to European wild bird-AIV H9N2. Both the ECE and MDCK cell system allowed efficient replication but the ECE system is considered as the better cultivation system for H9N2 viruses in order to get maximum amounts of virus within a short time period. In this study new strains of AIV H9N2 and H5N1 with significant genetic constitutions were described. Thus, continuous monitoring of the field samples, rapid reporting soon after outbreaks, molecular characterization to confirm the emergence of new reassortant strains and the biological properties to know its impact on the virulence are recommended. / Rokshana Parvin Molekulare Epidemiologie und biologische Charakterisierung von aviären Influenzaviren der Subtypen H5N1 und H9N2 Institut für Virologie Eingereicht im November 2014 Seiten 106, Abbildungen 7, Tabelle 1, Literaturangaben 339 , Publikationen 4 Schlüsselwörter: Aviäres Influenza Virus, H5N1, H9N2, Reassortment, Mutation, Replikation und Wachstumskinetik Einleitung Weltweit kommt es in der Geflügelproduktion durch Infektionen mit aviären Influenzaviren (AIV) zu hohen Morbiditäts- und Mortalitätsraten und damit verbunden zu hohen wirtschaftlichen Verlusten. Zu den bedeutenden AIV in der Geflügelwirtschaft werden die hoch pathogenen aviären Influenzaviren (HPAIV) des Subtyps H5N1 sowie AIV des Subtyps H9N2 gezählt. Letztere besitzen die Charakteristika von niedrigpathogenen aviären Influenzaviren. Durch diese Subtypen kommt es regelmäßig in vielen Ländern in Asien, im Nahen Osten und Europa zu wiederholten Krankheitsgeschehen. Dies bedingt die dringende Notwendigkeit von andauerndem Monitoring, Überwachung und Charakterisierung der zirkulierenden Viren. Ziele der Untersuchungen Die vorliegende Studie soll folgende drei Hauptfragestellungen beantworten: i) Molekulare Epidemiologie des HPAIV H5N1 bei Zugvögeln in Bangladesch, ii) Molekulare Charakterisierung von AIV des Subtyps H9N2 und iii) Biologische Eigenschaften von AIV des Subtyps H9N2. Materialien und Methoden Der erste Teil der Arbeit befasst sich mit zwei HPAIV Stämmen des Subtyps H5N1, welche im Monitoring Programm in Bangladesch von insgesamt 205 gepolten Kotproben, isoliert wurden. Die Charakterisierung der beiden Isolate erfolgte durch Vervielfältigung der acht Genomsegmente und nachfolgende phylogenetische Analysen. Der zweite Teil der Arbeit beschreibt die retrospektive Analyse eines AIV des Subtyps H9N2, welches von einer Geflügelproduktionsanlage in Bangladesch eingesandt wurde. Weiterhin wurden aus einer Geflügelmast- und Legehennenhaltung mit respiratorischer Symptomatik drei AIV des Subtyps H9N2 isoliert und charakterisiert. Auch hier wurde das gesamte Genom amplifiziert, kloniert und nachfolgend phylogenetisch analysiert. Im letzten Teil der Studie wurden vier europäische AIV H9N2 Isolate, von welchen 3 Isolate zur H9N2 Sublinie G1 gehören und ein Isolat von einem Wildvogel selektiert und in embryonierten Hühnereiern (EHE) und auf Madin-Darby canine kidney (MDCK) Zellen passagiert. Mittels 50% tissue culture infectious dose (TCID50), Hämagglutinationstest (HA) und RT-real-time-PCR (qRT-PCR) wurden von diesen so passagierten Viren die Vermehrungskinetik bestimmt. Die Morphologie der infizierten Zellen nach Infektion wurde mittels Immunfluoreszenztest analysiert. Eine Bestimmung der Amantadin Empfindlichkeit dieser Viren erfolgte mit einem ELISA. Ergebnisse Die beiden neuen HPAIV des Subtyps H5N1 von Zugvögeln können in die Clade 2.3.2.1 eingeordnet werden und clustern mit kürzlich aus Enten, Hühnern, Wachteln und Krähen isolierten AIV aus Bangladesch. Eine Verwandtschaft der Viren konnte auch auf Ebene der Aminosäure Sequenz gezeigt werden, obwohl einige einzigartige Aminosäure Austausche nachgewiesen wurden. Diese Austausche zeigen keine Verbindung mit bekannten konservierten Regionen der molekularen Determinanten der Viren. Die phylogenetische Analyse der AIV aus Bangladesch und Ägypten zeigt eine deutliche Verbindung mit den derzeit zirkulierenden AIV auf diesem geographischen Gebiet sowie die Verwandtschaft zu dem Isolat A/Quail/HK/G1/1997. Dies bestätigt, dass die in dieser Studie analysierten AIV zu der Subline G1 gehören. Alle sechs internen Gensegmente des AIV H9N2 aus Bangladesch zeigen eine hohe Sequenz Homologie mit einem HPAIV des Subtyps H7N3 aus Pakistan. Zusätzlich zeigt das interne Gene PB1 eine hohe Homologie auf Nukleinsäureebene zu einem derzeit in Bangladesch zirkulierenden HPAIV des Subtyps H5N1. Somit ist das AIV H9N2 aus Bangladesch als ein einzigartiges Isolat anzusehen, welches durch Reassortierung interne Gensegmente mit hochpathogenen AIV teilt. Im Gegensatz dazu, sind die internen Gene des AIV H9N2 aus Ägypten sehr ähnlich zu anderen Mitgliedern der Sublinie G1, welche keine Hinweise auf Reassorierung zeigen. Nur einzelne Punktmutationen konnten in den entsprechenden Gensegmenten nachgewiesen werden. In Hinblick auf die biologische Charakterisierung, konnte in den drei AIV H9N2 der Sublinie G1 vergleichsweise höhere Titer nachgewiesen werden als in einem europäischen AIV H9N2 Wildtypisolat. Insgesamt zeigten die in EHE passagierten Viren höhere Titer als die MDCK-Zell passagierten Viren. Schon nach einer Passage auf Zellkultur konnten einzelne Nukleotidaustausche in den HA, NA und NS kodierenden Gensegmenten nachgewiesen werden, wobei keine dieser Veränderungen einen Einfluss auf konservierte Regionen haben, die die Pathogenese oder Virulenz der Viren beeinflussen. Alle untersuchten H9N2 Viren sind sensitiv gegenüber Amantadin. Schlussfolgerungen Die vorliegende Studie zeigt erstmalig das Vorkommen von HPAIV H5N1 bei Zugvögeln in Bangladesch, welches als Haupteintragsquelle der neuen HPAIV H5N1 in der dortigen Geflügelhaltung angesehen wird. Das AIV H9N2 aus Bangladesch zeigt zwei unabhängige Reassortierungen mit HPAIV des Subtyps H7N3 und H5N1. Hingegen zeigt das ägyptische AIV H9N2 Punktmutationen, welche sehr typisch für diese Viren sind. Die hier untersuchten AIV H9N2 der Sublinie G1 zeigen im Vergleich zu einem europäischen AIV H9N2 eine höhere Replikationsrate. Eine Replikation der Viren konnte in EHE und MDCK-Zellen gezeigt werden, jedoch wird das EHE als das geeignetere System für die Kultivierung von H9N2 Viren betrachtet, da hier in einer kürzeren Zeitspanne mehr Virus produziert werden kann. Des Weiteren konnten in dieser Studie neue Isolate von AIV des Subtyps H9N2 und H5N1mit einem bedeutenden genetischen Aufbau beschrieben werden. Daher wird ein kontinuierliches Monitoring von Feldproben, unverzügliche Meldung von Ausbruchsgeschehen, die molekulare Charakterisierung zur Dokumentation eventuell auftretender neuer Reassortanten sowie Untersuchungen der biologischer Eigenschaften zur Virulenzbestimmung empfohlen.

Rokshana Parvin Molecular epidemiology and biological properties of avian influenza viruses of subtype H5N1 and H9N2 Institute of Virology Submitted in November 2014 Pages 106, Figures 7, Table 1, References 339, Publications 4 Keywords: Avian Influenza Virus, H5N1, H9N2, Reassortment, Mutation, Replication and Growth kinetics Introduction Avian influenza viruses (AIVs) are the major cause of significant disease outbreaks with high morbidity and mortality worldwide in domestic birds resulting in great economic losses. Especially the subtypes of highly pathogenic avian influenza viruses (HPAIV) H5N1 and low pathogenic avian influenza viruses (LPAIV) H9N2 became the most prevalent AIVs in poultry causing regular disease outbreaks in many countries of Asia, the Middle East and Europe and are still ongoing events. Therefore, continues monitoring, surveillance and characterization of the circulating viruses are of high priority. Objectives The current study was designed for three main objectives; i) Molecular epidemiology of the HPAIV H5N1 in migratory birds in Bangladesh, ii) Molecular characterization of the AIV subtype H9N2 and iii) Biological properties of the AIV subtype H9N2. Materials and methods In first the part of the investigations, two HPAIV H5N1 strains were confirmed from 205 pools of fecal surveillance samples in Bangladesh. The two isolated H5N1 viruses were characterized by genome amplification and sequence analysis of the all eight genome segments. In the second part of the investigations, a confirmed AIV H9N2 from a retrospective analysis derived from a poultry farm in Bangladesh was characterized. Furthermore, three AI-H9N2 viruses were isolated and characterized from a commercial broiler and broiler breeder flock with clinical respiratory manifestations in Egypt. Full length genome amplification, cloning, sequencing and comprehensive phylogenetic analyses were performed for all eight genome segments. In the final part of the study, four selected Eurasian lineage H9N2 viruses - three G1 sub-lineages H9N2 and one European wild bird H9N2 virus - were propagated in embryonated chicken eggs (ECE) and Madin-Darby canine kidney epithelial cell culture systems. The ECE-grown and cell culture-grown viruses were monitored for replication kinetics based on tissue culture infectious dose (TCID50), hemagglutination assay (HA) and quantitative real time RT-PCR (qRT-PCR). The cellular morphology after infections was analyzed by immunofluorescence assay and cellular ELISA was performed to screen the sensitivity of the viruses to amantadine. Results The two newly isolated HPAIV H5N1 strains from migratory birds belonged to clade 2.3.2.1 and clustered together with other recently isolated viruses in Bangladesh derived from ducks, chickens, quails and crow. The amino acid sequences were also genetically similar although, some unique amino acid substitutions were observed. These substitutions were not related to the known conserved region of the molecular determinants of the virus. The phylogenetic analyses of the isolated AIV H9N2 from Bangladesh and Egypt revealed their close relationship with their respective contemporary isolates and maintained ancestor relation with A/Quail/HK/G1/1997 confirming that all studied H9N2 belonged to G1 sub-lineage. All six internal gene segments of the Bangladeshi AIV H9N2 showed high sequence homology with the HPAIV subtype H7N3 from Pakistan. In addition, also the PB1 internal gene showed high nucleotide homologies with a recently circulating HPAI-H5N1 virus from Bangladesh. Thus, the Bangladeshi AIV H9N2 is genetically a unique strain which shares internal gene segments with different HPAI viruses and takes part in reassortment events. On the other hand, the internal gene segments of the Egyptian H9N2 viruses were similar to the other members of the G1 sub-lineage with no evidence of reassortment events. In this virus rather point mutations within their respective gene segments are observed. With regard to the biological characterization, the three G1-H9N2 viruses produced comparatively higher titer than the Eurasian wild type-AIV H9N2. Overall, the ECE-grown viruses yielded higher titers than cell culture-grown viruses. Following a single passage in cell culture, individual nucleotide substitutions were noticed in HA, NA and NS gene sequences but none of them are related to the conserved region that can alter virus pathogenesis or virulence. All of the studied H9N2 viruses were sensitive to amantadine. Conclusion The present study demonstrated for the first time the presence of HPAI H5N1 in the wild migratory bird population in Bangladesh and determine as one of the major cause to introduce the new clade of HPAIV H5N1 into the Bangladeshi poultry flocks. The Bangladeshi AIV H9N2 strain has exhibited two independent reassortment events with HPAIV of subtype H7N3 and H5N1.The Egyptian AIV H9N2 strains were limited to regular point mutations which is very common for AIVs. The G1-H9N2 viruses showed a higher replication profile when compared to European wild bird-AIV H9N2. Both the ECE and MDCK cell system allowed efficient replication but the ECE system is considered as the better cultivation system for H9N2 viruses in order to get maximum amounts of virus within a short time period. In this study new strains of AIV H9N2 and H5N1 with significant genetic constitutions were described. Thus, continuous monitoring of the field samples, rapid reporting soon after outbreaks, molecular characterization to confirm the emergence of new reassortant strains and the biological properties to know its impact on the virulence are recommended. / Rokshana Parvin Molekulare Epidemiologie und biologische Charakterisierung von aviären Influenzaviren der Subtypen H5N1 und H9N2 Institut für Virologie Eingereicht im November 2014 Seiten 106, Abbildungen 7, Tabelle 1, Literaturangaben 339 , Publikationen 4 Schlüsselwörter: Aviäres Influenza Virus, H5N1, H9N2, Reassortment, Mutation, Replikation und Wachstumskinetik Einleitung Weltweit kommt es in der Geflügelproduktion durch Infektionen mit aviären Influenzaviren (AIV) zu hohen Morbiditäts- und Mortalitätsraten und damit verbunden zu hohen wirtschaftlichen Verlusten. Zu den bedeutenden AIV in der Geflügelwirtschaft werden die hoch pathogenen aviären Influenzaviren (HPAIV) des Subtyps H5N1 sowie AIV des Subtyps H9N2 gezählt. Letztere besitzen die Charakteristika von niedrigpathogenen aviären Influenzaviren. Durch diese Subtypen kommt es regelmäßig in vielen Ländern in Asien, im Nahen Osten und Europa zu wiederholten Krankheitsgeschehen. Dies bedingt die dringende Notwendigkeit von andauerndem Monitoring, Überwachung und Charakterisierung der zirkulierenden Viren. Ziele der Untersuchungen Die vorliegende Studie soll folgende drei Hauptfragestellungen beantworten: i) Molekulare Epidemiologie des HPAIV H5N1 bei Zugvögeln in Bangladesch, ii) Molekulare Charakterisierung von AIV des Subtyps H9N2 und iii) Biologische Eigenschaften von AIV des Subtyps H9N2. Materialien und Methoden Der erste Teil der Arbeit befasst sich mit zwei HPAIV Stämmen des Subtyps H5N1, welche im Monitoring Programm in Bangladesch von insgesamt 205 gepolten Kotproben, isoliert wurden. Die Charakterisierung der beiden Isolate erfolgte durch Vervielfältigung der acht Genomsegmente und nachfolgende phylogenetische Analysen. Der zweite Teil der Arbeit beschreibt die retrospektive Analyse eines AIV des Subtyps H9N2, welches von einer Geflügelproduktionsanlage in Bangladesch eingesandt wurde. Weiterhin wurden aus einer Geflügelmast- und Legehennenhaltung mit respiratorischer Symptomatik drei AIV des Subtyps H9N2 isoliert und charakterisiert. Auch hier wurde das gesamte Genom amplifiziert, kloniert und nachfolgend phylogenetisch analysiert. Im letzten Teil der Studie wurden vier europäische AIV H9N2 Isolate, von welchen 3 Isolate zur H9N2 Sublinie G1 gehören und ein Isolat von einem Wildvogel selektiert und in embryonierten Hühnereiern (EHE) und auf Madin-Darby canine kidney (MDCK) Zellen passagiert. Mittels 50% tissue culture infectious dose (TCID50), Hämagglutinationstest (HA) und RT-real-time-PCR (qRT-PCR) wurden von diesen so passagierten Viren die Vermehrungskinetik bestimmt. Die Morphologie der infizierten Zellen nach Infektion wurde mittels Immunfluoreszenztest analysiert. Eine Bestimmung der Amantadin Empfindlichkeit dieser Viren erfolgte mit einem ELISA. Ergebnisse Die beiden neuen HPAIV des Subtyps H5N1 von Zugvögeln können in die Clade 2.3.2.1 eingeordnet werden und clustern mit kürzlich aus Enten, Hühnern, Wachteln und Krähen isolierten AIV aus Bangladesch. Eine Verwandtschaft der Viren konnte auch auf Ebene der Aminosäure Sequenz gezeigt werden, obwohl einige einzigartige Aminosäure Austausche nachgewiesen wurden. Diese Austausche zeigen keine Verbindung mit bekannten konservierten Regionen der molekularen Determinanten der Viren. Die phylogenetische Analyse der AIV aus Bangladesch und Ägypten zeigt eine deutliche Verbindung mit den derzeit zirkulierenden AIV auf diesem geographischen Gebiet sowie die Verwandtschaft zu dem Isolat A/Quail/HK/G1/1997. Dies bestätigt, dass die in dieser Studie analysierten AIV zu der Subline G1 gehören. Alle sechs internen Gensegmente des AIV H9N2 aus Bangladesch zeigen eine hohe Sequenz Homologie mit einem HPAIV des Subtyps H7N3 aus Pakistan. Zusätzlich zeigt das interne Gene PB1 eine hohe Homologie auf Nukleinsäureebene zu einem derzeit in Bangladesch zirkulierenden HPAIV des Subtyps H5N1. Somit ist das AIV H9N2 aus Bangladesch als ein einzigartiges Isolat anzusehen, welches durch Reassortierung interne Gensegmente mit hochpathogenen AIV teilt. Im Gegensatz dazu, sind die internen Gene des AIV H9N2 aus Ägypten sehr ähnlich zu anderen Mitgliedern der Sublinie G1, welche keine Hinweise auf Reassorierung zeigen. Nur einzelne Punktmutationen konnten in den entsprechenden Gensegmenten nachgewiesen werden. In Hinblick auf die biologische Charakterisierung, konnte in den drei AIV H9N2 der Sublinie G1 vergleichsweise höhere Titer nachgewiesen werden als in einem europäischen AIV H9N2 Wildtypisolat. Insgesamt zeigten die in EHE passagierten Viren höhere Titer als die MDCK-Zell passagierten Viren. Schon nach einer Passage auf Zellkultur konnten einzelne Nukleotidaustausche in den HA, NA und NS kodierenden Gensegmenten nachgewiesen werden, wobei keine dieser Veränderungen einen Einfluss auf konservierte Regionen haben, die die Pathogenese oder Virulenz der Viren beeinflussen. Alle untersuchten H9N2 Viren sind sensitiv gegenüber Amantadin. Schlussfolgerungen Die vorliegende Studie zeigt erstmalig das Vorkommen von HPAIV H5N1 bei Zugvögeln in Bangladesch, welches als Haupteintragsquelle der neuen HPAIV H5N1 in der dortigen Geflügelhaltung angesehen wird. Das AIV H9N2 aus Bangladesch zeigt zwei unabhängige Reassortierungen mit HPAIV des Subtyps H7N3 und H5N1. Hingegen zeigt das ägyptische AIV H9N2 Punktmutationen, welche sehr typisch für diese Viren sind. Die hier untersuchten AIV H9N2 der Sublinie G1 zeigen im Vergleich zu einem europäischen AIV H9N2 eine höhere Replikationsrate. Eine Replikation der Viren konnte in EHE und MDCK-Zellen gezeigt werden, jedoch wird das EHE als das geeignetere System für die Kultivierung von H9N2 Viren betrachtet, da hier in einer kürzeren Zeitspanne mehr Virus produziert werden kann. Des Weiteren konnten in dieser Studie neue Isolate von AIV des Subtyps H9N2 und H5N1mit einem bedeutenden genetischen Aufbau beschrieben werden. Daher wird ein kontinuierliches Monitoring von Feldproben, unverzügliche Meldung von Ausbruchsgeschehen, die molekulare Charakterisierung zur Dokumentation eventuell auftretender neuer Reassortanten sowie Untersuchungen der biologischer Eigenschaften zur Virulenzbestimmung empfohlen.

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.

For both influenza viruses and hepatitis C viruses, T cell responses to conserved antigens are one strategy for the human host to control the spread of infection. Such T cell responses can be generated with the use of viral vectored vaccines. Initially I show that the viral vectored vaccine MVA-NP+M1 can boost memory T cell responses to influenza A virus in adults aged over 50 years old. However within this group, MVA-NP+M1 had reduced immunogenicity in adults who were aged over 70 years old. The influenza virus-specific T cell responses comprised both CD4 and CD8 T cells, and were capable of secreting multiple Th1 cytokines. I then show that MVA-NP+M1 can be safely co-administered alongside seasonal influenza vaccine. The combination does not interfere with the peak T cell response that normally occurs 1 week following MVA-NP+M1. There was a statistically significant increase in antibodies to the H3N2 strain when the vaccines were co-administered, suggesting that the MVA-NP+M1 can act as an adjuvant. The efficacy of MVA-NP+M1 in humans had been previously evaluated in an influenza virus challenge study. I used a whole blood transcriptome approach to improve the classification of outcomes following influenza virus challenge. For subjects with laboratory-confirmed influenza, individuals with moderate/severe symptoms were found to have a distinct transcriptional signature comprising over 2,000 genes. I used a machine learning algorithm to reduce this variation down to just six genes (CCL2, SEPT4, LAMP3, RTP4, MT1G and OAS3). I validated this finding using expression data from an independently conducted challenge experiment. Data from these six genes was successfully able to predict symptomatic and asymptomatic cases with 89% and 100% accuracy respectively. To induce T cell responses to hepatitis C virus, I used the vaccines ChAd3-NSmut and MVA-NSmut in a prime-boost regimen. While the combination was highly immunogenic in healthy young adults, MVA-NSmut alone was unable to prime immune responses. The magnitude of T cell responses to the vaccine immunogen was correlated with the breadth of the T cell responses to different epitopes. Re-administration of the same two vaccines after a short time interval (8 weeks) did not improve upon previous peaks in T cell response. However with a longer time interval (> 34 weeks), some individuals were able to achieve higher frequencies of virus-specific T cells compared to the first round of vaccines. A whole blood transcriptome approach was used to study gene expression in volunteers vaccinated with ChAd3-NSmut and MVA-NSmut. Vaccination with MVA-NSmut results in a very strong, but relatively short-lived host gene expression signature. In contrast, the transcriptional response seen following ChAd3-NSmut was much less pronounced. A comparison of the functional analysis of gene lists from both vaccines showed that similar pathways were being activated and repressed.

Les virus influenza provoquent chaque année la grippe saisonnière qui peut toucher 5 à 15 % de la population. Les médicaments antiviraux sont un moyen important complémentaire à la vaccination pour le traitement et la prévention de la grippe. Actuellement, deux classes d’antiviraux ont été approuvées, l'une pour inhiber l'étape de décapsidation (l’inhibiteur du canal ionique M2), et l'autre pour empêcher la libération de néo-virions (l’inhibiteur de la neuraminidase). Cependant, de plus en plus de virus sont nativement résistants aux inhibiteurs de la protéine M2. Des virus résistants aux inhibiteurs de la neuraminidase ont également circulé durant les hivers 2008 – 2009. Le développement de nouveaux médicaments afin de substituer ou de compléter ces inhibiteurs est donc crucial dans la lutte contre les virus de la grippe. L’accent mis ces dernières années sur l’activité biologique des sucres (oligosaccharides/polysaccharides) montre une voie pour l’étude de l’activité antivirale d’une des plus importantes biosources. Dans le but d'évaluer le potentiel antigrippal des molécules dérivées de sucres, nous avons effectué un criblage à partir d'une bibliothèque de 245 composés de polysaccharides et d'oligosaccharides, dont la plupart proviennent d’algues et de végétaux supérieurs. Plusieurs molécules actives réparties dans différentes familles de sucres ont été mises en évidence. Parmi les candidats d'intérêt, l’oligosaccharide sulfaté 152, appartenant à la famille des arabinogalactanes de l'espèce Codium fragile, a présenté une activité inhibitrice vis-à-vis des deux virus influenza de type A et de type B in vitro. Le mécanisme d’action de cet oligosaccharide a été caractérisé. Il montre que les deux glycoprotéines de surface, l’hémagglutinine et la neuraminidase, sont les cibles virales de cette molécule / The annual seasonal flu caused by influenza viruses can affect 5 to 15 % of the population. In addition to vaccination, the use of antiviral drugs in the treatment and prevention allows the control of influenza virus infection. So far, two classes of antiviral drugs have been approved for influenza treatment, one to inhibit the uncoating step (M2 inhibitors), and the other to prevent the release of progeny virions (NA inhibitors). However, the emergence and circulation of M2 inhibitor resistant viruses at high frequencies have restricted the use of these inhibitors. Neuraminidase inhibitor resistance among circulating influenza viruses has emerged since the 2008 – 2009 season. The development of new classes of antiviral agents is crucial in the fight against influenza virus. In recent years, many molecules belonging to a large group of compounds known as carbohydrates (oligosaccharides/polysaccharides) have been revealed essential for various biological activities. The establishment of carbohydrate-based antiviral agents is, therefore, a highly promising strategy. In order to evaluate the potentially anti-influenza molecules derived from carbohydrates, we have performed a screening from a library of 245 polysaccharides and oligosaccharides. These compounds were extracted mainly from plants and algae. Several active molecules of different families have been identified. Among them, the sulphated oligosaccharide 152, belonging to the family of arabinogalactane, was found to be highly active toward both influenza virus A and B in vitro. This oligosaccharide was purified from the green algal species Codium fragile. The study of the 152 mechanism suggests that this oligosaccharide can cooperatively inhibit both viral HA binding activity and NA catalytic activity

Viral sequence data has great potential for answering questions about the epidemiological dynamics and evolution of viruses. Classical approaches have sought amino acid changes that alter pathogenesis or transmissibility by influencing a virus's ability to enter or replicate within cells. However, this approach rarely recognises the fundamental impact of heterogeneous host contact structures and existing immunological responses on viral transmission. This thesis draws heavily on ecological and immunological concepts to explore the epidemiological dynamics, diversity and evolution of viruses using molecular sequence data. A number of different research approaches and study systems are used in this thesis. I begin by describing a novel polyomavirus in a European badger, and apply phylogenetic techniques to analyze the evolutionary history of the Polyomaviridae. I subsequently describe a large metaviromic study in a population of wild mute swans, for which host demographic data are available. I describe nine new viral species and test whether age and season are associated with differences in abundance and prevalence of different viral taxonomic groups. The study highlights the potential of metaviromics for investigating viral epidemiological dynamics in natural populations. Influenza A viruses of avian origin (AIV) threaten human and animal health. Using phylogeographic methods, I reconstruct the spatial spread of an H5N8 virus at a regional scale, and investigate how bird density and migration shaped this dispersal. Despite the importance of acquisition of humoral immunity to different strains throughout the lifespan of wild birds for epidemiological dynamics, this topic is poorly understood. I assess the accumulation of immune responses to AIV with age in mute swans. I consider how ecological factors, including age-structured immunity, might have affected the epidemiology of an H5N8 outbreak in the population.

Protection from influenza A virus (IAV) challenge requires switched, high affinity Abs derived from long-lived memory B cells and plasma cells. These subsets are generated in germinal centers (GCs), hallmark structures of T helper cell-driven B cell immunity. A full understanding of the acute and persistent GC B cell reaction following respiratory IAV infection is lacking, as is the characterization of IAV-induced T follicular helper (TFH) cells that support GCs. Additionally, it remains unclear as to whether IAV-induced GC B cells are subject to control by regulatory T cells (Tregs). To address this, GC B cell and TFH cell responses were analyzed in mice following pulmonary challenge with IAV. Studies demonstrated that marked GC reactions were induced in lung-draining lymph nodes (dLNs), lung, spleen and nasal-associated lymphoid tissue (NALT), although the magnitude, kinetics, and isotype switching patterns of the response was site-specific, and largely depended on the magnitude of IAV-induced TFH cell populations. TFH cell magnitude peaked prior to that of GC B cells in all tissues, and TFH cells purified from dLNs generated IL-21 and IFN-gamma upon activation, although CD4+CXCR5- T effector cells produced higher levels of all cytokines. IgA+ GC B cells were infrequent in most sites, but composed a significant subset of the switched GC population in NALT. Further, splenectomized mice withstood a lethal recall challenge, suggesting the spleen to be unnecessary for long-term protection. Additionally, GC B cell populations were analyzed at distal time points to assess the understudied, persistent GC B cell response after IAV infection. Our analysis demonstrated that persistent GC B cell populations in mouse lungs directly correlated with infectious dose, pathogenicity of the virus, as well as the presence of long-term CD4+ T cell help. Finally, experiments showed that Tregs contribute to the control of GCs induced in the spleen by IAV challenge. This was demonstrated by a marked increase in the number of total and switched GC B cell numbers when Tregs were either depleted or disrupted in vivo proximal to IAV exposure.

Influenza A virus (IAV) is a major cause of serious respiratory illness worldwide, leading to approximately 5 million severe cases and 500,000 deaths per year. Given the disease severity, associated economic costs, and recent appearance of novel IAV strains, there is a renewed interest in developing novel and efficacious “universal” IAV vaccination strategies as well as therapeutic remedies. Previous studies from our laboratory have concentrated on IAV-specific CD8 T cell-mediated protection against IAV infection as IAV-specific CD8 T cells are needed for efficient clearance of virus. Recent studies highlight that immunizations capable of generating local (i.e., nasal mucosa and lung) tissue-resident memory T and B cells in addition to systemic immunity offer the greatest protection against future IAV encounters. Current IAV vaccines are designed to largely stimulate IAV-specific antibodies, but do not generate the lung-resident memory T and B cells induced during IAV infections. In order to effectively generate lung-resident memory populations, it is believed a local antigen depot is needed as tissue-resident memory formation is enhanced by the presence of local antigen. Recently, polyanhydride nanoparticles have been demonstrated to slowly release their contents at the site of inoculation serving as an antigen depot. However, the ability of an intranasal vaccination with polyanhydride nanoparticles to induce IAV-specific lung-resident immune responses and provide protection against subsequent IAV infection has not been determined. Here, I report on the intranasal administration of a biocompatible polyanhydride nanoparticle-based IAV vaccine (IAV-nanovax). IAV-nanovax is capable of providing protection against subsequent homologous and heterologous IAV infections in both inbred and outbred populations. My findings demonstrate that vaccination with IAV-nanovax promotes the induction of germinal center B cells within the lungs that are associated with both systemic IAV-specific IgG as well as local lung IAV-specific IgG and IgA antibodies. Furthermore, intranasal IAV-nanovax vaccination leads to a significant increase in IAV-specific CD4 and CD8 T cells within the lung vasculature as well as in the lung tissue. Most importantly, my studies demonstrate that IAV-nanovax induced lung-resident IAV-specific CD4 and CD8 T cells express canonical tissue-resident memory markers. This dissertation further explores a novel regulation pathway previously identified by our laboratory where plasmacytoid dendritic cells (pDCs) eliminate IAV-specific CD8 T cells early during high-dose and high-pathogenic IAV infections in a FasL:Fas (pDCs:CD8 T cell) dependent manner. However, recent studies suggest that B cells are the predominate lymphocyte to express FasL in mice. Here, I demonstrate that FasLpos B cells greatly outnumber FasLpos pDC within the lung draining lymph nodes (dLNs) during IAV infections. Interestingly, my results demonstrate the presence of two subsets, CD11cpos and CD11cneg, of FasL-expressing B cells that differentially influence the IAV-specific CD8 T cell response during high-dose IAV infections. While CD11cneg B cells kill IAV-specific CD8 T cells, contributing to lethality during high-dose IAV infections, CD11cpos B cells may instead be protective. In addition to the negative impacts of high-dose IAV infections, I also demonstrate that chronic ethanol (EtOH) consumption detrimentally impacts existing IAV-specific CD8 T cell memory responses. Here, my results reveal that chronic EtOH consumption causes a numerical loss in existing IAV-specific CD8 T cell memory responses. This numerical loss in existing IAV-specific CD8 T cell memory is associated with a reduction in cytotoxic activity within the lungs as well as an increase in morbidity and mortality during a secondary IAV challenge. Together, the results presented herein demonstrate the ability of a novel polyanhydride nanovaccine to induce robust pulmonary IAV-specific T and B cell responses and further our understanding of factors that can negatively impact IAV-specific CD8 T cells as well as protection against IAV infection. Overall these findings highlight the importance of IAV-specific CD8 T cells, as well as CD4 T cells and B cells, in providing protection against IAV infections.

HLA-DR1 transgenic (DR1 Tg) mice provide a model for evaluating the breadth and specificity of CD4 T cell responses that may develop in humans following influenza infection or vaccination. Recent studies identified a tremendously broad HLA-DR1-restricted CD4 T cell responses in DR1 Tg mice infected intranasally with influenza A/New Caledonia/20/99 (NC). In this study, our goals were to characterize B cell responses after NC infection in DR1 Tg mice and establish the correlation between B cell responses and CD4 T cell responses in this system. Influenza-specific B cell responses following virus administration were analyzed in DR1 Tg mice and in the genetically matched H-2b strain C57BL/10J (B10). Following intranasal (i.n.) NC infection, B cell responses in B10 mice featured strong IgG2b and IgG2c production and were typical of previously described B cell responses to a variety of mouse-adapted influenza strains. In contrast, B cell responses in DR1 Tg mice followed delayed kinetics and were strongly skewed to IgG1 production, suggesting the Th2 polarization of CD4 T cell responses. The different antibody isotype profile in DR1 Tg mice compared to B10 mice was evident in antibody secreting cells (ASCs) frequencies and in circulating Abs levels. Surprisingly, although DR1 Tg mice had lower influenza-specific Abs levels, they exhibited higher neutralizing Abs titers early in the response. B cell responses following intranasal infection of influenza A/Puerto Rico/8/1934 (PR8) or intramuscular vaccination of inactivated NC in DR1 Tg mice were different from the observed IgG1 bias after i.n. NC infection. After i.n. PR8 infection, B cell responses were similar in DR1 Tg mice and B10 mice, characterized by predominant IgM/IgG3 production. Additionally, following intramuscular administration of inactivated NC, B cell responses were skewed towards IgG2c production in both DR1 Tg mice and B10 mice, suggesting the Th1 polarization of CD4 T cell responses. A mechanistic understanding of IgG1/Th2 biased B cell responses and better neutralizing Abs production in DR1 Tg mice following i.n. NC infection may have implications for the optimal control of influenza infection.