Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that mediate host-responses to pathogens. In mammals, TLR ligands promote cellular activation and the production of cytokines. Several TLR ligands have been employed prophylactically for the control of bacterial or viral diseases in the mouse model. However, the TLR-mediated responses in chickens have not been well described. Importantly, the utility of TLR agonists for the control of viral pathogens, such as avian influenza virus (AIV), has not been fully explored in chickens. To this end, the studies described in this thesis characterized the kinetics of in vivo responses in chickens to the TLR4 ligand lipopolysaccharide (LPS) and the TLR21 ligand CpG ODN. It was demonstrated that both of these ligands induced the up-regulation of several immune system genes in the spleen, including those associated with pro-inflammatory and antiviral responses, as well antigen presentation. By harnessing the immunostimulatory properties of TLR ligands, it was also demonstrated that the prophylactic administration of either poly I:C (a TLR3 ligand), LPS or CpG ODN may confer immunity to a low pathogenic avian influenza virus, as determined by a reduction in both oropharyngeal and cloacal virus shedding in infected birds. Furthermore, transcriptional analysis of genes in the spleen and lungs identified interleukin (IL)-8, interferon (IFN)-α and IFN-γ as correlates of immunity. In conclusion, TLR ligands may modulate several aspects of the chicken immune system to induce an anti-viral state, thereby conferring immunity to AIV.

Cold-adapted (ca) live attenuated influenza vaccines (LAIVs) have been introduced as alternatives to existing inactivated influenza vaccines. The influenza A components of the FDA-approved ca LAIVs (Flumist®; Medimmune) have common internal genes derived from the donor strain A/Ann Arbor/6/60 ca and surface genes derived from current wild-type (wt) epidemic strains. The aim of this thesis was to investigate determinants of immunogenicity for reassortants of A/Ann Arbor/6/60 ca, using a range of immunological assays, including recently developed MHC tetramer techniques. From the study, the extent of viral replication in the respiratory tract of mice, the primary site of inoculation, was a key factor in determining ca vaccine immunogenicity. Replication was shown to be influenced by both viral surface Ags and the host MHC. The H3 ca reassortants CR6, CR18, CR29 and CR6-35* exhibited greater replication efficiency (as determined by their PFU:HAU ratios) than the H1 ca reassortants CR35 and CR6-35. The H3 ca reassortant CR6 caused a 3.79% loss in body weight but no losses were observed for the H1 ca reassortant CR35 and the ca H2N2 donor strain A/Ann Arbor/6/60 ca. Higher HI responses were detected after 3 weeks in groups infected with the H3 ca reassortant CR6 (GMT 80) than with the H1 reassortant CR35 (GMT 10) and the H2 ca donor strain A/Ann Arbor/6/60 ca (GMT 13). Recently developed techniques were used to evaluate specific T-cell response to ca LAIVs. Fluorescent-labelled tetramer is the key reagent for use in tetramer-based flow cytometry assays. The NP366-374 peptide of influenza A viruses comprises an immunodominant epitope that is highly conserved between subtypes. Tetramers developed for A/PR/8/34 (H1N1) were able to detect NP-specific cytotoxic T lymphocytes (CTLs) induced by A/Ann Arbor /6/60 ca (H2N2). An attempt to prepare the A/Ann Arbor/6/60 ca-specific-NP-tetramer is described. H-2Db monomers were successfully refolded with the peptide, but only 20% were able to form tetramers through biotin-streptavidin linkage, resulting in a poor capacity to stain. By contrast, an IFN-γ ICC assay developed in parallel demonstrated that peptide NP366-374 was able to restimulate A/Ann Arbor/6/60 NP ca-specific CTLs and secrete IFN-γ when tested in vitro. Specific-B and T cell responses induced in the lungs in response to infection by ca reassortants exhibited great variability that was determined by the growth characteristics of different viruses. Type I (CTL) responses were induced by low yielding ca reassortants, such as CR35 (H1N1). Viruses with enhanced growth characteristics, such as CR6 (H3N2), produced higher Type II (HA-specific Ab) responses. In addition, host factors, such as MHC type, were found to play an important role in responses to the same viruses. Susceptible mouse strains, such as C57BL/6, showed higher CTL but lower serum Ab responses than more resistant strains, such as BALB/c. Throughout this PhD project, a fine balance between the humoral and CMI, local and systemic immune responses induced by ca LAIVs was demonstrated. The need to assess local immune responses, in addition to serum antibody levels, for the evaluation of vaccine efficacy was an important conclusion of the thesis.

Numerous toxins and pathogens gain entry into mammalian cells using cell surface glycans. The Iyer group at Georgia State University is working on the development of glycoconjugates for the accurate detection of infectious agents. In this thesis, I have focused on the development of glycans to detect influenza virus and norovirus. In the first section, I have focused on influenza viruses. A panel of synthetic glycans was synthesized as receptor mimics for the specific capture of influenza viruses. The synthetic glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. This glycan printed microarray was evaluated for its ability to capture three strains of influenza viruses. The analytical limit of detection is ~10 pfu/ml, (plaque forming units/milliliter) which is clinical relevant as 102 viral particles are typically required to cause infection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using the microarray and determined the feasibility of this system to determine antiviral resistance for different strains. In addition to optical detection, I developed an electrochemical assay to rapidly detect influenza viruses. Here, we utilized an unique property of influenza viral surface enzyme, Neuraminidase (NA), which cleaves terminal N-Acetyl Neuraminic acid (sialic acid) from cell surfaces and proteins. We designed an electrochemical assay that uses glucose bearing sialic acid substrates. Glucose is released when exposed to viral NA or intact viruses. The released glucose can be detected using repurposed glucose meters. Thus, personal glucose meters that were designed to assist diabetics and prediabetics monitor blood glucose can potentially be used to detect pathogens. Using this approach, we have detected 19 unique strains of influenza viruses. We also demonstrated drug susceptibility using this assay. The limit of detection of this assay is 102 pfu/sample, which is clinically relevant. The results were validated plaque assays and polymerase chain reaction (PCR). In the second part of this thesis, I focused on norovirus detection. I developed a focused glycan microarray that comprised of a library of histo blood group antigens (HBGAs). The HBGAs were attached to a carrier protein and printed onto activated glass slides. A panel of norovirus virus like particles (VLPs) and strains that included different genogroups was exposed to the microarray. We found that different VLPs and strains give rise to unique binding patterns. When the binding pattern of VLPs for a particular strain were compared to the corresponding intact virus, the binding patterns didn't match well, presumably because the virus does not recognize the same antibody as the VLPs. Unfortunately, antibodies for the virus cannot be generated because the virus cannot be grown in a laboratory setting. Indeed, all norovirus samples are obtained from human challenge studies. I also used surface plasmon resonance (SPR) studies in an effort to determine the binding affinities. Divalent biotinylated H type glycans were synthesized and their binding affinities with different VLPs and viral strains were determined. Initial studies suggest that the binding affinities are strain specific. These results demonstrate that glycans can be used to capture and isolate norovirus, although more research is required to develop glycan based norovirus detection kits.

Deeper understanding and technological progress in materials physics demand exploration of soft and hard matter at their relevant length scales. This research focuses on the nanometer length scale investigation of structural changes required for membrane fusion in virus nanoparticles and nano-spectroscopic investigation of layered material surfaces implementing scattering type scanning near-field optical microscopy (s-SNOM). Spectroscopy and imaging experiments were deployed to investigate the chemical and structural modifications of the viral protein and lipid bilayer under various environmental pH variations. It has been shown that breakage of viral membrane could occur even without the presence of a targeting membrane, if the environment pH is lowered. This is in contrary to the current viral fusion model, which requires virus binding to a host cell membrane for forming the fusion pore to release the viral genome. The fusion inhibitor compound 136 can effectively prevent the membrane breakage induced by low pH. The chemical surface stability and degradation of black phosphorus (BP) under ambient conditions have been studied using s-SNOM. We found that the degraded area and volume on the surface of black phosphorus increase with time slowly at the start of degradation and enlarge rapidly (roughly exponentially) afterward and reach saturation growth following S-shaped growth curve (sigmoid growth curve). The theoretical model presented suggests that the degraded sites in the adjacent surrounding causes the experimentally observed exponential growth of degraded area at the initial stage. By studying the BP surfaces coated by Al2O3, boron nitride (BN) and hybrid BN/Al2O3 layers through the period up to 6 months, it has been concluded that ~5 nm thin hybrid layer of BN/Al2O3 helps the surface passivation of BP flakes of thickness ~30 nm. This is supported by the electrical characterization results of BP field effect transistor coated with a BN/Al2O3 layer. We have performed infrared nano-spectroscopy on muscovite mica exfoliated on silicon and silicon dioxide substrates. We show that the near-field profile in s-SNOM can penetrate down to several hundreds of nanometers and enable spectroscopy of buried structures. We found spectral broadening of mica as its thickness increases revealing clearly the effect of size on the absorption response.

Natural killer (NK) cells are lymphocytes of the innate immune system that play a major role in the destruction of both tumours and virally-infected cells. The cytotoxicity of NK cells is tightly controlled by signals received through activating and inhibitory receptors. NK cells express a variety of inhibitory receptors such as Ly49 receptors. Ly49 receptors bind to class I MHC molecules that expressed on normal cells. Using Ly49-deficient (NKCKD) mice we show that Ly49-KD NK cells successfully recognize and kill influenza virus-infected cells and that NKCKD mice exhibit better survival than wild-type mice. Moreover, influenza virus infection has a propensity to upregulate cell surface expression of MHC-I on murine lung epithelial cells in vivo. Significantly, we demonstrate increased lung damage of WT-mice versus NKCKD mice after influenza virus infection as determined by histological analyses. This data indicated that absence of Ly49 inhibitory NK receptors greatly enhances survival of infected mice.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19633号 / 医科博第71号 / 新制||医科||5(附属図書館) / 32669 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 小柳 義夫, 教授 斎藤 通紀, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The Influenza virus is a major human respiratory pathogen responsible for seasonal ‘flu’ outbreaks and sporadic global pandemics. The Influenza polymerase complex is necessary for viral RNA synthesis and full virulence and requires the assembly of three conserved subunits: PA, PB1 and PB2. A recombinant chimeric protein mimetic consisting of the N-terminus (20 amino acids) of PB1 fused to Maltose Binding Protein (MBP) and Tat Nuclear Localization Signal (NLS) was designed and purified with the aim of inhibiting the assembly of the polymerase by mimicking PB1. The cell-penetrating protein mimetic was shown to efficiently enter the cell nucleus and prevent assembly of the Influenza polymerase, thus inhibiting viral replication. When MDCK cells were incubated with the mimetic and subsequently challenged with Influenza A virus, viral replication decreased up to 98% at 50 µM. Using a nuclear extraction assay, the mimetic was shown to efficiently penetrate the plasma membrane and enter the host nucleus. GST pull-down assays showed that the mimetic interacts with PA. Molecular modeling was then employed to predict the improved hypothetical free energy of binding between PB1 and PA and determined two significant substitutions for PB1 threonine at position six: glutamic acid (T6E) and arginine (T6R). These mutations increased potency of the mimetic at 25 µM (71% for T6E and 77% for T6R compared to 36% for the native construct) and 12.5 µM (27% for T6E and 70% for T6R compared to 16% for the native construct), suggesting a more stable interaction with PA consistent with molecular modeling. Using various in vitro assays, the mimetic was shown to be non-toxic to host cells. Targeting critical protein-protein interactions using a peptide fused to a cell-penetrating carrier protein presents a novel and intriguing approach in designing anti-viral therapeutics. / Thesis / Master of Science in Medical Sciences (MSMS)

The COVID-19 pandemic has been an ongoing disaster that has devasted millions of lives. With the development of COVID-19 vaccines in late 2020, there was a potential for populations to gain artificial active immunity in order to prevent future outbreaks. However, despite successful clinical trials, millions of citizens have been hesitant to receive the COVID-19 vaccines (Khubchandani et al., 2021). Demographics of the most prominent US vaccine-hesitant populations consist of ethnic/racial minorities and Republicans groups (Khubchandani et al., 2021). Little information is known about COVID-19 vaccine hesitancy in colleges and universities. Colleges provide an elevated risk for infection through their communal residencies, the reemergence of campus activities, and continuous travel to home (Sharma et al., 2021). This study explored COVID-19 vaccine hesitancy in UCF college students and explored potential pathways to achieve higher vaccination rates. Potentially believed COVID-19 misinformation was also studied. A COVID-19 opinion survey was designed and distributed to the UCF college population. Two hypotheses were made for this study: (1) There is a significant effect on vaccination status among people of different political parties, field of study, living conditions, masking frequency, and scores on the knowledge-based questions portion. (2) There will be a significant effect on knowledge-based scores with political party and field of study. The results were analyzed using Chi-square, one-way ANOVA, or two-way ANOVA on SPSS. The results showed a significant effect on vaccination status in political parties, masking frequency in class, and scores on the knowledge-based survey questions. There was no significance with race/ethnicity and field of study. There was a significant effect on the knowledge-based survey questions with political party and field of study. Potential side effects and the vaccines being seen as ineffective were the top two reasons that students choose not to vaccinate.

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.