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The respiratory epithelium acts as both, a barrier of the respiratory tract to Nipah virus (NiV) entry and at the same time as a significant determinant of virus shedding. Both, for humans and pigs, replication in the respiratory tract epithelia is considered a major factor in transmission to other hosts. To understand why the virus constitutes a high-risk pathogen for livestock and humans, knowledge about
viral replication and host responses in relevant cells and tissues is crucial. Most in vitro studies, however, have been performed in conventional cell lines or non-differentiated lung cells. Only a few examples exist where Henipavirus infections have been investigated in fully-differentiated lung
epithelial cell models.
Thus, one aim of this thesis was to investigate infection, replication, spread and host protein dynamics of NiV in primary bronchial epithelial cells (BEC) cultivated at the air-liquid-interphase (ALI). By
immunofluorescence imaging, the NiV infection dynamics in BEC-ALI cultures were monitored over a 12 day time course, in order to provide detailed information about the infection process in the
respiratory epithelium of pigs and ferrets. Compared to undifferentiated primary BEC, the specific infectivity of NiV in BEC-ALI cultures was low. Infections remained focal and complete infection of the
cultures was not observed, even at 12 dpi. Analysis of viral titers and viral mRNA indicated a limited
virion release from the infected ALI-cultures while most of the newly synthesized NiV-RNA remained
cell associated. Immunofluorescence analysis of cross sections from infected ALI-cultures revealed
large infected areas that exhibited a strong cytopathic effect (CPE). Disruption of the epithelium
resulted in apical release of virus antigen-positive cell detritus while ciliated areas and basal cells were
less affected. From these data it was concluded, that NiV transmission could be supported by
exhalation of cell debris associated NiV and thus may contribute to rapid spread of infection in swine
populations.
A second aim was to explore the dynamics of host responses to NiV infection in differentiated BEC-ALI
culture and to assess whether this differs to conventional cell line data available from literature. Even
though strong CPE appeared in later phases of NiV infection, at least the porcine PBEC-ALI cultures
remained robust enough to allow protein sampling over 12 days infection course. Subsequent MS-based proteomics enabled unprecedent insight in complex cell culture response upon NiV infection.
Previous reports indicated a lack of efficient interferon type I induction in non-differentiated pig or
human BEC which were considered a prerequisite for efficient replication in the respiratory epithelium
and virusspread. In contrast to non-differentiated pig BEC (PBEC), in PBEC-ALI cultures multiple factors
involved in interferon responses were upregulated upon NiV infection. Thereby it was demonstrated
that NiV infection induced a robust innate immune response upon infection with elevated components of antigen processing and presentation resulting in the conversion from the constitutive proteasome to the immunoproteasome. In contrast to previous reports about NiV-infected non-differentiated
PBEC or endothelial cells, incomplete immunoproteasome formation and limitations in interferon
response could be excluded. Thus, a model is proposed in which NiV infection and spread in differentiated PBECs is slowed by potent innate immune responses to the virus infection. Overall, the
findings highlight the important role of the respiratory epithelium not only as a physical barrier to virus
infections but also indicate itsrole as a primary site of adaptive immune induction through NiV induced
antigen processing and MHC I presentation.
Finally, to allow functional studies of Henipaviruses at the BSL-2 biosafety level a recombinant CedPV
was generated and rescued. An imaging based screening and quantitative analysis pipeline was established to investigate the role of cellular factors and to screen for potential virus and host gene
directed inhibitory factors. Accordingly, different host and viral genes were targeted with a siRNA-pool
either targeting virus or selected cellular mRNAs followed by the infection with the CedPV and the
quantification of infected cells. With proof of concept of the siRNA screening pipeline, the recombinant
CedPV clone was used as a backbone to insert variousfluorescence reporter genesin order to optimize
the analysis workflow by allowing direct virus quantification in live, unstained samples. Consequently,
this thesis provides a valuable proof for future approaches related to the function of virus proteins,
influence of host-factors and virusreplication and Henipavirus-inhibitorscreens at low biosafety levels.
As the animal-to-human interface becomes increasingly narrow, transmission events of zoonotic pathogens between animals and humans become more and more probable. While SARS-CoV-2 already accomplished a spillover infection to humans and is responsible for the current pandemic, the bat H9N2 IAV with so far unknown zoonotic potential was only recently discovered. In order to identify I) the role and potential of a newly discovered, potentially pre-pandemic virus, such as the bat H9N2, or II) possible future prevailing virus mutant variants of an already known pandemic virus, such as SARS-CoV-2, it is important to characterize these emerging viruses in vivo as soon and as good as possible.
The first objective in this dissertation (Publications I and II) therefore deals with the characterization of bat H9N2 and the estimation of its zoonotic or even pandemic potential.
In Publication I, a general susceptibility of directly inoculated Egyptian fruit bats to bat H9N2 was confirmed by successful seroconversion, although exhibiting only moderate viral shedding. All three contact animals remained seronegative, though one contact bat showed slight lesions in the histopathological analysis.
Publication II further addressed the question of the zoonotic potential of this virus. Inoculation of day-old turkey hatchlings demonstrated moderate susceptibility to bat H9N2 infection with a measurable seroconversion, while day-old chicken hatchlings were not susceptible to bat H9N2. Ferrets proved to be highly susceptible to bat H9N2 with high viral shedding, a transmission efficiency rate of 100% to direct contact animals at 2 days post contact, but with only minimal clinical signs. Importantly, the virus demonstrated the ability to evade the MxA-restriction factor and to replicate efficiently in human lung tissue explants. Furthermore, seasonal IAV- and standard IAV-vaccines showed no cross reactivity against the bat-N2 protein in humans. Therefore, further research on such viruses is urgently needed in order to prevent a renewed pandemic situation in the future as caused by SARS-CoV-2.
The second objective in this dissertation dealt with the identification and characterization of emerging SARS-CoV-2 Variants of Concern (VOCs).
Therefore, in Publication III, competitive infection experiments were performed using the Syrian golden hamster, the ferret, and transgenic mouse models (K18-hACE2 and hACE2-KI). These studies revealed replicative and transmissive predominance of Alpha VOC over Beta VOC, but not over SARS-CoV-2 WT in the hamster model, although Beta VOC substantially replicated in the lungs of donor animals. In contrast, the Alpha VOC had an unambiguous replication and transmission advantage over WT SARS-CoV-2 in the ferret and both mouse models. A recombinant SARS-CoV-2 WT-SAlpha virus helped to assign the fitness advantage of this variant particularly to the spike protein-associated mutations.
In Publication IV, in vitro results inferred an early replicative fitness advantage of Omicron BA.1 over Delta VOC, although the opposite was observed in competitively inoculated hamsters, ferrets and naive hACE2-KI mice. In addition, Publication IV demonstrated a disadvantage in transmission for the VOC Omicron BA.1 over the Delta VOC and a lack of susceptibility of ferrets after a single infection with the VOC Omicron BA.1. An mRNA vaccination of K18-hACE2 mice caused a drastic reduction of infectious virus particles in organ material following an infection with a recombinant SARS-CoV-2 WT-SDelta, but not when challenged with the SARS-CoV-2 SOmicron BA.1 clone.
This dissertation includes numerous, comprehensive experimental studies that are generally important for the characterization of emerging, potentially pre-pandemic viruses and may provide crucial information about the future dominance of certain virus variants in an ongoing pandemic. Here, the need for the use of a variety of animal models becomes apparent. By characterizing and classifying potentially zoonotic strains, these methods will help to better prepare for potentially upcoming pandemics and, in the case of a zoonotic or even pandemic event, to better detect and understand the circulating strains and their evolution.
Until today, more than 100 years after its first description in Italy, the highly pathogenic avian influenza virus (HPAIV) has not lost its fearsome character for wild birds, poultry and humans. On the contrary, the number of outbreaks with high casualty rates in wild birds and poultry has multiplied in recent years and cases of zoonotic infections are also increasingly reported from HPAI endemic areas. The epidemiology of these infections is complex and also involves surface water and possibly sediments of shallow standing waters, which could play a role as a vector medium and/or virus reservoir. The goal of this project was to expand current knowledge of the influence of water on the spread of AIV. As part of this project, we were able to ...
1. ...improve AIV detection methods using real time RT-PCR in terms of sensitivity and breadth of viruses detected. In addition, we succeeded in economizing the procedure so that fewer resources are required and results are obtained faster (publication I: [173]).
2. ...develop an ultrafiltration-based enrichment method for AIV from surface water and evaluate it with field samples from HPAI outbreak areas in wild bird habitats (Wadden Sea coast of Schleswig-Holstein) and previously unaffected regions (Antarctic Weddell Sea) (publication II: [174]). Furthermore, protocols for testing different environmental sample matrices for AIV screening were tested and compared to results of passive monitoring by dabbing diseased or dead wild birds. AIV was detected in more than half (61%) of 44 water samples. We received additional sediment samples from 36 of the 44 water samples. In 18 of 36 of the sediments tested, as well as in 4.16% of 1705 fecal samples tested AIV was detected. However, the studies of the environmental samples mostly yielded only generic AIV detections, with viral loads in the range of the detection limit. This massively hampered further investigations for sub- and pathotyping. In contrast, 79.41% of 68 samples from passive monitoring showed high to very high HPAIV viral loads which also allowed sub- and pathotyping.
3. ...demonstrate in animal experiments that even very low titers (0.1 TCID50 ml-1) of HPAI viral infectivity in water can induce productive infection in susceptible but clinically largely resistant mallard ducks (publication III: [175]). Furthermore, we were able to develop evidence that there is a difference in virus spread that depends on the type of (contaminated) water source. This means that infections on poultry farms with inverted or nipple drinkers may follow a different course than infections in the wild, which are mediated via larger surface waters.
Overall, the results of this project highlight the important role of surface and drinking water, as well as aquatic sediments, in the spread of AIV. The methods developed here for AIV detection extend the possibilities for surveillance of AIV infections; however, passive remains superior to active surveillance of HPAIV infections in several aspects. Examination of various environmental samples did not yield a significant advantage in terms of an early warning system that would indicate the presence or spread of HPAIV in wild bird habitats prior to the occurrence of lethal infections in wild birds.
Lyssaviruses, the causative agents of rabies, are a long-known threat for animals and humans. To date, terrestrial rabies still accounts for tens of thousands of human deaths annually, notwithstanding ambitious vaccination campaigns targeting susceptible dog and wildlife populations that act as reservoirs for the prototypic rabies virus. Moreover, the continuing discovery of newly emerging virus species in hitherto unconcerned chiropteran hosts and geographic regions drive the expansion of the Lyssavirus genus by unveiling its actual variety, host range and distribution.In this work, the genetic diversity of three distinct lyssaviruses, namely EBLV-1, KBLV and RABV, was elucidated by in-depth genomic analyses to provide further insight into lyssavirus evolution. The generation of full-genome sequences from primarily bat-associated Danish EBLV-1 samples significantly increased the number of available Danish EBLV-1 genome sequences while phylogenetic and phylogeographic analysis revealed a stronger phylogeographic structure for the cluster A1 of the sublineage EBLV-1a than it was postulated in previous studies. In addition, the acquisition of a nearly complete genome sequence for the Kotalahti bat lyssavirus provided the basis for the classification of this putative new lyssavirus species as a recognized member of the genus. Furthermore, phylogenetic analysis revealed the affiliation of KBLV to a group of Myotis-associated lyssaviruses giving a deeper insight into the shared evolutionary history of lyssaviruses co-evolving with particular bat species. Moreover, a deep-sequencing approach was utilized to assess the high genetic diversity of vaccine virus populations, uncovering three independent patterns of single nucleotide variants (SNVs) that became selected in ERA-related vaccine-induced cases. However, no apparent influence of the genetic diversity of vaccine viruses on microevolutionary processes like a potential reversion to virulence or a species-specific adaptation of the vaccine virus strains could be detected, leaving the question for the cause of rabies induction in the affected animals unanswered. Lastly, the successful implementation of a hybridization capturing system for the generation of full-genome sequences and deep-sequencing variant analyses of RABV and KBLV samples was demonstrated for a diagnostic bait set, highlighting the versatility and consistency of this approach to assess the genetic spectrum of known and novel lyssavirus species while setting the basis for its application and optimization in upcoming projects.In conclusion, as shown by the studies in this work, the investigation of lyssavirus genomes at the sub-consensus, full-genome and population level remains crucial to assess the complexity of lyssavirus evolution, as it provides an indispensable source of information to cover the diversity of the genus and understand evolutionary dynamics on a long-term and microevolutionary scale.
Technological advances in light microscopy have always gone hand in hand with unprecedented biological insight. For microbiology, light microscopy even played a founding role in the conception of the entire discipline. The ability to observe pathogens that would otherwise evade human observation makes it a critical necessity and an indispensable tool to infectious disease research. Thus, the aim of this thesis was to optimize, extend, and functionally apply advanced light microscopy techniques to elucidate spatio-temporal and spatio-morphological components of bacterial and viral infection in vitro and in vivo.
Pathogens are in a constant arms race with the host’s immune system. By finding ways to circumvent host-mediated immune responses, they try to evade elimination and facilitate their own propagation. The first study (publication I) demonstrated that the obligate intracellular pathogen Coxiella burnetii is not just able to infect natural killer (NK) cells, but is actually capable of surviving the harsh degradative conditions in the cytotoxic lymphocyte’s granules. Using live-cell imaging of reporter-expressing Coxiella burnetii, the transient NK cell passage was closely monitored to provide detailed spatio-temporal information on this dynamic process in support of a range of static analyses. Bacterial release from NK cells was pinpointed to a time frame between 24 to 48 hours post-infection and the duration of release to about 15 minutes.
The second approach (publications II-V) aimed at shedding light on the greater spatio-morphological context of virus infection. Thus far, most studies investigating the distribution or tropism of viruses in vivo have used conventional immunohistochemistry in thin sections. Omitting the native spatial context of the infection site in vivo inherently bears the risk of incomplete description. While the microscopic tools and sample preparation protocols needed for volumetric 3D immunofluorescence imaging have recently been made available, they had not gained a foothold in virus research yet. An integral part of this thesis was concerned with the assessment and optimization of available tissue optical clearing protocols to develop an immunofluorescence-compatible 3D imaging pipeline for the investigation of virus infection inside its intact spatio-morphological environment (publication II). This formed the basis for all subsequent volumetric analyses of virus infection in vivo presented here. Consequently, this thesis provided a valuable proof of concept and blueprints for future virus research on the mesoscopic scale of host-pathogen interactions in vivo (publications II-V), using rabies virus (RABV; publications II-IV) and the newly-emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; publication V) as infection models for the nervous system and the respiratory tract, respectively.
Applying and further improving this volumetric 3D imaging workflow enabled unprecedented insights into the comprehensive in vivo cell tropism of RABV in the central (CNS) (publication III) and peripheral nervous system (PNS) (publication IV). Accordingly, differential infection of CNS-resident astrocytes by pathogenic and lab-attenuated RABV was demonstrated (publication III). While either virus variant showed equal capacity to infect neurons, as demonstrated by quantitative image analysis, only pathogenic field RABVs were able to establish non-abortive infection of astrocytes via the natural intramuscular inoculation route. A combined 3D LSFM-CLSM workflow further identified peripheral Schwann cells as a relevant target cell population of pathogenic RABV in the PNS (publication IV). This suggested that non-abortive infection of central and peripheral neuroglia by pathogenic RABV impairs their immunomodulatory function and thus represents a key step in RABV pathogenesis, which may contribute significantly to the establishment of lethal rabies disease.
Finally, utilizing the full volumetric acquisition power of LSFM, a further refined version of the established 3D imaging pipeline facilitated a detailed mesoscopic investigation of the distribution of SARS-CoV-2 in the respiratory tract of the ferret animal model (publication V). Particularly for this newly-emerged pathogen of global concern, in-depth knowledge of host-pathogen interactions is critical. By preserving the complete spatio-morphological context of virus infection in the ferret respiratory tract, this thesis provided the first specific 3D reconstruction of SARS-CoV-2 infection and the first report of 3D visualization of respiratory virus infection in nasal turbinates altogether. 3D object segmentation of SARS-CoV-2 infection in large tissue volumes identified and emphasized a distinct oligofocal infection pattern in the upper respiratory tract (URT) of ferrets. Furthermore, it corroborated a preferential replication of SARS-CoV-2 in the ferret URT, as only debris-associated virus antigen was detected in the lower respiratory tract of ferrets, thus providing crucial information on the spatial distribution of SARS-CoV-2.
Ebolaviruses are dependent on host cell proteins for almost all steps in their viral life cycle. While some cellular factors with crucial roles in the ebolavirus life cycle have been identified, many of them remain to be identified or fully characterised. This thesis focuses on the characterisation and identification of host cell interactions of the highly pathogenic Ebola virus (EBOV), probing host-virus interaction at various stages of the viral life cycle. Beginning with viral budding, the function of a recently proposed late domain motif within the EBOV matrix protein VP40 was examined using an EBOV transcription and replication-competent virus-like particle (trVLP) system. Although this motif has been suggested to interact with the endosomal sorting complex required for transport (ESCRT), we could show that this late domain motif does not contribute to EBOV budding.
While many host cell proteins have been identified so far that are important for viral budding, only a few proteins are known that are necessary for EBOV RNA synthesis. Thus, to identify host proteins that are involved in viral replication and transcription, we performed a genome-wide siRNA screen in the context of an EBOV minigenome assay. Using this approach, we identified several proteins that appear to be important for viral RNA synthesis or protein expression. Two of the most prominent hits in our screen were CAD (Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase) and NXF1 (nuclear RNA export factor 1). CAD catalyses the first three steps in the de novo pyrimidine biosynthesis, while NXF1 is the main nuclear export protein for cellular mRNAs. In subsequent characterisation studies, using a range of life cycle modelling systems as well as molecular analyses, we could demonstrate that the canonical function of CAD during the pyrimidine biosynthesis is necessary for EBOV replication and transcription. In contrast to this, for NXF1 we discovered a so-far unknown function: Again, by applying different life cycle modelling alongside with molecular assays, we provided evidence that the EBOV nucleoprotein recruits NXF1 into inclusion bodies, the site of EBOV RNA synthesis, where it binds viral mRNAs to export them from these structures. Importantly, for both CAD and NXF1 we were able to recapitulate key data in the context of live EBOV infection, confirming their roles in the viral life cycle.
Both of these identified host factors are promising targets for antiviral therapies and indeed de novo pyrimidine synthesis is emerging as a possible antiviral target for a number of viruses. Similarly, as we could show NXF1 to be important in the life cycle of the highly pathogenic Junín virus, this raises the possibility that disruption of this interaction may result in broad-spectrum antiviral activity. Moreover, for an increasing number of negative-sense RNA viruses inclusion bodies as site of viral RNA synthesis are described to have a liquid organelle character. Therefore, our findings on NXF1 also provide an intriguing model to explain how negative-sense RNA viruses in general overcome this obstacle and export viral mRNAs from inclusion bodies.
The advances in high-throughput sequencing technologies have revolutionized the possibilities for pathogen identification in cases of unknown disease origin. Diagnostic metagenomics allows the unbiased and simultaneous detection of almost all nucleic acids in a clinical sample, with the potential to provide pivotal insights into otherwise undeterminable causes of human or animal disease.
In this thesis, possibilities, pitfalls and the suitability of Ion Torrent and Illumina sequencing platforms for comprehensive use in diagnostic metagenomics were assessed and optimized procedures developed. Clinical field samples, undiagnosable by standard diagnostics, were taken as real-life examples for the investigations. The results show that cross-contamination due to index swapping and run-to-run-carryover constitute a major issue on Illumina platforms, severely compromising the correct interpretation of results for clinical specimens. In contrast, Ion Torrent platforms did not display any form of cross-contamination, however, the commercial library preparation method is less efficient. Combining the advantages of both platforms, customized Y adapters, facilitating highly efficient library preparation, were developed for Ion Torrent sequencing and applied in further experiments. The obstacles of strongly degraded RNA in formalin-fixed paraffin-embedded samples were identified and the workflow adapted to meet the requirements of smaller fragments. Additionally, it was shown that adequate sampling is a very important step, if not the most important step, in the workflow, as well as subsequent validation of the obtained results in terms of causation. The achievements in this study allow other researchers the application of a sensitive and optimized diagnostic metagenomics workflow.
Furthermore, the investigations on the clinical samples resulted in the discovery of a novel respirovirus with putative zoonotic potential, the first description of Borna disease virus 1 in human organ transplant recipients, and the discovery of a very distantly related novel ovine picornavirus. These discoveries build a basis for further research and expand the knowledge regarding new and emerging viruses.
The highly oncogenic alphaherpesvirus Marek’s disease virus (MDV) causes immense economic losses in the poultry industry. The main targets of in vivo MDV infection are primary B and T lymphocytes. The cytolytic infection of B cells leads to depletion of lymphoid cells results in severe immunosuppression. Infected B cells recruit and activate T cells. The close interaction between B cells and T cells enables efficient intercellular transfer of MDV. During infection of T cells, the virus enters a latent state. Infection of T cells can lead to transformation of these cells and formation of lymphoma, which manifest in various visceral organs. This study aimed at the characterization of the proteomes of MDV-infected lymphocytes during the lytic and latent phases of infection.
Previous in vitro studies concerning the MDV pathogenesis and host-virus interactions have been mainly conducted with primary fibroblasts or kidney cells, due to the short lifespan of primary lymphocytes in cell culture. Recently, a cultivation system has been established that extents the lifespan of primary lymphocytes through the addition of cytokines to the growth medium. This allowed the infection of B cells in vitro and to conduct quantitative proteomic analysis of primary lymphocytes. Infection with GFP labelled virus recombinants allowed the isolation of infected cells by FACS for the proteome analysis of MDV infected B lymphocytes. An efficient quantitative proteomic workflow was developed, which consisted of a filter-aided (FASP) digest of the extracted proteins, followed by differential dimethyl chemical labeling of the peptides for quantitative evaluation prior to LC-MALDI TOF/TOF mass spectrometry. Only few alterations of the protein and transcript expression profiles were observed after infection of primary B cells with the very virulent RB-1B and the live-attenuated vaccine strain CVI988/Rispens. Relevant changes in relative protein levels were found for only twelve and six interesting host proteins after RB1B and CVI988 infection, respectively. However, the regulations were confirmed by inspection of the spectra from all experiments. The identified candidates play a role in immune response, translation and inflammatory response.
To confirm the potential infection markers, RNA-seq analysis of three biological replicates of each RB-1B -, CVI988- and mock-infected B cells was performed. Eighty expressed MDV transcripts could be identified, which were associated with lytic infection. The same MDV proteins were identified after infection with RB-1B or CVI988. However, transcriptome and proteome analysis of MDV-infected primary B cells showed only poor correlation. This indicates that the changes in protein expression profiles are mostly due to posttranscriptional events. Infection marker candidates were identified by the RNA-seq analysis, for which the gene expression was altered by MDV infection. Although almost 12,000 transcripts were identified, only few transcript levels changed markedly after MDV infection. The biological processes immune response, apoptotic process, signal transduction, cell migration and response to virus were enriched after MDV infection. The RNA-seq results confirm the observation that alterations of protein levels early after MDV infection are rare.
Most notably, MDV induces transformation of lymphocytes leading to malignant T-cell lymphomas in visceral organs with mortalities of up to 100 %. While several factors involved in MDV tumorigenesis have been identified, the transformation process is not fully understood. Therefore, we set out to fill this knowledge gap using proteome analysis of transformed T-cells ex vivo. In addition, the role of the viral telomerase RNA during transformation was assessed by comparison of tumors that had formed after infection with WT-virus or a telomerase RNA negative mutant. A major obstacle for tumor proteome analyses is the preparation of sufficient amounts of homogenous tumor tissue, as tumors appear with a dispersed morphology in the affected organs. The quantitation of cell types within the tumors indicated varying portions of hepatocytes, connective tissue, and CD3+ lymphocytes even with the same virus strain in different animals. However, the ∆vTR-induced tumors contained lower levels of hepatocytes and higher levels of CD3+ lymphocytes compared to WT tumors in all tested tumor samples. Thus, ∆vTR tumors were chosen for determination of differences in protein expression profiles of tumors and naïve T cells for their lower content of liver cells. We developed a workflow for the proteome analysis of T cell tumors from livers of MDV-infected chickens. Samples included laser capture micro-dissected tissue cuts from tumors and surrounding healthy liver tissue as well as naïve T-cells prepared from thymus. To enable quantitative proteome analysis, samples were digested using the FASP protocol and peptides were isotope-coded by differential dimethyl labeling. To improve proteome analysis peptides were fractionated by preparative isoelectric focusing prior to nano-HPLC MALDI/TOF-TOF mass- spectrometric analysis.
Proteomic analyses of LCM dissected ΔvTR tumor compared to naïve T cells, the main targets of transformation, identified nineteen potential transformation markers but again only minor changes in relative levels were observed. Several of the identified markers could also be verified by RT-qPCR on transcript level. The identified transformation candidates were associated with nucleosome assembly, regulation of transcription, inflammatory response, immune response and oxidation-reduction process.
However, further functional analyses are necessary to fully elucidate the role of the identified markers during MDV infection and transformation.
The virosphere comprises all known and unknown viruses in our ecosystems. Advanced sequencing technologies in combination with metagenomic analysis have become a key tool for exploring this global diversity of viruses. However, discovery of novel viruses and comparative analyses are often based on small sequence fragments or lack biological context, which restricts a proper classification. In this study advanced genomic methods were used that included comprehensive knowledge of viral genomes along with supporting biological metadata in order to identify and classify viruses at different levels of genetic relationships. In a first example, the genetic background of vaccine-induced rabies cases was revealed by analyzing and comparing the genetic diversity of viral populations. Furthermore, the fundament for a taxonomic reclassification of orthopoxviruses was established on basis of a wide scale genomic analysis. In addition, novel neurotropic mamastroviruses from sheep and cattle were classified as members of a single species that provided evidence of interspecies transmission. Finally, two putative novel species of alphaherpesviruses and orthopoxviruses were identified. These examples are based on field cases that provide substantial corresponding clinical metadata and information of host-pathogen interactions. The analyses, therefore, puts taxonomic classification into biological and epidemiological context, rather than addressing generic phylogenetic relationships. Furthermore, the presented work demonstrates that a universal approach for virus classification is neither feasible nor reasonable as analyses must be adjusted the nature of the addressed virus. All results with impact on the current taxonomic classification will be or are already reported to the International Committee on Taxonomy of Viruses. In conclusion, this thesis contributed to the classification concepts of viruses and expanded the knowledge of virosphere diversity.
Next Generation Sequencing (NGS)-technologies developed very fast in recent years and is used widely in current research areas. The aim of this study was to use NGS (i) for the identification of pathogens in outbreaks and (ii) for the identification of virulence-relevant sequencepolymorphisms when comparing whole genome sequences. Therefore, a previous developed workflow was used to identify a new virus of the family Bornaviridae. The generation of whole genome sequences elucidated the molecular epidemiological connection of infection of variegated squirrels (Sciurus variegatoides) and three human cases of fatal encephalitis. By generating the whole genome sequence of a Porcine Epidemic Diarrhea Virus (PEDV) in Germany it was possible to find difference compared to circulating high virulent strains in the USA. This led to potential virulence marker to distinguish strain in the USA and Germany. Connections between sequence variation and virulence were further investigated for the bovine viral diarrhea virus 2c (BVDV-2c), cowpox viruses (CPXV) and classical swine fever virus (CSFV). Here, for a highly virulent BVDV-2c strain a mixture of different genome structure variants could be found. The majority of these genomes harbors a duplication within the p7/NS2 coding region and might cause a high virulence. For CPXV virus isolated of different hosts were analyzed and a correlation between genome sequence and the A-type inclusion body phenotype could be found. Furthermore, several deletion/insertion events were detected which might influence the virulence of these strains. Finally, the virus population of CSFV strains in pigs was characterized. However, the population of the inoculum as well as of acute-lethal and chronically infected animals gave no indication that the virus itself causes the different types of disease outcome. In conclusion, this thesis shows the great potential of NGS for virus identification and characterization. Furthermore, it makes the identification of potential virulence marker possible which subsequently can be analyzed by reverse genetics.