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In temperate regions, climate warming alters temperature and precipitation regimes. During winter, a decline in insulating snow cover changes the soil environment, where especially frost exposure can have severe implications for soil microorganisms and subsequently for soil nutrient dynamics. Here, we investigated winter climate change responses in European beech forests soil microbiome. Nine study sites with each three treatments (snow exclusion, insolation, and ambient) were investigated. Long-term adaptation to average climate was explored by comparing across sites. Triplicated treatment plots were used to evaluate short-term (one single winter) responses. Community profiles of bacteria, archaea and fungi were created using amplicon sequencing. Correlations between the microbiome, vegetation and soil physicochemical properties were found. We identify core members of the forest-microbiome and link them to key processes, for example, mycorrhizal symbiont and specialized beech wood degraders (fungi) and nitrogen cycling (bacteria, archaea). For bacteria, the shift of the microbiome composition due to short-term soil temperature manipulations in winter was similar to the community differences observed between long-term relatively cold to warm conditions. The results suggest a strong link between the changes in the microbiomes and changes in environmental processes, for example, nitrogen dynamics, driven by variations in winter climate.
Background: Methanogenic archaea represent a less investigated and likely underestimated part of the intestinal tract microbiome in swine.
Aims/Methods: This study aims to elucidate the archaeome structure and function in the porcine intestinal tract of healthy and H1N1 infected swine. We performed multi-omics analysis consisting of 16S rRNA gene profiling, metatranscriptomics and metaproteomics.
Results and discussion: We observed a significant increase from 0.48 to 4.50% of archaea in the intestinal tract microbiome along the ileum and colon, dominated by genera Methanobrevibacter and Methanosphaera. Furthermore, in feces of naïve and H1N1 infected swine, we observed significant but minor differences in the occurrence of archaeal phylotypes over the course of an infection experiment. Metatranscriptomic analysis of archaeal mRNAs revealed the major methanogenesis pathways of Methanobrevibacter and Methanosphaera to be hydrogenotrophic and methyl-reducing, respectively. Metaproteomics of archaeal peptides indicated some effects of the H1N1 infection on central metabolism of the gut archaea.
Conclusions/Take home message: Finally, this study provides the first multi-omics analysis and high-resolution insights into the structure and function of the porcine intestinal tract archaeome during a non-lethal Influenza A virus infection of the respiratory tract, demonstrating significant alterations in archaeal community composition and central metabolic functions.
Hepatitis E virus (HEV) is emerging worldwide as a zoonotic pathogen that has remained largely undetected for decades, if not centuries. Its enormous success can be attributed to the wide range of host species, which can transmit the virus to humans, depending on the viral genotype. As a result, HEV is likely to remain a challenge even when the remaining hepatitis viruses (HAV, HBV, HCV), which are transmitted exclusively between humans, are under control. Although millions of HEV infections occur each year, little is known about this puzzling pathogen. One major issue in HEV research is the lack of reliable model systems. Established animal models are inefficient, expensive, or simply not representative of human HEV. On the other hand, cell culture systems are limited by the slow growth of the virus and inefficient replication and infection. The aim of this work is to with deepen the understanding of zoonotic HEV in animal hosts in Germany. For this purpose, a molecular and phylogenetic characterization of HEV sequences from rabbits and swine was conducted. A novel subtype of the zoonotic genotype HEV-3 was identified in a rabbit sample, further emphasizing the role of rabbits as HEV host species and possible reservoir of zoonotic HEV infections in Germany. On the other hand, a molecular biological screening of pigs and wild boars in Mecklenburg-Western Pomerania indicates a wide range of HEV-3 subtypes circulating in swine in north-east Germany. Furthermore, an optimized replicon system was established in order to enable characterization of various HEV sequences by reverse genetics. As a proof of concept, two rabbit HEV derived replicons were compared with two established, cell culture adapted HEV strains. The influence of different regions of the nonstructural protein on HEV replication was determined and quantified. In particular, a system was established, to reproducibly compare different strains and genotypes. This refined replicon system will enable the characterization of further HEV sequences and thus expand the knowledge on the determinants of the viral life cycle.
Mixotrophic and heterotrophic protists hold a key position in aquatic microbial food webs. Whereas they can account for the bulk of bacterivory in pelagic systems, the potential structuring effect of these consumers on bacterial communities is far from clear. We conducted short-term grazing experiments to test for the overall impact on bacterial community structure and possible prey preferences of phagotrophic protists. The protist taxa selected for this study include three mixotrophic flagellates, comprising two obligate- and one facultative mixotroph, and one phagoheterotrophic flagellate lacking phototrophic capacity. Bacterioplankton from seven different lakes were enriched and used to represent semi-natural prey communities. Our study demonstrated protist strain specific impacts on bacterial community composition linked to grazing. The three mixotrophs had variable impacts on bacterial communities where the two obligate mixotrophs exhibited lower grazing rates, while showing a tendency to promote higher bacterial diversity. The phagoheterotroph displayed the highest grazing rates and structured the bacterial communities via apparent selective grazing. Consistent selectivity trends were observed throughout the experiments, such as the apparent avoidance of all flagellates of Actinobacteria, and high grazing on dominant Burkholderiales taxa. However, there was no consistent “fingerprint” of mixotrophic grazing on prey communities, but the structuring impact rather seemed to depend on the trophic mode of the individual protist taxa, i.e. their dependence on phototrophy vs. phagotrophy. Our findings highlight the differential structuring impact of protist taxa on bacterial communities which may have important ecological implications, for example during periodic dominance of obligate mixotrophic bacterivores in changing lake ecosystems.
Mass spectrometry-based Proteome analysis of porcine cells infected with African swine fever virus
(2023)
ASFV, a highly contagious, pathogenic and lethal pathogen of swine, poses a major threat to domestic and wild suids worldwide as neither vaccines nor treatments are available. Compared to other well-characterized similarly complex viruses like herpesviruses or adenoviruses, the understanding of ASFV biology is poor.
To improve the understanding of ASFV biology, following the establishment of a robust protocol for the isolation of primary monocyte-derived porcine macrophages (moMΦ) and their infection with ASFV for mass spectrometry (MS)-based proteome analysis was performed.
Under both conditions, naïve and infected, the isolated cells showed cell type-specific characteristics like phagocytosis and antigen presentation and protein expression patterns, including the expression of swine leucocyte antigens and CD markers. Furthermore, moMΦ could be reproducibly infected with ASFV isolates of different genotypes and pathogenicity.
The ASFV protein expression patterns in moMΦ correlate well with those observed in established cell lines at transcript and protein level. The expression of 27 ASFV proteins was confirmed at the protein level. Among them, 9 members of multi-gene families (MGF) and 12 novel open reading frames (nORFs) were recently predicted based on transcription start site mapping.
The direct comparison of closely related ASFV genotype II isolates revealed no virulence-associated protein expression patterns beyond those expected based on the genome sequences of the isolates.
Using different MS quantification strategies, it was shown that ASFV affects both static protein expression levels and protein synthesis. These changes in protein expression impact proteins and pathways known to be targeted by ASFV, including CD-markers, ER-stress and cell death pathways, and cellular antiviral responses. Beyond these observations that further validated the moMΦ infection model, novel effects of the ASFV infection on the cellular proteome were noticed.
These effects include the decreased expression levels of cathepsins, especially cathepsins D (CTSD), H (CTSH) and L (CTSL) as well as the transient activation of MAPK14/p38 prior to its strong downregulation. In addition to MAPK14/p38 further members of the MAPK14/p38 signaling pathway, like MAPKAPK2, were affected by ASFV infection.
As these modulations of the cellular proteome would in general result in decreased pro-inflammatory responses, it did stand out that the synthesis of interferon-response related genes including MX1 and ISG15 evaded the ASFV-induced global reduction of protein synthesis. In contrast, the synthesis of genes involved in RNA processing and splicing was significantly impaired. In total, the regulations of individual host proteins assessed in the context of the whole cellular proteome integrate well with each other and other cellular responses to ASFV infection and may help to improve the understanding of host-virus interactions.
Overall, this thesis provides novel insights into the expression of ASFV-encoded ORFs of different isolates and the host response to ASFV infection. It points out that the current knowledge of the ASFV coding capacity, temporal protein expression patterns, protein functionality, post-translational modifications and host interactions is still sketchy as many aspects of ASFV replication have yet to be understood. The established moMΦ-model to study ASFV infections in vitro provides a powerful tool for future applications to increase the understanding of ASFV biology.
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.
The role of cell-penetrating peptides in the induction of T cell responses by virus-like particles
(2023)
Many viral structural proteins can self-assemble into virus-like particles (VLPs). VLPs can serve as an effective vaccine or be used as a vaccine platform. One of these structural proteins is the hepatitis B virus core antigen (HBcAg), which appears to be suitable as an antigen carrier due to its high immunogenicity. HBcAg has a major immunodominant region (MIR) that is presented on the surface of the VLPs after self-assembly. Foreign antigens can be inserted into this region. Since HBcAg VLPs, unlike the Hepatitis B virus (HBV), do not have an envelope, they are not able to penetrate cell membranes efficiently. As an extracellular antigen, HBcAg VLPs primarily induce a strong humoral immune response.
In the present study, we investigated the extent to which HBcAg can be modified to also elicit an enhanced cellular, particularly a cytotoxic, immune response. A cytotoxic CD8+ T cell response is predominantly induced by intracellular antigens. Therefore, our goal was to increase the cell penetration capacity of VLPs. We aimed to achieve this by fusing cell-penetrating peptides (CPPs) to HBcAg. CPPs can spontaneously penetrate cell membranes to enter the cytoplasm of cells. To guarantee that the CCPs were localized to the surface of the VLPs, we fused CPPs to the N-terminus of HBcAg. The CCPs were followed by a tag to allow the purification of VLPs. The T cell epitopes, against which the induced CTL should be directed, were derived from the Large T antigen and inserted into the MIR of HBcAg. Finally, we fused fluorescent proteins to the C-terminus of HBcAg to track the entry of VLPs into cells.
Modifications of HBcAg may lead to reduced stability or altered structure of VLPs. To analyze the stability of VLPs, we used nanoscale differential scanning fluorimetry (nanoDSF) analysis. This revealed that the N-terminal fusion of CPPs or the tag to HBcAg does not reduce VLP stability. However, some peptides incorporated into the MIR had a significant effect on the structure and stability of the VLPs. While the incorporation of a Flag-tag or a peptide from ovalbumin had no negative effect on VLP stability, the incorporation of peptides representing T cell epitopes of Large T antigen interfered with VLP formation. Denaturation and reassembly of the aggregates significantly improved the homogeneity of the VLPs, and the C-terminal addition of arginine-rich domains enhanced stability.
Using live cell imaging and flow cytometry, we demonstrated that HBcAg VLPs functionalized with CPP exhibited up to 40% more efficient penetration into professional antigen-presenting cells (JAWS II) than HBcAg VLPs without CPP. This resulted in the increased presentation of integrated T cell epitopes by dendritic cells. In vivo, we detected significantly increased induction of SV40 Large T antigen-specific CTL in mice immunized with CPP-conjugated VLPs compared to unconjugated VLPs.
In this study, we demonstrated that a stronger cellular immune response can be induced by CPP-functionalized HBcAg VLPs than with the unmodified HBcAg VLPs in vitro as well as in vivo. This discovery may have positive implications for future vaccine development where an enhanced cellular component of the immune response is desirable.
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.
Posttranslational modifications are involved in the regulation of virtually all cellular processes, including immune response, nevertheless, they are also targets manipulated by invading pathogens. The first investigated example is protein citrullination which is an important posttranslational modification that acts on a multitude of processes like supervision of cell pluripotency and rheumatoid arthritis. Citrullination of targeted arginine residues is performed by the Peptidylarginine deiminase. Within the first published manuscript, being part of this thesis, it was possible to show the use of this posttranslational modification by the human pathogen Porphyromonas gingivalis to facilitate innate immune evasion at three distinct level. P. gingivalis was demonstrated to citrullinate proteins by Porphyromonas peptidylarginine deiminase resulting in diminished phagocytosis and subsequent killing by neutrophils. Furthermore, it was shown that citrullination of histone H3 enables P. gingivalis to survive in neutrophil extracellular traps and incapacitate the lysozyme-derived peptide LP9.
The second investigated posttranslational modification is ubiquitination and its role in respiratory tract infections. Ubiquitination is the covalent attachment of a small protein that consisting of only 76 amino acids to the ε-amino group of lysine residues to posttranslational modify proteins. Acute infections of the lower respiratory tract such as viral and bacterial co-infections are among the most prevalent reasons of fatal casualties worldwide. Therefore, the interactions between host and pathogens resulting in the impairment of the hosts immune response and immune evasion of the pathogens, need to be elucidated. To get new insights in the infection driven changes in protein polyubiquitination and alterations in the abundance of ubiquitin E3 ligases involved in ubiquitination, cellular proteomes were monitored in detail by high resolution mass spectrometry. Therefore, the epithelial cell lines 16HBE14o- (Manuscript II) and A549 (Manuscript III) were co-infected with influenza A virus H1N1 and Streptococcus pyogenes or Staphylococcus aureus or with influenza A virus H1N1 and Streptococcus pneumoniae, respectively. Here, it could be shown in 16HBE14o- cells that co-infection of epithelial cells is not characterized by decreased cell survival and that observable effects on the proteome and ubiquitinome are mostly additive rather than synergistic. S. pyogenes infection affected the mitochondrial function, cell-cell adhesion, endocytosis and actin organization. Viral infection affected mRNA processing and Rho signaling. Viral and bacterial co-infection was detected to affect processes that were already affected by both of the corresponding single infections. No further pathways were strongly affected by the co-infection. A similar result has been observed in A549 cells co-infected IAV and S. pneumoniae. Overrepresented gene ontology terms depict the sum of those observed in the viral and bacterial single infection. Moreover, no significant change in cell survival upon co-infection compared to single bacterial infection was noticed for A549 cells either. This led to the suggestion that co-infection of investigated epithelial cells under examined conditions possesses additive rather than synergistic effect and thus, may not worsen the outcome of the infection within the studied conditions. Infections in other systems, may provide varying results and thus should be examined in future studies.
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.