Biology subjects

Schusser, B.

Publications and source records attributed to Schusser, B..

13 recordsLinked to original sources

bioRxiv2026

Opposing roles of ACE2 and TMPRSS2 in IBV Beaudette infection

Infectious bronchitis virus (IBV) is an economically important avian gammacoronavirus. IBV uses sialic acids and heparan sulfate for cellular attachment, but the host proteins governing its infection remain poorly defined. Here, we investigated the roles of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) during IBV Beaudette (IBV BD) infection. ACE2 knockout impaired IBV replication in DF-1 cells, whereas ACE2 reconstitution restored infection. Furthermore, ACE2 overexpression enhanced infection of DF-1 and primary chicken embryo kidney cells, supporting the role of chicken ACE2 as a proviral host factor. In contrast, TMPRSS2 exerted a restrictive function on IBV BD infection as demonstrated in TMPRSS2 KO DF-1 cells and upon pharmacological inhibition, which significantly increased IBV BD infection in DF-1 and Vero cells. RNA sequencing revealed significant changes in lipid metabolism and membrane-related processes following TMPRSS2 knockout and pharmacological inhibition. These findings highlight the opposing roles of ACE2 and TMPRSS2 in IBV BD infection, emphasizing the combined impact of proviral host factors, protease activity, and the cellular membrane environment during gammacoronavirus infections.

microbiology↗

bioRxiv2026

Virus-Like Particles: The Next Frontier in Livestock Gene Editing

Pigs and chickens are not only the most important livestock species for global food production but also serve as key model organisms in various research disciplines. The pig is widely used in translational research due to its anatomical and physiological similarity to humans, providing valuable insights into immunology, metabolism, and disease mechanisms. In contrast, the chicken has become an essential model for studies related to poultry health, animal welfare, and developmental biology. Its externally developing embryo offers exceptional accessibility for experimental manipulation. Recent advances in genome editing technologies, particularly CRISPR/Cas9, have further expanded the potential of these species for functional genomic studies, although the efficient delivery of such tools remains a major challenge. By using virus-like particles (VLPs), we have been able to overcome this limitation. Here, we evaluated VLPs as delivery vehicles for genome engineering tools in pigs and chickens, two key livestock species at the human-animal interface. VLP-mediated delivery enabled efficient Cre recombination and high CRISPR/Cas9 editing rates in porcine cells, organoids, and oocytes, particularly when multiplexed. In chickens, VLPs supported robust Cre recombination and Cas9-mediated editing in cell culture, tracheal organ cultures, and in ovo. Reporter VLPs and dCas9 VLPs further demonstrated the versatility of this platform across porcine and avian systems. Together, these findings establish VLPs as an efficient and time-saving strategy for gene editing in livestock, with relevance for animal health, agricultural productivity, and translational One Health research.

genetics↗

bioRxiv2025

Unraveling new characteristics of γδT cells using scRNA-seq in TCR KO chicken

The characterization of T cells in chickens has proven to be challenging, primarily due to the lack of specific markers to differentiate classical Th1, Th2, and Th17 subsets. Furthermore, chickens possess a notably high proportion of {gamma}{delta} T cells, making them a unique model for investigating the poorly understood role of these cells not only in chickens but also in mammals. To gain deeper insights into the functions and characteristics of the {beta} and {gamma}{delta} T cell subsets in chickens, whole transcriptome analysis (WTA) on CD3+ single cells isolated from wild type (WT), TCR C{beta} knockout (KO) and TCR C{gamma} KO chickens at embryonic day (ED) 18, day (d) 3, and d14 post-hatch was conducted. The results indicate that {gamma}{delta} T cells exhibit cytolytic activity in both TCR C{beta} KO and WT chickens. A distinct cluster of {gamma}{delta} T cells expressing genes associated with the interferon pathway was revealed in TCR C{beta} KO chickens, which may contribute to the severe phenotype in these chickens, which is characterized by severe inflammation of spleen, gut and stomach. Additionally, novel gene markers were identified that precisely define the {beta} and {gamma}{delta} T cell subsets. These findings provide new insights into chicken T cell biology and contribute to a better understanding of the mechanisms underlying the severe phenotype observed in TCR C{beta} KO chickens.

immunology↗

bioRxiv2025

Compensatory Mechanisms in γδ T Cell-Deficient Chickens Following Salmonella infection

Avian {gamma}{delta} T lymphocytes are highly abundant in the intestinal mucosa and play a critical role in immune defense against infectious diseases in chickens. However, their specific contributions to infection control remain poorly understood. To investigate the role of {gamma}{delta} T cells and their possible compensation, we studied wild-type and {gamma}{delta} T cell knockout chickens following infection with Salmonella Enteritidis. Bacterial loads in the liver, cecal content, and cecal wall were quantified. Immune cell populations in blood, spleen, and cecum were analyzed using flow cytometry. Immune gene transcription in sorted T cell subsets and cecal tissue was measured by RT-qPCR. Strikingly, chickens lacking {gamma}{delta} T cells had significantly higher bacterial loads in the liver and more extensive Salmonella invasion in the cecal wall during the early stages of infection compared to wild-type birds. In the blood, infected {gamma}{delta} T cell knockout chickens displayed a significantly increased percentage of CD25+ NK-like cells. In both blood and tissue, infected wild-type chickens demonstrated an increased absolute number of CD8++ {gamma}{delta} T cells. Conversely, {gamma}{delta} T cell knockout chickens exhibited an augmented cell count of a CD8++CD4- non-{gamma}{delta} T cell population after infection, which might include {beta} T cells. At 7 days post infection (dpi), gene expression analysis revealed elevated transcription of the activation marker IL-2R and proinflammatory cytokines (IL-17A, IFN-{gamma}) in CD8++CD4- non-{gamma}{delta} T cells from {gamma}{delta} T cell knockout chickens compared to CD8++ {gamma}{delta} T cells from wild-type birds. By 12 dpi, these differences diminished as transcription levels increased in {gamma}{delta} T cells of wild-type animals. Our findings demonstrate that {gamma}{delta} T cells play a role in early immune protection against Salmonella Enteritidis infection in chickens. In later stages of the infection, the {gamma}{delta} T cells and their functions appear to be replaced by other cells.

immunology↗

bioRxiv2025

Type I and type III interferon receptor knockout chickens: Novel models for unraveling interferon dynamics in influenza infection

The rapid cross-species transmission of highly pathogenic avian influenza presents a significant zoonotic threat. Elucidating the avian interferon (IFN) system, the primary antiviral defense in chickens, is critical for controlling the virus at its source and preventing its spillover into humans and other species. We engineered type I (IFN-/{beta}) and type III (IFN-{lambda}) IFN receptor knockout chickens to dissect the role of IFNs in viral infections. Results revealed that type I IFN predominantly modulates innate immune cell populations, T cell subsets, and their contribution to antibody production following immunization under physiological conditions. In ovo and in vivo challenge experiments utilizing diverse influenza A virus strains demonstrated strain-specific roles of both IFN-/{beta} and IFN-{lambda} in orchestrating viral pathogenesis, immunological responses, and tissue-tropism effects. Notably, type I IFN was particularly crucial in the initial defense mechanisms against H3N1 avian influenza A virus infection. These novel models offer unprecedented insights into avian IFN biology within the context of avian influenza, which is essential for developing more effective strategies to prevent and control this public health challenge.

immunology↗

bioRxiv2024

Regulation of B cell migration to the Bursa of Fabricius by CCR7 and cell adhesion molecules in chicken embryonic development

The development of functional B lymphocytes during chicken embryogenesis relies on a series of tightly regulated processes. Precursor B cells migrate from the spleen via the blood to the bursa of Fabricius, where they colonize the bursal follicles to undergo further maturation and differentiation. To better understand the molecular mechanisms underlying early B cell migration in the chicken embryo, transcriptome analysis of B cells isolated from the spleen, blood, and bursa at embryonic days (ED) 12, ED14, and ED16 was performed. These findings suggest that sphingosine-1-phosphate (S1P) and its receptors regulate B cell presence in the bloodstream, while CCR7 and CXCR4 guide B cells to the bursa. Additionally, integrins and cell adhesion molecules, such as PECAM1, appear to facilitate transendothelial migration into the bursal mesenchyme. This study highlights a coordinated interplay between chemokines, integrins and cell adhesion molecules involved in B cell recruitment and colonization of the bursa microenvironment. These findings enhance our understanding of early B cell migration and shed light on the mechanisms governing B cell trafficking during chicken embryonic development.

immunology↗

bioRxiv2023

Unraveling the role of γδ T cells in the pathogenesis of an oncogenic avian herpesvirus

Mareks disease virus (MDV) is an oncogenic alphaherpesvirus that causes deadly T cell lymphomas in chickens. MDV is highly cell associated which allows the virus to evade antibody-mediated virus neutralization. Therefore, T cell-mediated immune responses are thought to be crucial for combating this deadly pathogen. In chickens, gamma delta ({gamma}{delta}) T cells represent a major population with up to 50% of all peripheral T cells. However, their role in MDV pathogenesis and tumor formation remains poorly understood. To investigate the role of {gamma}{delta} T cells in MDV pathogenesis, we infected genetically modified chickens that lack {gamma}{delta} T cells (TCR C{gamma}-/-) with very virulent MDV. Strikingly, disease and tumor incidence were highly increased in the absence of {gamma}{delta} T cells, indicating that {gamma}{delta} T cells play an important role in the immune response against MDV. In the absence of {gamma}{delta} T cells, virus replication was increased by up to 89-fold in the thymus and spleen, both potential sites of T cell transformation. Taken together, our data provide the first evidence that {gamma}{delta} T cells play an important role in restricting MDV replication, pathogenesis and tumors caused by this deadly pathogen. Author Summary{gamma}{delta} T cells are the most abundant T cells in chickens, but their role in fighting pathogens remains poorly understood. Mareks disease virus (MDV) is an important veterinary pathogen, causes one of the most frequent cancers in animals and is used as a model for virus-induced tumor formation. Our study revealed that {gamma}{delta} T cells play a crucial role in combating MDV, as disease and tumor incidence was drastically increased in the absence of these cells. {gamma}{delta} T cells restricted virus replication in the key lymphoid organs, thereby decreasing the likelihood of causing tumors and disease. This study provides novel insights into the role of {gamma}{delta} T cells in the pathogenesis of this highly oncogenic virus.

microbiology↗

bioRxiv2023

Reinstatement of RIG-I in chickens via genetic modification reveals new insights into the dynamic evolution of avian immune sensors

Retinoic acid-inducible gene I (RIG-I) activates mitochondrial antiviral signaling proteins, initiating the antiviral response. RIG-I and RNF135, a ubiquitin ligase regulator, are missing in domestic chickens but conserved in mallard ducks. The chickens RIG-I loss was long believed to be linked to increased avian influenza susceptibility. We reinstated both genes in chickens and examined their susceptibility to infection with an H7N1 avian influenza virus. Uninfected RIG-I-expressing chickens exhibited shifts in T and B cells. At the same time, the H7N1 infection led to severe disease, persistent weight loss, and increased viral replication compared to wild-type chickens. The simultaneous expression of RIG-I and RNF135 potentiated the RIG-I activity and was associated with exacerbated inflammatory response and increased mortality without influencing virus replication. Additional animal infection experiments with two other avian influenza viruses validated these findings. They confirmed that the harmful effects triggered by RIG-I or RIG-I-RNF135-expression require a minimum degree of viral virulence. Our data indicate that the loss of RIG-I in chickens has likely evolved to counteract deleterious inflammation caused by viral infection and highlight an outcome of restoring evolutionary lost genes in birds. Significance StatementThe evolutionary loss of a crucial innate immune sensor like RIG-I in domestic chickens and its presence in closely related avian species such as ducks has long puzzled researchers. We genetically reinstated RIG-I in chickens, alongside its ubiquitination factor, RNF135, to uncover their roles in responding to influenza virus interactions in chickens. Our findings suggest that the loss of RIG-I in chickens may have occurred as an adaptive strategy to mitigate harmful inflammation associated with influenza infection. We shed light on an outcome of reinstating evolutionarily lost genes in birds and open new avenues for understanding immune responses in vertebrates.

immunology↗

bioRxiv2023

Nociception in chicken embryos, Part III: Analysis of movements before and after application of a noxious stimulus

Many potentially noxious interventions are performed on chicken embryos in research and in the poultry industry. It is therefore in the interest of animal welfare to define the point at which a chicken embryo is capable of nociception. The present part III of a comprehensive study examined the movements of developing chicken embryos with the aim of identifying behavioral responses to aww noxious stimulus. For this purpose, a noxious mechanical stimulus and a control stimulus were applied. The recorded movements of the embryos were evaluated using the markerless pose estimation software DeepLabCut and manual observations. After the application of the noxious stimulus, a significant increase in beak movement was identified in 15- to 18-day-old embryos. In younger embryos, no behavioral changes related to the noxious stimulus were observed. The results indicate that noxious stimuli at the beak base evoke a nocifensive reaction in chicken embryos starting at embryonic day 15.

animal behavior and cognition↗

bioRxiv2023

Nociception in chicken embryos, Part I: Analysis of cardiovascular responses to a mechanical noxious stimulus

While it is assumed that chicken embryos acquire the ability for nociception during the developmental period in the egg, an exact time point has not yet been specified. This study aimed to determine the onset of nociception during embryonic development in chicken. Changes in blood pressure and heart rate (HR) in response to a mechanical noxious stimulus at the base of the beak versus a light touch on the beak in chicken embryos between embryonic days (EDs) 7 and 18 were examined. Mean arterial pressure (MAP) was the most sensitive parameter for assessing cardiovascular responses. Significant changes in MAP in response to a noxious stimulus were detected in embryos at ED16 to ED18, while significant changes in HR were observed on ED17 and ED18. Infiltration anesthesia with the local anesthetic lidocaine significantly reduced reactions in MAP on ED18, so the cardiovascular changes can be assumed to be nociceptive responses.

physiology↗

bioRxiv2023

Nociception in chicken embryos, Part II: Embryonal development of electroencephalic neuronal activity in ovo as a prerequisite for nociception

Chicken culling is forbidden in Germany since 2022, male/female selection and male elimination must be brought to an embryonic status prior to the onset of nociception. The present study evaluated the ontogenetic point when noxious stimuli potentially could be perceived/processed in the brain in ovo. EEG-recordings from randomized hyperpallial brain sites were recorded in ovo and noxious stimuli were applied. Temporal and spectral analyses of the EEG were performed. The onset of physiological neuronal signals could be determined at developmental day 13. ERP/ERSP/ITC analysis did not reveal phase-locked nociceptive responses. Although no central nociceptive responses were documented, adequate EEG-responses to noxious stimuli from other brain areas cannot be excluded. The extreme stress impact on the embryo during the recording may overwrite the perception of noniceptive stimuli. The results suggest developmental day 13 as the earliest embryonal stage being able to receive and process nociceptive stimuli.

neuroscience↗

bioRxiv2023

Knockout of αβ but not γδ T cells in chickens is associated with high cytotoxicity and deficiency of regulatory and helper T cells

The availability of genetically modified mice has facilitated the study of mammalian T cells. No model has yet been developed to study these cells in chicken, an important livestock species with a high availability of {gamma}{delta} T cells. To investigate the role of {gamma}{delta} and {beta} T cell populations in birds, we generated chickens lacking these T cell populations. This was achieved by genomic deletion of the constant region of the T cell receptor {gamma} or {beta} chain, leading to a complete loss of either {gamma}{delta} or {beta} T cells. Our results show that a deletion of {beta} T cells but not {gamma}{delta} T cells resulted in a severe phenotype in knockout chickens. The {beta} T cell knockout chickens exhibited granulomas associated with inflammation of the spleen and the proventriculus. Immunophenotyping of {beta} T cell knockout chickens revealed a significant increase in monocytes and the absence of CD4+ T cells and FoxP3+ regulatory T cells compared to wild type chickens. In addition, we observed a significant decrease in immunoglobulins, B lymphocytes, and changes in the bursa morphology. Our data reveal the consequences of T cell knockouts in chickens and provide new insights into their function in vertebrates. Significance statementThe lack of genetically modified chickens has severely limited research in avian immunology compared to other animal models. Here, we report the generation of two T cell knockout chicken lines that will contribute significantly to the understanding of T cell biology as a very important research model as well as an important livestock species. The generated animals reveal the function of different T cell populations in chickens and will help to better understand the role of these cells during the interaction with various pathogens in birds.

immunology↗

bioRxiv2020

Resources for genome editing in livestock: Cas9-expressing chickens and pigs

Genetically modified animals continue to provide important insights in biomedical sciences. Research has focused mostly on genetically modified mice so far, but other species like pigs resemble more closely the human physiology. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR/Cas9. Here, we report for the first time the generation of Cas9 transgenic chickens and pigs that allow in vivo genome editing in these two important agricultural species. We demonstrated that Cas9 is constitutively expressed in all organs of both species and that the animals are healthy and fertile. In addition, we confirmed the functionality of Cas9 for a number of different target genes and for a variety of cell types. Taken together, these transgenic animal species expressing Cas9 provide an unprecedented tool for agricultural and biomedical research, and will facilitate organ specific reverse genetics as well as cross-species comparisons. Significance statementGenome engineering of animals is crucial for translational medicine and the study of genetic traits. Here, we generated transgenic chickens and pigs that ubiquitously express the Cas9 endonuclease, providing the basis for in vivo genome editing. We demonstrated the functionality of this system by successful genome editing in chicken and porcine cells and tissues. These animals facilitate organ specific in vivo genome editing in both species without laborious germ line modifications, which will reduce the number of animals needed for genetic studies. They also provide a new tool for functional genomics, developmental biology and numerous other applications in biomedical and agricultural science.

molecular biology↗