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Tick-borne encephalitis virus (TBEV) is a neurotropic member of the genus Flavivirus. It may transmit to humans through the bite of an infected tick or consumption of unpasteurized dairyproducts, and causes tick-borne encephalitis (TBE). TBE constitutes a significant health burden in Eurasia, with more than 10,000 cases reported every year. In this thesis, I have investigated the role of the innate immune response in restricting infection in the central nervous system (CNS), identified virulence factors and developed a new model system to study the structural proteins of TBEV.

Viral tropism is important for understanding underlying mechanisms of pathology. In the first part,we combined whole-brain imaging with single nuclei RNA-sequencing after infection of wildtype (WT) and interferon (IFN) α/β receptor knockout (Ifnar^-/-) mice by Langat virus (LGTV), a low-virulent model for TBEV. We found that absence of type I IFN signaling changes viral tropism and leads to an impaired inflammatory response. For neurons, astrocytes, and microglia we also compared the response to LGTV infection in vivo with the response of primary monocultures infected in vitro. Primary cells are often used for mechanistic studies of neurotropic viruses, but we found limited overlap in altered pathways between in vivo and in vitro, which emphasizes the role of cellular crosstalk in shaping the transcriptional response to infection in the brain.

The second part addresses viral determinants of pathogenicity. By comparing disease progression induced by different TBEV strains in a mouse model, we identified TBEV 93/783 as a highly virulentstrain belonging to the European subtype. We could show that two unusual amino acid substitutions in the envelope (E) protein of 93/783 enhanced neurovirulence and contributed to pathogenesis. To facilitate further studies of the structural proteins of TBEV, we generated and thoroughlycharacterized a chimeric virus with the pre-membrane (prM) and ecto-E protein of TBEV 93/783 in the genetic background of LGTV. The chimeric virus shows similar growth kinetics as the parental LGTV in vitro but is less pathogenic in our mouse model. Meanwhile, it remained neurovirulent and structurally similar to TBEV, making it a useful tool for studying the structural proteins of TBEV under lower biosafety conditions. Taken together, these findings deepen our understanding of what determines the outcome of tick-borne flavivirus infection and the utility of the available model systems for studying disease mechanisms. 

This thesis leverages the power of single-cell RNA and ATAC sequencing to enhance our understanding of the innate and adaptive immune systems in higher mammals. The primary focus is on the transcriptional networks that guide the activation and differentiation of human primary CD4+ T cells into Th1, Th2, Th17, and iTreg subsets, using a GMP-based protocol and ex vivo/in vitro approaches. Additionally, computational models for gene regulatory network (GRN) inference and analysis were employed to elucidate gene regulation using a data-driven, multi-omics approach. This research also encompasses viral response-related studies to provide a comprehensive view of the immune response, specifically targeting the central nervous system (CNS) upon TBEV infection and lung tissues during SARS-CoV-2 infection.

In Paper 1, a multi-omics linear and non-linear approach is developed to predict gene popularity using a large number of high-throughput sequencing datasets. We show that additional omics layers are beneficial to construct GRNs capable of accurately predicting gene popularity. In Paper 2, a comprehensive atlas of human primary CD4+ T cell activation and differentiation is created using in vitro cell differentiation and single-cell RNA and ATAC sequencing. Novel gene regulatory dynamics of JUNB are identified, and a new probabilistic approach based on Markov chains for GRN analysis and interpretation is introduced. In Paper 3, the connection between type I interferon response in the mouse brain and TBEV infection is explored using single nuclei RNA sequencing. In Paper 4, the role of intrinsic resistance factors in human COVID-19 susceptibility is investigated using both single-cell and bulk RNA sequencing, and identifies SERPINS as critical regulators of the process.

The findings of this thesis contribute significantly to the understanding of transcriptional networks governing human CD4+ T cell differentiation and activation. This work aims to improve therapy and demonstrate the efficacy of NGS and computational tools in deciphering the transcriptional networks involved in various viral infections.