3D-Imaging Based Analysis of Pathogens Distribution in Infected Organs and Tissues

Abstract

Tissue optical clearing (TOC) and fluorescence imaging have significantly advanced 3D imaging of biological samples. However, few studies have applied these techniques in infection biology although they provide valuable information that can enhance our understanding of spatiotemporal dynamics in infections and disease progression. This thesis addresses this gap by utilizing these techniques to analyze optically cleared tissue samples from animals experimentally infected with rabies virus (RABV) or severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), in order to gain deeper insights into virus replication and pathogenicity in the brain and lung. To achieve this, image segmentation and quantification pipelines, including the use of AI, for the extraction of comparable data on cell tropism and the spatiotemporal development of infection processes were developed. Previous work demonstrated that intramuscular (i.m) inoculation of mice with highly virulent field RABV strains led to the infection of both neurons and astrocytes in the brain, of which the latter may significantly shape the host response to RABV infection. Accordingly, the first aim was to investigate whether astrocyte tropism of field viruses is a late phase phenotype or whether it already occurs in early phases before the onset of clinical signs. Immunofluorescence imaging of optically cleared mouse brain sections confirmed astrocyte infection at early time points of brain invasion and indicates that astrocyte tropism of RABV indeed could shape host reactions to infection already at those early time points of infection. The second aim was to investigate whether resistance of rRABV DogA to interferon (IFN) response, mediated by the phosphoprotein (P), is a prerequisite for CNS infection from peripheral sites and astrocyte tropism in the brain. Using a recombinant virus mutant, rRABV DogA-IFNmut, which has impaired type I interferon antagonistic activity, age-dependent courses of brain infection following i.m. inoculation were observed. In 4-week-old mice, the infection proved lethal, while older mice recovered despite showing clinical signs. While these data demonstrated insufficient block of the virus by the interferon system in peripheral tissues, they also suggest a significant role of the type I interferon response in restricting virus spread and astrocyte tropism in the brain. The third aim was to explore the effect of increased glycoprotein (G) cell surface expression that was previously observed in cell culture on the virulence of field RABV DogA. Based on the hypothesis that de-regulated G transport to non-synaptic plasma membrane sites results in the release of extracellular virus from neurons and hence recognition by the immune system, the mutant rRABV DogA-NTN, that has three cell culture-adaptive mutations in G was used. Although delayed pathogenesis and requirement of higher viral loads to induce clinical signs were observed, no detectable increase in surface expression of the protein was found in neurons, despite visualized changes in intra-neuronal G distribution. These results indicate that differences in surface transport regulation between standard cell lines and neurons in vivo exist. Involved mechanisms, however, remain unclear. Finally, to effectively manage SARS CoV-2 infection, a thorough understanding of the immune dynamics within the lungs is needed. Consequently, TOC combined with 3D imaging techniques was used to investigate viral infection and the corresponding immune response in the lungs of experimentally infected hamsters over a 7-day period. This approach enabled high-resolution visualization of cellular interactions, revealing a spatially localized antiviral response. Infected epithelial cells displayed an upregulation of major histocompatibility complex II (MHC II), highlighting immune system activation. Monocyte-derived macrophages played a crucial role in viral clearance; however, their presence was also associated with postinfection endothelial damage, illustrating their dual role in both defending against the virus and contributing to tissue injury. Furthermore, early signs of tissue repair were observed alongside inflammatory and necrotizing processes, potentially laying the groundwork for longterm alterations in lung structure and function. These findings provide valuable insights into the complexities of immune responses in viral infections and may inform future therapeutic strategies