Studies on the neurotropism of the highly pathogenic Nipah and Ebola virus
Nipah and Ebola viruses are highly pathogenic, zoonotic agents that are associated with high case fatality rates. While Nipah virus (NiV) is known to predominantly target the central nervous system (CNS), neurological complications from Ebola virus (EBOV) infections became apparent during the outbre...
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| Format: | Doctoral Thesis |
| Language: | English |
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Philipps-Universität Marburg
2024
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| Online Access: | PDF Full Text |
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| Summary: | Nipah and Ebola viruses are highly pathogenic, zoonotic agents that are associated with high case fatality rates. While Nipah virus (NiV) is known to predominantly target the central nervous system (CNS), neurological complications from Ebola virus (EBOV) infections became apparent during the outbreak in West Africa (2013-2016). Due to their pathogenicity and the need for biosafety level 4 facilities for research, our understanding of underlying neuropathogenic mechanisms is limited. Especially how these viruses initially establish infection in the CNS is unclear. Limited autopsy data also restricts our knowledge of neuropathology in humans, necessitating reliance on animal models. However, the use of such models is challenging and costly to perform under adequate safety conditions, prompting the exploration of alternative methods. This project employed primary murine tissue to investigate NiV and EBOV neurotropism. In vitro and ex vivo key model systems were established to investigate the hematogenous pathway as a potential entry route into the central nervous system and to evaluate the susceptibility of different brain cells and regions. Furthermore, by preparing cultures from either wildtype or interferon receptor alpha/beta knockout mice (IFNAR-/-), the role of type I interferons was investigated, as they seem to play a crucial role for disease outcome after systemic infection as well as for the local brain-specific immune responses. In addition, in vitro models using human immortalized cell lines were established to investigate the transferability to infections in humans.
To explore the hematogenous route as a potential entry path, primary murine brain endothelial cells were isolated and characterized along with murine and human in vitro models of the blood-brain barrier. Endothelial cells derived from human and murine sources exhibited susceptibility to NiV and EBOV infections, resulting in cytopathic effects and impaired barrier function. These findings suggest that NiV and EBOV may utilize this route to penetrate the CNS. Interestingly, no disparities in susceptibility to NiV and EBOV were observed among endothelial cells isolated from either wild type or IFNAR-/- mice. This suggests that, at the cellular level, a functional type I interferon response alone is insufficient to constrain NiV and EBOV infections in brain endothelial cells.
To identify potential target cells for NiV and EBOV within the CNS and to assess their role during infection, a primary murine neuron culture (N), and a neuroglial mixed culture (NG) was isolated. Moreover, ex vivo organotypic brain slices (BS) were established to investigate viral spread in a neuronal network. The suitability of these models for neurotropic virus infections was first evaluated under biosafety level 2 conditions using vesicular stomatitis virus, which exerts a high neurotropism. The virus was able to infect the primary cultures and replicated efficiently, which proved the suitability of the models. However, virus replication in BS was only efficient in tissue from IFNAR-/- mice and led to the release of several cyto- and chemokines and significant tissue damage. This indicates that in BS, where the neuronal network is preserved, more effective antiviral responses can occur. NiV was able to infect the N and NG cultures from both mouse strains. Notably, viral replication was comparatively more efficient in the N culture, accompanied by pronounced cytotoxicity, suggesting a potential role of other brain cell types in offering protection or highlighting neurons as important drivers of NiV replication. In BS, infection primarily manifested in small foci, although areas of widespread infection were also observed. Viral replication was more robust in slices derived from IFNAR-/- mice, underscoring the significance of type I interferons in controlling viral spread. Over time, NiV infection induced tissue damage and secretion of numerous mediators, with generally higher levels observed in slices from IFNAR-/- mice. Similarly, EBOV exhibited infection in N and NG cultures, as well as in BS. In BS, infection was sporadic, forming small foci, often accompanied by astrogliosis near infected areas. Viral replication was constrained in slices from both mouse strains, nevertheless infection prompted tissue damage and the release of cytokines and chemokines, which was more pronounced in slices from IFNAR-/- mice. Altogether, the relatively confined infection of NiV and EBOV in these cultures could contribute to their persistence in the CNS. Surprisingly, the choroid plexus appeared highly susceptible to both viruses, emphasizing the blood-liquor barrier as a potential entry and persistence site for these pathogens. Interestingly, IL-17a and IL-9 were found in increased concentrations following infections with all tested viruses in BS derived from both mouse strains. As both mediators have been associated with neuroinflammatory diseases, further analysis whether they play a decisive role in NiV and EBOV neuropathology should be undertaken.
Overall, these findings confirm the appropriateness of the models for investigating the neuropathogenicity of NiV and EBOV. The insights gleaned from this study align with previous observations from human autopsies and animal models, highlighting the utility of these models as alternative methods for investigating NiV and EBOV neurotropism. Nevertheless, further in-depth analyses are necessary to gain a comprehensive understanding of how the viruses enter the brain and how immune responses promote or diminish infection and tissue damage. The established models lay a groundwork for future investigations, such as visual spatial genomics to further explore the neurotropism and neuropathology of NiV and EBOV. |
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| Physical Description: | 178 Pages |
| DOI: | 10.17192/z2024.0176 |