Nipahvirus-induzierte Zytokinantwort in porcinen und humanen respiratorischen Epithelzellen

Das hochpathogene Nipahvirus (NiV), das zu dem Genus Henipavirus in der Familie Paramyxoviridae gehört, führt seit 1998 zu Ausbrüchen in Südostasien. Dabei verursacht das Virus schwere Erkrankungen in verschiedenen Spezies und hohe Letalitätsraten beim Menschen, wogegen es keine Behandlungsmöglichke...

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Bibliographic Details
Main Author: Elvert, Mareike
Contributors: Maisner, Andrea (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2020
Online Access:PDF Full Text
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The highly pathogenic Nipah virus (NiV), which belongs to the genus Henipavirus in the family Paramyxoviridae causes outbreaks of infection in Southeast Asia since 1998. NiV induces severe diseases in various species and high mortality rates in humans, while no treatment or vaccination is available. For these reasons, NiV is classified as a biological safety level 4 (BSL-4) pathogen. Due to severe course of disease, NiV is listed on the R&D Blueprint list of priority diseases of the World Health Organization (WHO). This list contains viruses prioritized for further research. During the first outbreak in Malaysia and Singapore in 1998/1999, NiV was transmitted from the virus reservoir, fruit bats, to pigs. Infected pigs showed a severe symptomatic inflammation of the lung and transmitted the virus also to humans via secretions. Infected humans developed severe encephalitis but rarely showed respiratory symptoms or virus shedding in airway secretions. Because the genome sequences of NiV isolates from pigs and humans do not differ, the variations in the respiratory involvement indicate host species-specific differences. Our research group already revealed that the entry receptor for NiV (Ephrin-B2) is differently expressed in human and porcine respiratory epithelial cells, which also influenced the infection efficiency. The aim of this work was to identify further host factors that influence NiVMalaysia infection in airway epithelia. Therefore, infection studies in primary human and porcine respiratory epithelial cells were performed. NiV replication, the release of infectious virus particles and innate immune responses, namely the induction of interferons (IFNs), interferon-stimulated genes (ISGs) and proinflammatory cytokines, were investigated. NiV infection of human and porcine epithelial cells from different parts of the lung (trachea, bronchia, small airways) showed that all epithelial cells reacted with an upregulation of type III IFN, especially IFN-λ2,3 (human) or IFN-λ3 (porcine). Both, human and porcine bronchial epithelial cells showed higher IFN responses to NiV compared to epithelial cells from the trachea or small airways. In addition to quantitative differences in the IFN response in cells from different parts of the lung, this work revealed clear species-specific differences in cytokine induction. Direct comparative studies of NiV-infected human and porcine bronchial epithelial cells (HBEpC and PBEpC) showed that, despite similar replication rates (measured via the intracellular viral RNA content), the type III IFN and ISG responses in PBEpC were significantly reduced compared to HBEpC. The release of infectious virus particles (virus titres in the cell supernatant) was increased in PBEpC. This suggests that NiV induces a relatively low antiviral response in porcine bronchial epithelial cells and replicates productively. The infection studies of this work have further shown that, despite the limited IFN and ISG response, NiV infection induced a prominent expression of proinflammatory cytokines (IL-6, IL-8) in porcine bronchial epithelial cell cultures. Altogether, the comparative infection studies in this work provide the first clear evidence that differences in cytokine response are important host species-specific factors that could influence NiV infection in the human and porcine lung. Based on these results, the differences observed in vivo in respiratory symptoms and virus shedding in respiratory secretions might be explained as follows: In humans, NiV infection in the lung triggers a moderate proinflammatory response and induces an efficient IFN-dependent antiviral immune response, which in turn limits virus production. In contrast, NiV infection in pigs causes a pronounced inflammatory response in the lung. The IFN-dependent inhibition of virus replication is limited, which may explain the prominent respiratory symptoms in pigs and the productive virus shedding via airway secretions. Since IFN-λ appears to play an important role in the antiviral immune response in bronchial epithelia, the effects of cyclosporine A (CSA) were analyzed, which was recently found to cause an IFN-λ dependent inhibitory effect on MERS coronaviruses. In fact, CSA treatment of primary human and porcine bronchial epithelial cells resulted in type III IFN upregulation and a strong inhibition of NiV infection. Wheter CSA is a possible therapeutic approach to inhibit NiV infection in the lung, should be tested in the future in a more complex primary cell culture models containing larger intact epithelial layers with diverse epithelial and submucosal cell types. Therefore, in the last part of this work, a method to generate lung slices, so-called porcine ex vivo lung slices cultures, was established. The lung slices prepared from fresh pig lungs were vital for several days and infection with recombinant NiVeGFP could be successfully detected by autofluorescent NiV-positive cells in the bronchial lumen and the lung tissue. Thus, a system is available for future NiV inhibitor studies in a complex ex vivo cell model.