Einfluss von Wirtsfaktoren auf die Nipahvirus-Infektion humaner und porciner Bronchial-Epithelzellen

Zusammenfassung Das hochpathogene, BSL-4 klassifizierte Nipahvirus (NiV) ist in der Lage, unterschiedliche Säugerspezies zu infizieren, darunter auch das Schwein und den Menschen. In Schweinen kommt es nach der NiV-Infektion zur Ausbildung einer akuten, respiratorischen Erkrankung und das Virus kan...

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Bibliographic Details
Main Author: Sauerhering, Lucie
Contributors: Maisner, Andrea (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2014
Online Access:PDF Full Text
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Summary Nipah virus (NiV) is a biosafety level 4 (BSL-4) classified paramyxovirus that is highly pathogenic for humans, pigs and other mammals. During the first outbreak in Malaysia, pigs developed a prominent bronchial infection with harsh cough and virus transmission via airway secretions was frequent. In contrast, human infections were generally characterised by severe encephalitis without major involvement of the respiratory tract. In this study we wanted to analyse the molecular basis of the differences in NiV replication in respiratory epithelial cells of pigs and humans. To study the NiV-infection of the respiratory tract of humans and pigs, we isolated primary bronchial epithelial cells (PBEpC) from pig lungs and used commercially available human bronchial epithelial cells (HBEpC). Infection studies of PBEpC and HBEpC showed that both cell types are infectable with NiV. However, human cells were more permissive to NiV infection. Furthermore, NiV is able to upregulate its own receptor (Ephrin B2) in infected cells which can facilitate the virus spread. Since it is known that there are 100 % amino acid identity between pig and human NiV isolates (AbuBakar, 2003), it can be hypothesized that host factors are responsible for the differences in airway infections. To analyse this, we studied different host factors which are important during the replication cycle of NiV. First the proteolytic activation of the fusionprotein was analysed. This is an important prerequisite for fusion of the viral membran with the host membran and thus, for entering of the target cell. It is known that the two proteases cathepsin L and/or cathepsin B are nessessary for activation of the fusionprotein (Pager et al., 2006 und Diederich et al., 2012). In the first part analysis of protease activity inhibitor studies lead to the result that cathepsin B is the predominant host cell protease in both, porcine and human bronchial epithelial cells. Another important host factor is the expression of the viral receptor (ephrin B2 and ephrin B3) which is essential for viral entry and spread of infection by cell-to-cell fusion (synyctia formation). So in the second part we compare ephrin B2 and ephrin B3-mRNA-levels in PBEpC and HBEpC. Both, porcine and human bronchial epithelial cells express ephrinB2. Interestingly, ephrin-B3 is only expressed in human cells. It is not only the additional ephrin-B3 expression that distinguishes the two cell types. Quantitative real-time-PCR analysis furthermore showed that HBEpC express much higher levels of ephrin-B2 than the porcine cells. Additional expression of human ephrinB2 causes a more efficient cell-cell-fusion in PBEpC and more NiV-N-mRNA is found in the supernatant of infected cells. So far, the data suggest that the receptor expression (more ephrinB2 and additional ephrinB3) supports a more efficient NiV replication in respiratory epithelial cells in the human lung, compared to replication in the airways of pigs. In 30 % of infected humans some respiratory clinical signs were found. So in the third part of this PhD thesis we compare the ephrin B2-level of different human donors to find out the variability of ephrin B2 expression in different individuals. We found that ephrin B2-levels differ in HBEpC of different donors and that less ephrin B2 leads to a slower NiV-infection. So the ephrin B2 levels are essential for an efficient NiV infection and may explain the different clinical outcomes in human infections.