Etablierung eines infektiösen Minigenomsystems für Marburg Virus basierend auf Virus-ähnlichen Partikeln

Marburg and the closely related Ebola virus make up the family filoviridae. Both viruses are causative agents of severe, mostly fatal hemorrhagic fever in humans and non human primates. Working with such pathogens is limited to laboratories with highest safety standards (BSL-4). In the present study, a model system for Marburg virus has been established to analyze viral transcription, morphogenesis, budding and infectivity under BSL-1 conditions. This approach is based on the transfection of plasmids encoding all viral proteins as well as a minigenome into target cells. The minigenome is composed of a luciferase reporter gene flanked by nontranscribed leader and trailer sequences of the viral genome. The minigenome can be recruited into nucleocapsid-like structures by the nucleocapsid proteins NP, VP35, VP30 and L. Upon interaction with VP40, VP24 and GP, the nucleocapsid-like structures can bud from the plasma membrane as enveloped infectious virus-like particles (iVLPs). Purified iVLPs from supernatant can be used for the infection of target cells. Detection of the reporter signal in target cells demonstrates successful infection and can be documented in an easy, fast and standardized way. The amount of transfected plasmids encoding the viral proteins have been titrated individually and variations in intracellular concentrations of the proteins, reporter gene activity, iVLP release, and infection of target cells by the released iVLPs were recorded. The results show that different combinations of viral nucleocapsid proteins are necessary for optimal reporter gene activity, iVLP release or infectivity of released iVLPs. Titration of plasmids encoding the viral matrix protein VP40 indicated that this protein plays a critical role in down regulation of transcription/replication of the minigenome and initiation of viral budding. The established iVLP system was also tested for its suitability as a screening tool to determine neutralizing titers of anti-Marburg virus sera and to screen small molecule libraries to detect anti-Marburg virus substances. In the later case it turned out that the iVLP system could be easily adapted to serve for highthroughput screening assays. Finally, the iVLP system was used to test the effect of a semen-derived protein on the infectivity of Marburg virus that can be sexually transmitted. Taken together, the iVLP system can be used as a reliable model to investigate viral transcription, budding, infectivity and to screen for antiviral substances.