Establishment and application of an infectious virus-like particle system for Marburg virus.

The highly pathogenic Marburg virus (MARV) can only be investigated in high containment laboratories, which is time consuming and expensive. To investigate the MARV life cycle under normal laboratory conditions, an infectious virus-like particle (VLP) system was developed. The infectious VLP system is based on the T7-polymerase driven synthesis of a MARV-specific minigenome that encodes luciferase and is transcribed and replicated by the simultaneously expressed MARV nucleocapsid proteins NP, VP35, L and VP30. Transcription of the minigenome resulted in luciferase activity and replication resulted in encapsidated minigenomes. The encapsidated minigenomes, together with the viral matrix proteins VP40 and VP24 and the surface glycoprotein (GP), formed VLPs at the plasma membrane. Among the released pleomorphic VLPs, filamentous particles of 200-400 nm in length showed the highest capacity to induce reporter activity upon infection of target cells. To characterize the infectious VLP system, the intracellular concentration of one of the components was titrated, while all others were held constant. Intracellular concentrations of nucleocapsid proteins that resulted in highest replication and transcription activities also yielded VLPs with the highest ability to induce luciferase activity in target cells. High intracellular levels of VP40 maximized the release of VLPs, but reduced their ability to induce luciferase activity in target cells. The intracellular concentration of GP positively correlated with its incorporation into VLPs and their infectivity. Finally, we demonstrated that the infectious VLP system was suitable for rapid screening of neutralizing antibodies directed against MARV.