Recombinant Marburg Virus Expressing EGFP Allows Rapid Screening of Virus Growth and Real-time Visualization of Virus Spread

Marburg virus (MARV) and the closely related Ebola virus (EBOV) belong to the filovirus family and cause a severe hemorrhagic fever in humans, with mortality rates up to 90%. Currently, there is no approved vaccine or antiviral treatment. Filoviruses have a nonsegmented negative-sense RNA genome encoding 7 structural proteins. Four of these proteins constitute the nucleocapsid complex, containing the nucleoprotein (NP), the viral polymerase L, the polymerase cofactor VP35, and the viral protein VP30 in close association with the viral genome (for review, see [1]). Cytoplasmic inclusions, which are thought to represent active sites of viral replication, are present as large aggregates in filovirus-infected cells. These inclusions are formed by all 4 nucleocapsid proteins, with NP being the driving force for aggregation due to self-assembly of NP [2, 3]. NP interacts with VP35, VP30, and L, either directly or via a linker protein, thereby redirecting the nucleocapsid proteins into cytoplasmic aggregates [4–8]. Rescue systems to recover infectious virus from full-length complementary DNA (cDNA) clones have been established for both MARV and EBOV [9–13]. These techniques were used to generate recombinant forms of EBOV, derived from isolates of the Zaire ebolavirus (ZEBOV) species, containing the enhanced green fluorescent protein (EGFP) gene within an additional transcription unit (ATU), which provide a sensitive and quantitative readout for antiviral drug screening assays and virus spread studies [14–21]. EGFP was efficiently expressed over 10 passages, confirming the stability of the EBOV constructs [15]. In this study, we rescued a recombinant MARV from a clone containing an ATU encoding EGFP. This clone allows for the visualization of MARV spread in infected cells and was used to assess the localization of EGFP and nucleocapsid proteins in infected cells.