Ebola Virus Life Cycle Modeling In Vivo

ABSTRACT Sporadic outbreaks of Ebola virus (EBOV) and related filoviruses pose a grave threat for worldwide health populations, especially those of Western African nations. The largest outbreak of EBOV in 2013 caused nearly 13,000 deaths and had case fatality rates of up to 90%. Despite its near half-century since its emergence in human populations, there is only one vaccine and only two recently approved therapeutic treatments for Ebola virus disease (EVD). Detailed molecular study of EBOV biology is necessary to rapidly advance antiviral development. The largest impediment to detailed molecular study of EBOV is the requirement for high containment facilities when handling infectious virus. As an alternative, many groups utilize the well-established life-cycle modeling of both minigenome and transcription and replication virus-like particles (trVLP) systems to assess molecular viral mechanisms under biosafety level-2 (BSL-2) conditions. To this end, we have generated a penta-cistronic minigenome (5XMG) construct that contains four of the viral open reading frames and a reporter gene. The minigenome system remains BSL-2 due to its inability to replicate unless in the presence of the replication "helper" proteins: NP, VP30, VP35, and L. While this is valuable as a safety mechanism, it inhibits the use of EBOV modeling in animals. It would be invaluable to have a murine model in which to study the dynamics of EBOV under BSL-2 conditions in vivo. The proposed study will generate two mouse models for studying EBOV dynamics using trVLPs by supplementing the necessary helper proteins in trans. We would achieve this through two specific aims: (Aim 1) In Vivo EBOV Life Cycle Modeling with trVLPs via EBOV Helper Protein Expression by mRNA-Lipid Nanoparticles (LNPs); (Aim 2) In Vivo EBOV Life Cycle Modeling with trVLPs Supported by EBOV Helper Protein Expression via Transgenesis. In this model, the proteins missing from our poly-cistronic minigenome (EBOV VP30, VP35, and L) would be expressed in mice via either mRNA encapsulated LNPs or as integrated, Cre-recombinase dependent mouse transgenes. Expression of the viral helper proteins will replicate our 5XMG to amplify and create additional trVLPs that would bud off, infecting additional cells in the animal's tissues. Importantly, the proposed system would be a self-contained model for infection with minigenome-containing trVLPs but will not generate any infectious virus. These tools can have many applications including the assessment of antiviral host responses as well as being a platform for testing antiviral therapies like vaccines and small molecule inhibitors.