Development and application of filovirus reverse genetics systems for antiviral compound screening

Filoviruses are a rapidly expanding group of human pathogens known for their potential to cause severely debilitating disease with a high mortality rate. Several members of the family Filoviridae can cause filovirus disease but most outbreaks in the last five decades were attributable to the well-known Ebola virus and Marburg virus, the first filovirus to be discovered. Treatment options for Ebola virus disease and Marburg virus disease remain limited and are largely focused on early supportive care and symptomatic treatment due to a lack of effective therapeutics. Because the high risk associated with Ebola and Marburg virus research necessitates the use of a biosafety level 4 facility to study replication-competent viruses, most research investigating possible virus inhibitors has been performed using non-infectious alternatives. However, especially for Marburg virus, the variety in available systems is very limited, hindering the scientific progress that can be made. The primary objective of the work presented here was therefore to develop new virus alternatives for Marburg virus to help close the gap with Ebola virus in terms of available research tools. Additionally, we aimed to optimize the newly developed tools for use in compound-screening applications and subsequently employ them to identify novel filovirus inhibitors, both for Marburg virus and Ebola virus. A first virus alternative that was developed is a new Marburg virus minigenome system that is regulated by the natively expressed RNA Polymerase II, unlike all existing systems, which are controlled by the exogenous T7 polymerase. We illustrate here some of the characteristics of this new system and highlight its usability in different cell lines. Additionally, we also demonstrate the usefulness of this new tool for compound screening and discuss its added benefit over existing systems. However, while this new system represents an interesting addition to the repertoire of available Marburg virus alternatives, the inherent limitations of minigenome systems still necessitate the availability of additional research tools to allow a more comprehensive study of filoviruses. Therefore, we also developed a more advanced system that makes use of a biologically contained Marburg virus. By creating a cell line that constitutively expresses one of the essential virus genes and simultaneously removing this gene from the virus genome, a recombinant virus can be made that is safe to handle because it is unable to replicate in regular cells. Conversely, in the specific cell line expressing the missing gene, this virus experiences a near-normal replication cycle, allowing the study of almost all aspects of the virus life cycle. While biological containment had already been shown to be possible for Ebola virus, we demonstrate here that, with the correct modifications and optimizations, comparable systems can also be made for other filoviruses. Additionally, we describe the optimization and validation of the resulting biologically contained Marburg virus system and an equivalent Ebola virus system as tools for high-throughput compound screening. Lastly, we used our newly developed assays to screen close to 5,000 compounds for their antiviral activity, resulting in the identification of several compounds that show strong selectivity towards virus inhibition and represent interesting leads for the development of new filovirus antiviral therapies.

Jaar van publicatie:2021

Toegankelijkheid:Open