High-throughput, luciferase-based reverse genetics systems for identifying inhibitors of Marburg and Ebola viruses

Abstract

Marburg virus (MARV) and Ebola virus (EBOV), members of the family Filoviridae, represent a significant challenge to global public health. Currently, no licensed therapies exist to treat filovirus infections, which cause up to 90% mortality in human cases. To facilitate development of antivirals against these viruses, we established two distinct screening platforms based on MARV and EBOV reverse genetics systems that express secreted Gaussia luciferase (gLuc). The first platform is a mini-genome replicon to screen viral replication inhibitors using gLuc quantification in a BSL-2 setting. The second platform is complementary to the first and expresses gLuc as a reporter gene product encoded in recombinant infectious MARV and EBOV, thereby allowing for rapid quantification of viral growth during treatment with antiviral compounds. We characterized these viruses by comparing luciferase activity to virus production, and validated luciferase activity as an authentic real-time measure of viral growth. As proof of concept, we adapt both mini-genome and infectious virus platforms to high-throughput formats, and demonstrate efficacy of several antiviral compounds. We anticipate that both approaches will prove highly useful in the development of anti-filovirus therapies, as well as in basic research on the filovirus life cycle.

Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

“…As shown in Fig. 3C, we used rEBOV/ZsG virus in Huh7 cells to measure the antiviral effect of 6azaU, a known inhibitor of viral replication (Crance et al, 2003;Morrey et al, 2002;Pyrc et al, 2006;Smee et al, 1987;Uebelhoer et al, 2014). Consistent with our previous report (Uebelhoer et al, 2014), 6azaU used at the maximum concentration tolerated without toxicity significantly reduced ZsG signal: 94% and 97% signal reduction at 62.5 μM and 125 μM concentrations of 6azaU, respectively…”

“…Historically, the most common approach for introducing a reporter gene into the filoviral genome has been to insert an additional transcription unit into any of the 6 intergenic regions (Albariño et al, 2013b;Ebihara et al, 2007;Hoenen et al, 2013;Schmidt et al, 2011;Schudt et al, 2013;Towner et al, 2005;Uebelhoer et al, 2014). Unfortunately, recombinant viruses generated using this approach exhibited attenuated phenotypes in animal models (Ebihara et al, 2007) or Genome of recombinant EBOV in the viral complementary sense, with the 7 ORFs depicted in the 5 0 to 3 0 orientation.…”

“…In addition, Huh7 cells minimize selection of variant viruses generated by reverse genetics (Tsuda et al, 2015). To rescue the wild-type recombinant EBOV (rEBOV), we co-transfected Huh7 cells with the full-length genomic plasmid, with codon-optimized support plasmids expressing EBOV NP, L (or inactive L; see below), VP35, and VP30, and with another plasmid expressing a codon-optimized version of the T7 RNA polymerase (Uebelhoer et al, 2014). For the negative control, we built a new plasmid expressing an inactive form of the EBOV L polymerase (L-inac), in which the GDN motif was removed from the L ORF.…”

“…Several reports also describe modifying the genomes of EBOV and MARV by inserting reporter genes, such as those coding for fluorescent proteins and luciferase (Albariño et al, 2013b;Ebihara et al, 2007;Hoenen et al, 2013;Schmidt et al, 2011;Schudt et al, 2013;Towner et al, 2005;Uebelhoer et al, 2014).…”

Potential clinical treatment for Ebola pandemic

“…However, these experimental treatments have no recorded human safety trials. Some drugs approved for human use for other indications, including chloroquine [6], estrogen receptor (clomiphene and toremifene) [7], ion channel blocker (amiodarone, dronedarone and verapamil) [8] and imatinib [6], have shown activity against Ebola virus infection or entry in vitro or in rodent models. These drugs would be candidates for treating EHF, alone or in combination with other antiviral drugs.…”