Identification of novel Ebola virus inhibitors using biologically contained virus

Development and optimization of biologically contained Marburg virus for high-throughput antiviral screening

“…Because codon optimization seemed necessary to reach sufficient VP30 expression, we hypothesized that the failure to rescue MARV could be attributed to an inadequate balance or lack of support proteins (VP35, NP and L). Therefore, all support plasmids were also codon-optimized and different plasmid ratios were evaluated, including the condition previously used for the rescue of biologically contained EBOV by our group, the conditions for MARV rescue previously described by Albariño et al and Enterlein et al, as well as the balances used in the iVLP system of Wenigenrath et al, with or without the structural support plasmids [12, 13, 19, 26]. These conditions were evaluated in Huh-7-MARV-CO-VP30, BHK-21-MARV-CO-VP30 and BSR-T7/5-MARV-CO-VP30 cells, using both genomic and antigenomic MARV constructs, totaling more than 100 individual rescue attempts, which were all unsuccessful.…”

“…To address these issues, we decided to attempt virus rescue directly in Vero E6 cells. Vero E6-MARV-CO-VP30 and Vero E6-MARV-VP30 cells were transfected in 6-well format, using twice the amount of transfection reagent (6:1) and the plasmid ratios used by us for the rescue of biologically contained EBOV or those described by Albariño et al (three replicates each) [13, 26]. On day eight post-transfection, respectively one and five cluster(s) (~20 cells) of eGFP-positive cells appeared in two replicates of the Vero E6-MARV-CO-VP30 cells transfected with the conditions described by Albariño et al [26].…”

“…A lentiviral construct containing MARV VP30 linked to a blasticidin S-deaminase (BSD) resistance gene by means of an internal ribosomal entry site (IRES) cassette was used to create stably transduced cell lines expressing VP30. The MARV lentivirus was made by modifying the existing EBOV VP30 constructs previously described in [13]. Assembly of PCR fragments with the NEBuilder HiFi DNA assembly cloning kit (New England Biolabs) was used to replace the EBOV VP30 by a (codon-optimized) MARV VP30 gene.…”

“…In Vero E6 cells transduced with a regular MARV VP30-construct, overall eGFP expression levels did not rise above the background noise observed in non-infected control wells, although occasionally small clusters of eGFP-expressing cells could be observed, indicating that virus growth is possible in this cell line but only limitedly (Supplementary Figure S3). We previously also established a cell line expressing EBOV VP30 [13]. Of note, when infecting these cells with MARV-VP30-eGFP, equally distributed eGFP expression could be observed in all replicates, albeit at a very weak efficiency, making it difficult to distinguish eGFP-positive cells correctly from background noise (Supplementary Figure S3).…”

“…Although the functioning of VP30 has not been fully resolved, it is known to act as an enhancer of virus transcription and it has been shown to be indispensable for virus replication [11, 12]. Based on this design, we have previously shown that it is possible to perform high-throughput screening of antiviral compounds using biologically contained EBOV, by creating suitable containment cell lines in which EBOV VP30 is stably integrated into the host genome via lentiviral transduction [13]. In this study, we show that through a series of modifications the rescue of biologically contained virus is also possible for MARV.…”