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| | Inhibition of Infectivity of Recombinant VSVs Expressing Glycoproteins from Zaire Ebolavirus, Sudan Ebolavirus, Bundybugyo Ebolavirus, and Lassa Arenavirus |
| Description: | Recombinant vesicular stomatitis viruses (VSVs) (serotype Indiana) expressing eGFP and EBOV, SUDV, or BDBV GP in place of VSV G, as well as those expressing RFP and LASV GP in place of VSV G (rVSV-EBOV/SUDV/BUDV/LASV GP) were produced, recovered, and amplified as described previously (Miller et al., EMBO J., 31: 1947-1960 (2012); Wong et al., J. Virol., 84: 163-175 (2010); Ng et al., Virology, 468-470: 637-646 (2014); Geisbert et al., PLoS Med., 2:e183 (2005)). The EBOV, SUDV, BDBV and LASV GP genes encoded by these viruses were derived from the following isolates: Genbank accession numbers NP_066246, YP_138523, YP_003815435, and ADY11070, respectively.The infectivity of the rVSVs expressing different viral glycoproteins in place of VSV G and the effect of added inhibitor were measured as follows. Vero or U2OS cells were seeded at 300,000 cells/ml in 50 μl in 96-well black plates with clear bottoms. After 24 hrs, cells were treated with compounds in 3-fold serial dilution series starting at 200 μM, followed by infection 1 hr later with appropriate rVSVs (e.g., rVSV-EBOV GP, rVSV-SUDV GP, rVSV-BDBV GP, or rVSV-LASV GP). The MOI ( 0.1 infectious units/cell) was chosen to keep the infection percentage between 40 and 60%. Cells were fixed with 4% formaldehyde at 12-14 hr post infection for 30 min prior to staining with Hoechst 33342 for 15 min at room temperature. Nuclei, and individual eGFP or RFP-positive cells were counted using a Cytation 3 automated fluorescence microscopy cell imager equipped with GFP, DAPI, and Texas Red filter cubes (BioTek Instruments, Inc., Winooski, Vt.). An example with PPZ2 (Chembridge #6179974) is shown in FIG. 2. The analog exhibits a high degree of selectivity for inhibition of infection by rVSV-EBOV GP (squares) as compared to rVSV-LASV GP (circles), and the nuclei count (triangles) remains nearly constant, reflecting low cytotoxicity of the compound.The rVSV assay was also used to compare the potency of MBX 3574 and MBX 3587 vs. rVSV-EBOV GP (squares), rVSV-SUDV GP (triangles), and rVSV-BDBV GP (circles) in FIG. 3. MBX 3574 and MBX 3587 are potent inhibitors of infection of Vero cells by rVSV carrying each of the three GP proteins, demonstrating a broad spectrum of anti-filoviral activity. |
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