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| | Inhibition of Authentic Filovirus Infections in Cell Culture Assays |
| Description: | The authentic filoviruses Ebola virus/H. sapiens-tc/COD/1995/Kikwit-9510621 (EBOV/Kik-9510621; EBOV-Zaire 1995 ) (Jahrling et al., J. Infect. Dis., 179 Suppl 1:S224-234 (1999)), Sudan virus/H. sapiens-gp-tc/SDN/1976/Boniface-USAMRIID 111808 (SUDV/Bon-USAMRIID 111808; SUDV-Boniface 1976 ) (Anonymous, Ebola haemorrhagic fever in Sudan, 1976. Report of a WHO/International Study Team. Bull World Health Organ., 56(2): 247-270 (1978)), and Marburg virus/H. sapiens-tc/DEU/1967/Hesse-Ci67 (MARV/Ci67) (Towner et al., PLoS Pathog., 4(11): e1000212 (2008)) were used in these studies under BSL-4 containment and procedures. Vero cells were pre-treated with the inhibitor compound added to each well (or in replicate wells) in a dilution series (typically two-fold diluted, beginning with 25 or 50 μM as the highest concentration) for 1 hour prior to addition of EBOV, SUDV or MARV at a multiplicity of infection (MOI) of 1 diluted in culture media. After a 1 hr incubation with virus, in the presence of inhibitor compound, virus inoculum was removed and replaced with fresh culture media containing compounds in a dilution series (typically two-fold diluted, beginning with 25 or 50 μM as the highest concentration). At 48 h post-infection, cells were fixed with formalin, and blocked with 1% bovine serum albumin. EBOV-, SUDV- or MARV-infected cells and uninfected controls were incubated with EBOV GP-specific mAb KZ52 (Lee et al., Nature, 454:177-182 (2008)), SUDV GP-specific Ab 3C10 (Herbert et al., M Bio, 6: e00565-15 (2015)), or MARV GP-specific mAb 9G4 (Swenson et al., FEMS Immunol. Med. Microbiol., 40: 27-31 (2004)). Cells were washed with PBS prior to incubation with either goat anti-mouse IgG or goat anti-human IgG conjugated to Alexa 488. Cells were counterstained with Hoechst 33342 stain (Invitrogen), washed with PBS and stored at 4° C. Infected cells were quantitated by fluorescence microscopy and automated image analysis. Images were acquired at 20 fields/well with a 20× objective lens on an Operetta high content device (Perkin Elmer, Waltham, Mass.). Operetta images were analyzed with a customized algorithm built from image analysis functions available in Harmony software. Evaluation of the effects of MBX 3574 and MBX 3587 on the infectivity of authentic EBOV, SUDV, and MARV is shown in FIG. 5. Other analogs were analyzed in the same manner to confirm efficacy against infectious filoviruses. |
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