Fig 1.
IFN-α2a does not clear KFDV infection in A549 cells.
A549 cells were infected with a 11 TCID50 units (MOI of 0.00001) of the indicated virus, and either treated or mock-treated with 2, 000 U/mL of IFN-α2a (designated as P0). Monolayers were passaged when untreated controls reached CPE of 90%, 96 and 48 hpi for KFDV and VSV-GFP, respectively and 2, 000 U/mL of IFN-α2a was either added or omitted (P1) and after 72 hpi for KFDV and 48 hpi for VSV-GFP. This procedure was repeated again for passage 2 (P2). (A) Before each passage, supernatants were harvested for titration by TCID50 assay determination on BHK-21 cells. The averages and standard deviation from three biological replicates are shown graphically and expressed in log10 scale TCID50/mL. Statistical significance is denoted as * P < 0.1, ** P < 0.05, *** P < 0.01. (B) Cell monolayers were visualized with light microscopy prior to passaging. Mock, non-IFN treated/infected controls. UI, Un-infected controls.
Fig 2.
IFN-α2a does not clear KFDV infection in BHK-21 cells.
BHK-21 cells were infected with a 11 TCID50 units (MOI of 0.00001) of the indicated virus, and either treated or mock-treated with 2, 000 U/mL of IFN-α2a (designated as P0). Monolayers were passaged when untreated controls reached CPE of 90%, 96 and 48 hpi for KFDV and VSV-GFP, respectively and 2, 000 U/mL of IFN-α2a was either added or omitted (P1) and after 72 hpi for KFDV and 48 hpi for VSV-GFP. This procedure was repeated again for passage 2 (P2). (A) Before each passage, supernatants were harvested for titration by TCID50 assay determination on BHK-21 cells. The averages and standard deviation from three biological replicates are shown graphically and expressed in log10 scale TCID50/mL. Statistical significance is denoted as * P < 0.1, ** P < 0.05, *** P < 0.01. (B) Cell monolayers were visualized with light microscopy prior to passaging Cell monolayers were photographed prior to passaging. Mock, non-IFN treated/infected controls. UI, Un-infected controls.
Fig 3.
Screening interferon-α/β subtypes against KFDV.
Cultures of A549 cells were pre-treated (grey bars) 24 hours prior to infection or post-treated (black bars) 1 hour after infection with 1, 000 U/mL of IFN-α (B2, C, D, F, G, H2, I, J1, K, WA, 2a, 2b or 4b), IFN-β (beta-1) and a recombinant IFN-α (Universal) subtypes and infected with KFDV at a MOI of 1. Supernatants were harvested for each treatment after 72 hours of incubation and quantified (expressed in log10 scale TCID50/mL) on BHK-21 (ATCC) when the mock-treated control cells displayed CPE near 100%. Pre-infection treatment experiments were assayed in two biological replicates and post-infection treatment experiments were assayed in three biological replicates; the resulting averages and standard deviations are presented. Mock, Mock-treated with IFN. UI, Un-infected control. IFN-α2b was excluded from 24-hour pre-infection treatment. * Significant compared to mock-treated samples (P < 0.1). *** Significant compared to mock-treated samples (P < 0.01).
Table 1.
Antiviral activity of interferon-α/β against the cytopathology of KFDV and VSV-GFP.
Table 2.
Antiviral activity of interferon-α/β against KFDV and VSV-GFP virion production.
Fig 4.
KFDV NS5 impedes the cellular antiviral state.
(A) VeroE6 (ATCC) cells were transfected with plasmid encoding KFDV NS proteins and Ebola virus VP24 and treated with 1, 000 U/mL of Universal IFN, 24 hpi. After a 24-hour incubation period, cells were infected with VSV-GFP (MOI of 2) and, pictures were taken with fluorescent (top panel) and light (bottom panel) microscopy 24 hours later. (B) VeroE6 (ATCC) cells were transfected with KFDV NS5-pCAGGS and treated with 1, 000 U/mL of commercially available type I IFNs, 24 hpi. After a 24-hour incubation period, cells were infected with VSV-GFP (MOI of 2) and, 24 hours later, the virus-containing supernatants were harvested for virus quantification. Dark grey bars indicate experiments in which cells were un-transfected. Light grey bars indicate NS5-expressing cells. Mock, no IFN treatment of cells lacking NS5 expression (dark gray bar) and with NS5 expression (light gray bar); UI represents uninfected/un-treated cells. Universal IFN controls included VP24-pCAGGS as anti-IFN control. The graph represents the log10 scale TCID50/mL averages and standard deviations from three biological repetitions. ***, Significant difference of NS5-expressing cells compared to VP24-expressing cells (P < 0.01).