Antiviral activity of interferon-α/β against KFDV and VSV-GFP virion production.

<p>Antiviral activity of interferon-α/β against KFDV and VSV-GFP virion production.</p

Antiviral activity of interferon-α/β against the cytopathology of KFDV and VSV-GFP.

<p>Antiviral activity of interferon-α/β against the cytopathology of KFDV and VSV-GFP.</p

IFN-α2a does not clear KFDV infection in BHK-21 cells.

<p>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.</p

Screening interferon-α/β subtypes against KFDV.

<p>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).</p

KFDV NS5 impedes the cellular antiviral state.

<p><b>(A)</b> 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>(B)</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).</p

IFN-α2a does not clear KFDV infection in A549 cells.

<p>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.</p