Detection of virus in organs.

<p>Groups of 20 mice were infected with 2×10³ TCID₅₀ per animal of either KFDV P9605 or AHFV Zaki-1 via footpad inoculation. Two additional control groups of 5 mice each were inoculated with serum-free MEM. Five mice from each group were sacrificed at days 2, 4, 6, and 8 post-infection, and the controls groups were sacrificed at the end of study. Organs were harvested and homogenized as described in the Materials and Methods, and homogenates were titrated on BHK21 cells by limiting dilution. Results for the lung (A), brain (B), kidney (C), spleen (D), liver (E), and plasma (F) are shown. Each symbol represents one animal. Background cut-offs of 10¹ TCID₅₀ mg⁻¹ (lung in [A]), 10² TCID₅₀ mg⁻¹ (brain, kidney, spleen, and liver in [B–E]), and 10³ TCID₅₀ ml⁻¹ (plasma in [F]) were applied to organ and plasma titrations based on the control groups, and are shown as dashed lines on the lower axes of the respective graphs. Note in (F) that no animals were viremic. Each symbol represents one animal, and geometric means are indicated by horizontal black lines.</p

Hematologic abnormalities induced by KFDV P9605 and AHFV Zaki-1 infection.

<p>(A) Total RBC counts from whole blood. (B) Hemoglobin (Hb) levels from whole blood. (C) Hematocrit levels from whole blood. (D) Platelet counts from whole blood. Each symbol represents one animal, and arithmetic means are indicated by horizontal black lines.</p

Virulence of KFDV and AHFV in mice.

<p>Groups of 5 mice were infected via footpad inoculation with 2×10³ TCID₅₀ per animal of either KFDV P9605, AHFV 2003, or AHFV Zaki-1. (A) Survival curve. Death of an animal is indicated by a step down on the curve. (B) Temperature. Mice were implanted with subdermal transponders as detailed in the Materials and Methods, and the temperature was monitored daily. (C) Weight change. Mice were weighed daily, and the change in weight is shown as a percentage of the initial study weight on the day of infection. Error bars in (B) and (C) represent standard deviations.</p

<i>In vitro</i> growth kinetics of tick-borne flaviviruses.

<p>BHK21 cells were infected with either OHFV Guriev, KFDV P9605, AHFV 2003, or AHFV Zaki-1 at an MOI of 0.01, and supernatant (A) and cell-associated (B) fractions were harvested daily for 5 days. Titers are expressed as TCID₅₀ ml⁻¹, and error bars represent standard deviations.</p

Differential cytokine induction by KFDV P9605 and AHFV Zaki-1.

<p>Organ homogenates were analyzed by Bio-Plex Pro Mouse Cytokine Assay as described in the Materials and Methods. Results for IL-6, IL-10, IFN-γ, MCP-1, and TNF-α are shown for the brain (A), kidney (B), and spleen (C). All cytokine concentrations were normalized to the amount of the respective tissue that was homogenized in each sample. Error bars represent standard deviations. Statistical significance is indicated by <b>*</b> for <i>p</i><0.05 and <b>**</b> for <i>p</i><0.01.</p

White blood cell (WBC) abnormalities induced by KFDV P9605 and AHFV Zaki-1 infection.

<p>(A) Total WBC counts from whole blood. Each symbol represents one animal. (B) Lymphocyte counts from whole blood. Each symbol represents one animal. Arithmetic means in (A) and (B) are indicated by horizontal black lines. (C) Composite graph showing average absolute counts of lymphocytes, neutrophils, monocytes, eosinophils, and basophils from 5 mice per day for each virus, and 10 uninfected control animals. (D) Composite graph showing average population proportions for lymphocytes, neutrophils, monocytes, eosinophils, and basophils from 5 mice per day for each virus, and 10 uninfected control animals.</p

Immunohistochemical findings in BALB/c mice experimentally infected with KFDV P9605 and AHFV Zaki-1.

<p>(A and B) Immunohistochemistry of the brain. (A) Mock. (B) Foci of positive immunostaining for KFDV P9605 (brown) within neurons and some astrocytes at day 8 post-infection. (C and D) Immunohistochemistry of the kidney at day 4 post-infection. (C) Mock. (D) AHFV Zaki-1-infected mouse, viral antigen (brown) can be found scattered in the cortex and the medulla. Positive immunostaining for AHFV Zaki-1 in mesangial cells of the glomeruli (inset). (E and F) Immunohistochemistry of the liver on day 6 post-infection. (E) Mock. (F) AHFV Zaki-1-infected mouse. AHFV Zaki-1 immunostaining (brown) in sinusoids and Kupffer cells (inset).</p

Activity of luciferase-containing pseudotyped virus and virus-like particles in the entry assay.

<p>ND, not determined; VLPs do not encode an expressed transgene to allow titer determination.</p>a<p>Pseudotyped virus particles or VLPs were pelleted through sucrose and resuspended in 0.01 volumes of the original culture volume. A total of 0.2 ml of virus was used per assay with 10⁶ cells and values were measured after 3 h of incubation.</p>b<p>Virus was prepared as above and used to infect HEK293-mCAT-1 cells. Titer was determined by limiting dilution using GFP reporter expression to count infected cells. Values for titer and entry assay are the mean±SD of 3 independent experiments.</p

Validation of luciferase virus entry assay for ZEBOV pseudotypes and VLPs.

<p>(A) The luciferase entry assay was performed using EVP (open bars) or VSVP (solid bars) or ZEBO-VLP (diagonally hatched bars) on HEK293-mCAT-1 cells in the presence of anti-Ebola virus neutralizing antibody (KZ52, 0.3 µg/ml) or a control antibody of irrelevant specificity (anti-HA, 12CA5; Roche, 0.3 µg/ml). Data were normalized to luciferase activity in cells incubated with untreated virus. Each data point represents the mean±SD of 3 independent experiments. (B) Luciferase entry assays were performed using VSVP, EVP, or FrVP on HEK293-mCAT-1 cells in the presence of either 20 mM ammonium chloride (open bars) or 50 nM bafilomycin A1 (solid bars). Effect of ammonium chloride was also tested on ZEBO-VLP. Data were normalized to luciferase activity for untreated cells. Each data point represents the mean±SD of 3 independent experiments. (C) Kinetics of entry was measured for EVP (open squares), VSVP (closed squares), and ZEBO-VLP (open circles). Each was incubated with HEK293-mCAT1 cells at room temperature. After 10 min, cells were washed to remove unbound virus and incubated at 37°C. At indicated time intervals, aliquots of cells were withdrawn, and luciferase signals were measured. For each virus, luciferase activity at different time points was normalized to the maximum luciferase activity for that virus. Each data point represents the mean±SD of 2 independent experiments.</p

PI3K is important for early step(s) during ZEBOV infection.

<p>Vero-E6 cells were incubated with LY294002 (50 µM) or Akt inhibitor (10 µM) for 2 h in the presence of virus. Unbound virus and drug was then removed and cells were cultivated. After 10 d, cells were fixed in formalin and stained with crystal violet; plaques were then counted. Effect of the drugs on VSV (Indiana strain) infection was also tested similarly, except that plaques were counted 2 d after inoculation of cells. Open bars, ZEBOV; solid bars, VSV (* <i>p</i><0.01).</p