Article Figures & Data
Figures
- FIG 1
Atypical Ebola virus disease in a nonhuman primate. (a to c) Clinical health scores, rectal temperatures, and percent weight changes for NHP B5, Control 1, and Control 2. Clinical scores for the survivors are presented as mean values, and temperature and weight change are presented as mean values ± standard deviations (SD) (error bars). (d to f) Viral RNA loads (genome equivalents [GEQ] per milliliter or gram) in blood, swabs (oral, nasal, and rectal swabs), and various tissues. For swab samples, symbol shapes indicate type of sample and colors indicate NHP, consistent with other panels (i.e., blue, NHP B5; red, Control 1; orange, Control 2; gray, survivors). (g) Infectious virus loads (median tissue culture infectious dose per gram) in various tissues. LN, mesenteric lymph node; SI, small intestine. (h) Histopathology (i, iii, and v) and immunohistochemistry (ii, iv, and vi) in brain stem, stomach, and mesenteric lymph node samples. A single focus of inflammation was observed (arrow) in the brain stem (i), composed primarily of cells with histiocytic morphology. This lesion was associated with viral antigen (ii). Lesions in the stomach were composed of multiple cell types, including neutrophils, histiocytes, and multinucleated giant cells in both the submucosa (asterisk) and lamina propria (arrow) (iii); viral antigen was observed primarily in histiocytic cells (arrow) and in a few multinucleated cells (arrowhead) (iv). Central accumulations of neutrophils surrounded by a rim of histiocytes (arrowhead) and scattered multinucleated giant cells (arrows) were often observed within the mesenteric lymph node lesions (asterisk) (v), and viral antigen could be observed within all cell types within the lesion (vi). Bars, 100 μm (i and ii) and 50 μm (iii to vi).
- FIG 2
Clinical chemistry, hematology, and serology from NHP B5. Blood/plasma samples obtained from NHP B5, Control 1, Control 2, and all survivors at various time points throughout infection were analyzed for the indicated clinical chemistry (a), hematology (b), or serology (c) parameters. Data for the survivors (n = 14) are presented as mean values ± SD. Normal ranges for each clinical chemistry or hematology parameter are indicated in gray. ALT, alanine aminotransferase; IU/L, international units/liter; ALP, alkaline phosphatase; GLOB, globulin; ALB, albumin; AMY, amylase; BUN, blood urea nitrogen; TBIL, total bilirubin; CRE, creatinine; WBC, white blood cells; NEU, neutrophils; LYM, lymphocytes; MON, monocytes; PLT, platelets; RBC, red blood cells; IgM, immunoglobulin M; IgG, immunoglobulin G.
- FIG 3
Cytokine profile disturbances in NHP B5. Plasma samples obtained from NHP B5, Control 1, Control 2, and all survivors at various time points throughout infection were analyzed for the indicated cytokine levels. Data for the survivors (n = 14) are presented as mean values ± SD. IFN-γ, gamma interferon; TNF-α, tumor necrosis factor alpha; IL-1RA, interleukin-1 receptor antagonist; IL, interleukin; MIG, monokine induced by IFN-γ; MIF, macrophage migration inhibitory factor; MCP-1, monocyte chemoattractant protein 1; ITAC, IFN-inducible T-cell alpha chemoattractant; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; G-CSF, granulocyte colony-stimulating factor.
- FIG 4
Genome mutations in virus from NHP B5. (a and b) Viral RNA isolated from the virus inoculum or the indicated blood or tissue samples was subjected to next-generation sequencing. All mutations that occurred at a frequency of 5% or higher across the entire genome (a) or within the GP coding sequence (b) are indicated by heatmap, with precise frequencies provided for the latter. Mutations are defined by the change in nucleotide at a given position on the genome using single-letter lowercase code and, where applicable, the change in amino acid at a given position within the specified protein using single-letter uppercase code. (c) A diagram of the GP1-GP2 heterodimer with amino acid sequence from the internal fusion loop highlighted. Mutations T544I, E545D, and D552N are indicated. G546 and N550 are highlighted in red and indicate critical amino acids within the CA45 epitope. DPI, days postinfection; LN, mesenteric lymph node; SP, signal peptide; RBS, receptor binding site; GC, glycan cap; MLD, mucin-like domain; IFL, internal fusion loop; HR, heptad repeats; TM, transmembrane region.
- FIG 5
E545D mutation in GP prevents CA45 binding and neutralization. (a) Reactivity of CA45 and KZ52 to the wild-type (WT) EBOV GP ectodomain (GPΔTMWT) or GP ectodomains containing the E545D (GPΔTME545D) or D552N (GPΔTMD552N) mutation was measured by ELISA. Median effective concentration (EC50) values are indicated for each curve. Data are presented as the mean optical density at 650 nm (OD650) ± SD from two replicates. (b) Replication-incompetent VSV expressing a luciferase reporter and pseudotyped with either WT EBOV GP (GPWT) or GP containing the E545D (GPE545D) mutation was incubated with the indicated antibodies at various concentrations before being used to infect Vero cells (60,000 cells/well) at a MOI of 0.04. Luciferase activity was measured 24 h later and used to calculate percent neutralization ± SD. Data are representative of three replicates.
- FIG 6
E545D mutation in GP enhances virus growth and contributes to virulence. (a) Vero E6, A549, Huh7, or Tb1.Lu cells were infected with recombinant EBOV expressing EGFP and wild-type (WT) GP (EBOV-EGFP-GPWT) or a GP containing the E545D mutation (EBOV-EGFP-GPE545D) at a MOI of 0.05 and EGFP fluorescence was measured at the indicated time points after infection. Data are presented as the mean relative fluorescence unit (RFU) ± SD from three technical replicates. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (b) Virulence was assessed in ferrets infected with a target dose of 1,000 TCID50 of either EBOV-GPWT (n = 8) or EBOV-GPE545D (n = 6). Percent survival, clinical score, body temperature, and percent weight change are presented. Clinical score data are presented for individual animals, while temperature and weight change are presented as mean values ± SD. Survival curves were significantly different (**, log rank, P = 0.0053; **, Gehan-Breslow-Wilcoxon, P = 0.0051). (c) Viral RNA levels in infected ferrets were assessed by RT-qPCR and are presented as genome equivalents (GEQ) per milliliter. Histograms indicate mean values ± SD, and dots indicate values from individual animals. **, P = 0.0015; #, animals not sampled.
Supplemental Material
TABLE S1
Study outline. Outline of experiments involving all animals analyzed in this study, including NHP B5, as well as the surviving animals and control animals. Group designation, along with study ID and animal ID are indicated. Age, sex, and weight at the start of the study are provided, as well as the EBOV inoculation dose and experimental treatment regimen. Outcome is indicated in the final column. Download Table S1, DOCX file, 0.01 MB.
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TABLE S2
Clinical history of NHP B5. The clinical features of disease observed in NHP B5 throughout the experiment are detailed, according to day postinfection (DPI). Download Table S2, DOCX file, 0.01 MB.
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FIG S1
In situ hybridization and additional histopathology in NHP B5. (a) In situ hybridization revealed positive staining in the meninges (i) which often corresponded to large cells with histiocytic morphology (ii, arrow). Positive staining in the stomach (iii) was associated with a lesion (iv) in both the submucosa (arrow) and lamina propria (arrowhead). In the mesenteric lymph node, RNA was detected within lesions (v) that were primarily associated with large cells with histolytic morphology (vi). In the spleen, positive staining was limited to small areas within the white pulp germinal centers (vii, arrows) and was often observed adjacent to the central artery (viii). Scale bars represent 100 μm (i), 20 μm (ii and viii), 200 μm (iii and vii), 50 μm (iv and vi), and 500 μm (v). (b) Histopathology (i, iii, v, and vii to ix) and immunohistochemistry (ii, iv, and vi) in brain stem, stomach, mesenteric lymph node, small intestine, and lung samples. Nonsuppurative meningitis was observed (i, arrows), and viral antigen was detected in these areas (ii, arrows). In the sections of stomach examined, a single focus of inflammation was observed (iii) in the submucosa (asterisks) and extending into the lamina propria (arrow). In the mesenteric lymph node, there were multifocal areas of pyogranulomatous inflammation (v, arrows) that were associated with the presence of viral antigen (vi). In the small intestine, the lamina propria exhibited increased numbers of inflammatory cells, including lymphocytes, plasma cells, and neutrophils (vii). There are numerous necrotic cells with shrunken cytoplasm and pyknotic nuclei (vii, arrow), and siderophages (vii, arrowheads) are scattered throughout, indicating previous hemorrhage. In the lung, multifocal areas of pulmonary hemorrhage and congestion were observed (viii). At a higher magnification, evidence of interstitial pneumonia was present, characterized by alveolar septa that are hypercellular and expanded by infiltration of inflammatory cells, including neutrophils and macrophages (ix, arrow). Areas of edema (ix, asterisk) and hemorrhage were also observed in the lung. Virus antigen was not detected by immunohistochemistry in the small intestine and lung samples. Scale bars represent 20 μm (vii), 50 μm (ix), 100 μm (i and ii), 200 μm (iii and iv), and 500 μm (v, vi, and viii). Download FIG S1, TIF file, 2.9 MB.
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FIG S2
Additional clinical chemistry parameters from NHP B5. Blood/plasma samples obtained from NHP B5, Control 1, Control 2, and all survivors at various time points throughout infection were analyzed for the indicated clinical chemistry parameters. Data for the survivors are presented as mean values ± SD. Normal ranges for each clinical chemistry or hematology parameter are indicated in gray. GLU, glucose; TP, total protein; Na+, sodium; K+, potassium; Phos, inorganic phosphate; Ca2+, calcium. Download FIG S2, TIF file, 0.3 MB.
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FIG S3
Additional cytokine levels in NHP B5. Plasma samples obtained from NHP B5, Control 1, Control 2, and all survivors at various time points throughout infection were analyzed for the indicated cytokine levels. Data for the survivors are presented as mean values ± SD. IL, interleukin; MIP, macrophage inflammatory protein; IP-10, interferon-inducible protein 10; RANTES, regulated-on activation normal T-cell expressed and secreted; MDC, macrophage-derived chemokine; HGF, hepatocyte growth factor; VEGF, vascular endothelial growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor. Download FIG S3, TIF file, 0.6 MB.
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FIG S4
Additional next-generation sequencing (NGS) data. Viral RNA isolated from the virus inoculum or the indicated blood or tissue samples was subjected to next-generation sequencing. (a) All mutations across the entire genome in samples from NHP B5 that occurred at a frequency of 2% or higher. (b) The nucleotide mutation at genome position 7672 was polymorphic, with both g-to-a and g-to-c mutations identified, both resulting in amino acid mutation E545D. The top two rows of the heatmap show nucleotide mutation frequency for each individual nucleotide mutation, while the bottom row shows the combined frequency. (c) All mutations across the entire genome in blood samples from control or surviving NHPs that occurred at a frequency of 5% or higher. (d) All mutations within the GP coding sequence in blood samples from control or surviving NHPs that occurred at a frequency of 5% or higher. Mutations are defined by the change in nucleotide at a given position on the genome using single-letter lowercase code. DPI, days postinfection; LN, mesenteric lymph node; T, terminal time point. Download FIG S4, TIF file, 1.2 MB.
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FIG S5
E545D mutation reduces CA45 binding kinetics. (a) Bio-layer interferometry sensograms depicting the binding kinetics of CA45 with the wild-type (WT) EBOV GP ectodomain (GPΔTMWT) or GP ectodomains containing the E545D (GPΔTME545D) or D552N (GPΔTMD552N) mutation. (b) Binding kinetics parameters for each GP construct, including on-rate (kon), off-rate (koff), and KD. Download FIG S5, TIF file, 0.5 MB.
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FIG S6
E545D mutation in GP enhances virus growth. Vero E6, A549, Huh7, or Tb1.Lu cells were infected with recombinant EBOV expressing EGFP and wild-type (WT) GP (EBOV-EGFP-GPWT) or a GP containing the E545D mutation (EBOV-EGFP-GPE545D) at a MOI of 0.01, and EGFP fluorescence was measured at the indicated time points after infection. Data are presented as the mean relative fluorescence unit (RFU) ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Download FIG S6, TIF file, 0.2 MB.
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FIG S7
Clinical chemistry and hematology parameters in ferrets. Blood/plasma samples obtained at various time points from ferrets infected with 1,000 TCID50 of either EBOV-GPWT or EBOV-GPE545D were analyzed for the indicated clinical chemistry (a) or hematology (b) parameters. Data are presented for individual animals. ALT, alanine aminotransferase; ALP, alkaline phosphatase; TBIL, total bilirubin; AMY, amylase; GLOB, globulin; ALB, albumin; BUN, blood urea nitrogen; CRE, creatinine; GLU, glucose; TP, total protein; Na+, sodium; K+, potassium; Phos, inorganic phosphate; Ca2+, calcium; WBC, white blood cells; NEU, neutrophils; LYM, lymphocytes; MON, monocytes; PLT, platelets; RBC, red blood cells. The dotted line indicates the upper or lower detection limit for the assay. Download FIG S7, TIF file, 1.2 MB.
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