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. 2004 Oct;78(19):10370-7.
doi: 10.1128/JVI.78.19.10370-10377.2004.

Analysis of Human Peripheral Blood Samples From Fatal and Nonfatal Cases of Ebola (Sudan) Hemorrhagic Fever: Cellular Responses, Virus Load, and Nitric Oxide Levels

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Analysis of Human Peripheral Blood Samples From Fatal and Nonfatal Cases of Ebola (Sudan) Hemorrhagic Fever: Cellular Responses, Virus Load, and Nitric Oxide Levels

Anthony Sanchez et al. J Virol. .
Free PMC article

Abstract

Peripheral blood samples obtained from patients during an outbreak of Ebola virus (Sudan species) disease in Uganda in 2000 were used to phenotype peripheral blood mononuclear cells (PBMC), quantitate gene expression, measure antigenemia, and determine nitric oxide levels. It was determined that as the severity of disease increased in infected patients, there was a corresponding increase in antigenemia and leukopenia. Blood smears revealed thrombocytopenia, a left shift in neutrophils (in some cases degenerating), and atypical lymphocytes. Infected patients who died had reduced numbers of T cells, CD8(+) T cells, and activated (HLA-DR(+)) CD8(+) T cells, while the opposite was noted for patients who survived the disease. Expression levels of cytokines, Fas antigen, and Fas ligand (TaqMan quantitation) in PBMC from infected patients were not significantly different from those in uninfected patients (treated in the same isolation wards), nor was there a significant increase in expression compared to healthy volunteers (United States). This unresponsive state of PBMC from infected patients despite high levels of circulating antigen and virus replication suggests that some form of immunosuppression had developed. Ebola virus RNA levels (virus load) in PBMC specimens were found to be much higher in infected patients who died than patients who survived the disease. Similarly, blood levels of nitric oxide were much higher in fatal cases (increasing with disease severity), and extremely elevated levels (>/=150 microM) would have negatively affected vascular tone and contributed to virus-induced shock.

Figures

FIG. 1.
FIG. 1.
Differential staining of peripheral blood smears (Wright's stain). Shown on the top row of images are low-magnification pictures of blood cells from an uninfected patient (A), and Ebola-S virus-infected patients with nonfatal (B) and fatal (C) disease outcomes (samples taken during the acute phase). The arrows in A point to platelets (dramatically reduced or absent in B and C). The bottom row shows higher-magnification images of differentiating and abnormal cells seen in acute-phase blood smears from Ebola-S patients: (D) plasmacytoid lymphocytes, (E) plasma cell, (F) normal neutrophil (top) and lymphoblast (bottom), (G) pseudo-Pelger cells, and (H) neutrophil with fragmented nucleus.
FIG. 2.
FIG. 2.
Flow cytometry (lymphocyte phenotyping) of PBMC samples obtained from fatal (A) and nonfatal (B) cases of Ebola-S disease. Average values were plotted as a percentage of gated cells (side scatter × CD24+ for B, T, and CD8+ T cells; side scatter × CD3+ for CD8+ HLA-DR+ and CD8+ CD38+). The numbers of specimens for each group are shown in parentheses in the legend. Arrows indicate the average values derived from four samples taken from healthy volunteers (Special Pathogens Branch workers). ND, not done. Asterisks indicate statistically significant differences between fatal and nonfatal cases (t < 0.05). The bars on the graph indicate standard errors.
FIG. 3.
FIG. 3.
Relative expression of cytokines, Fas antigen, FasL, and Ebola-S virus GP. Average values were determined from quantitative PCR (TaqMan) assays performed on samples from 48 patients with fatal disease (A) and 70 patients with nonfatal disease (B). Asterisks indicate statistically significant differences between fatal and nonfatal cases (t < 0.05). Black arrows indicate levels for 25 uninfected patients, and white arrow indicate levels for five healthy volunteers (Special Pathogens Branch workers). The bars on the graph indicate standard errors.
FIG. 4.
FIG. 4.
Relative expression of cytokines, Fas, FasL, and Ebola-S virus GP determined for three patients with severe Ebola-S disease (fatal and nonfatal) (A, B, and C) and one with mild disease (D). Black arrows indicate levels for 25 uninfected patients, and white arrows indicate levels for five healthy volunteers (Special Pathogens Branch workers).
FIG. 5.
FIG. 5.
Ebola-S virus antigen levels in blood compared with levels of relative Ebola-S virus GP gene expression in PBMC samples. Comparisons were made only on the basis of antigen levels (antigen sum optical density) regardless of time after disease onset. The bars on the graph indicate standard errors. Differences between fatal and nonfatal cases were found not to be statistically significant (t > 0.05).
FIG. 6.
FIG. 6.
NO levels in peripheral blood of Ebola-S virus-infected patients. A comparison of average NO concentrations determined for fatal and nonfatal cases is shown. The dashed line indicates a “normal” level, determined from the serum of five healthy volunteers (Special Pathogens Branch workers). Asterisks indicate statistically significant difference between fatal and nonfatal cases (t < 0.05). The bars on the graph indicate standard errors.

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