Research ArticleEMERGING INFECTIONS

A conserved transcriptional response to intranasal Ebola virus exposure in nonhuman primates prior to onset of fever

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Science Translational Medicine  28 Mar 2018:
Vol. 10, Issue 434, eaaq1016
DOI: 10.1126/scitranslmed.aaq1016
  • Fig. 1 Overview of the separation of animals and analysis performed.

    (A) Table showing the different animals (rows) and the different days after exposure (columns). White squares signify that the animal was not polymerase chain reaction (PCR)–positive at that time point (n.d., not detectable), blue squares indicate that they were RT-qPCR–positive, and gray squares mean that the RT-qPCR results were below the limit of quantification. The number of plus signs in the gray boxes represents the number of RT-qPCR replicates that were positive. Hashed boxes indicate no data for this time point. (B) Comparison of the four groups and their survival curves. The x axis is the days after exposure (DPE), and the y axis is the percent survival. The green line is the Normal group [nonhuman primates (NHPs) 1, 2, and 12], the blue line is the Delayed group (NHPs 3, 5, 8, and 10), the red line is the Late group (NHPs 4, 6, and 9), and the black line is the No Response group (NHPs 7 and 11). (C) Overview of the RNA quantification analysis performed on the animals. The x axis is the days after exposure, and the y axis is the different animals organized by outcome groups (Normal, green; Delayed, blue; Late, red).

  • Fig. 2 Blood chemistry data for animals over time.

    (A) Blood urea nitrogen (BUN), (B) creatinine (CRE), (C) calcium (CA), (D) gamma-glutamyl transferase (GGT), (E) alanine aminotransferase (ALT), (F) aspartate aminotransferase (AST), and (G) alkaline phosphatase (ALP). Normal, green; Delayed, blue; Late, red; and No Response, black.

  • Fig. 3 Comparison of leading pathways and upstream regulators.

    Overview of the most strongly differentially regulated pathways in the viremic animals. (A) Overview of the samples analyzed (large circles) that were symptomatic (red circles) with the number of biological replicates denoted in the circles. (B) Significantly differentially regulated pathways. The darker purple indicates a strong up-regulation of the pathways (positive z score), and the green means a down-regulation of the pathways (negative z score). The P values represent whether there is a significant number of genes in the pathway that are differentially expressed as determined by Ingenuity Pathway Analysis (IPA) through a Fisher’s exact test. *P < 0.05, **P < 10−3, and ***P < 10−6. (C) Upstream regulators from IPA. Purple denotes up-regulation, and green denotes down-regulation (log fold change). (D) Breakdown of the interferon response to the type of interferon responsive genes. The x axis is the specific types (type I, II, or III only) and overlap between the different responses. The green bars are samples from the Normal group, blue bars are samples from the Delayed group, and red bars are samples from the Late group. The different patterns correspond to different days after exposure.

  • Fig. 4 Late-state cytokine response conservation.

    Analysis of the conservation of the cytokine response. (A) Correlation plot using only the cytokine genes. The rows and columns are the same and represent a single group at a single time point. The dendogram is colored based on a k-means clustering with two centers. The first color bar at the top shows the groups (Normal, green; Delayed, blue; Late, red). The second color bar indicates when the groups become symptomatic with orange for symptomatic and with purple for those animals still in the incubation period. (B) Venn diagram of cluster 2 samples with cytokine genes that are differentially expressed. Spots lacking a value indicate 0 genes.

  • Fig. 5 Early and modest immune response at day 3 after exposure in Delayed animals.

    Analysis of the NanoString samples with only the genes identified as being up-regulated at an early time point (day 3 after exposure, Delayed). (A) MA plot of the 178 up-regulated genes at day 3 after exposure in Delayed animals. Each point represents a gene. The x axis is the log2 of the base mean counts, and the y axis is the log2 fold change relative to day 0. (B) MA plot for the same genes at day 6 after exposure. (C) Principal components analysis (PCA) of the 178 up-regulated genes across all samples. K-means clustering was performed on all principal components with k = 3 to determine the clustering. The PCA plot is showing the first two principal components. The colors represent the different clusters.

  • Fig. 6 Analysis of the pre-viremic ISG response.

    (A) Analysis of the onset of the interferon-stimulated gene (ISG) response. Large circles represent samples that were used in the analysis with red denoting symptomatic samples. The numbers indicate how many samples were used. (B) Comparison of the onset of viremia to induction of the group of ISGs. The days after exposure are on the x axis. The left y axis is the PCR values (black line), and the right is the number of genes annotated as ISGs that are up-regulated (blue line). (C) Similar plot for the Delayed animals, (D) Late animals, and (E) No Response animals.

  • Fig. 7 Correlation of NHP and human ISGs.

    Analysis of the fold induction correlation with regard to the ISG response in NHPs to human fatalities. (A) Correlation plot of all ISGs that are identified as being differentially regulated at any time point in the NHP data set. The x axis is the log2 fold change for the Normal animals at day 6 after exposure, and the y axis is the log2 fold change for the Delayed animals at day 10 after exposure (red points and line) or the Late animals at day 21 after exposure (blue points and line). The lines are linear fits. (B) Correlation of the human fatal cases (log2 fold change compared to controls) on the x axis and the fold change of the Late group (log2 fold change compared to controls) at day 21 after exposure. The line represents a linear fit of the data. (C) Correlation plot similar to (B) but with only the early ISGs shown. (D) Table of the genes in (C) with their comparison of the log2 fold change in humans and NHPs. Reported P values are from a correlation test.

  • Fig. 8 Host response to infection modeled relative to fever onset.

    Alignment and modeling of gene expression across the four NHP groups. (A) Example gene (ISG15) expression (log2 fold change, y axis) in the NanoString data set relative to day after exposure (x axis). (B) Expression of the same example gene (log2 fold change, y axis) relative to the onset of a fever (0) on the y axis. The green line is the Normal animals, the blue line is the Delayed animals, and the red line is the Late animals. (C) Logistic model fit for the expression of ISG15 relative to the onset of fever. The points are the mean log2 fold change for a group at its time relative to fever onset, and the solid line is the model fit for the fold change. (D) Comparison of the early ISG expression (black lines) logistic model fit relative to the onset of fever (x axis) to many cytokine genes (red lines). The dashed line is for a reference of when the gene expression crosses the threshold of a log2 fold change > 2.

  • Table 1 Comparison of the ISG response, quantifiable viremia, and fever.

    DPE, days post exposure; N.D., not detectable.

    GroupISG+PCR+Fever (DPE)
    Normal6 DPE6 DPE7.1 ± 0.99
    Delayed6 DPE10 DPE9.46 ± 0.06
    Late14 DPE21 DPE19 ± 0.74
    No ResponseN.D.N.D.N.D.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/434/eaaq1016/DC1

    Fig. S1. Comparison of animals before exposure.

    Fig. S2. Comparison of MCV between groups.

    Fig. S3. Correlation of cytokine expression in symptomatic animals.

    Fig. S4. Example genes from NanoString in Delayed animals.

    Fig. S5. Interferon response in NanoString CodeSet.

    Fig. S6. Histogram of deviances from curve fitting.

    Table S1. Cytokine expression in RNA-seq.

    Table S2. Early expressed genes in the Delayed group.

    Table S3. Normalized NanoString counts.

  • Supplementary Material for:

    A conserved transcriptional response to intranasal Ebola virus exposure in nonhuman primates prior to onset of fever

    Emily Speranza, Sandra L. Bixler, Louis A. Altamura, Catherine E. Arnold, William D. Pratt, Cheryl Taylor-Howell, Christina Burrows, William Aguilar, Franco Rossi, Joshua D. Shamblin, Suzanne E. Wollen, Justine M. Zelko, Timothy Minogue, Elyse Nagle, Gustavo Palacios,* Arthur J. Goff,* John H. Connor*

    *Corresponding author. Email: gustavo.f.palacios.ctr{at}mail.mil (G.P.); arthur.j.goff.civ{at}mail.mil (A.J.G.); jhconnor{at}bu.edu (J.H.C.)

    Published 28 March 2018, Sci. Transl. Med. 10, eaaq1016 (2018)
    DOI: 10.1126/scitranslmed.aaq1016

    This PDF file includes:

    • Fig. S1. Comparison of animals before exposure.
    • Fig. S2. Comparison of MCV between groups.
    • Fig. S3. Correlation of cytokine expression in symptomatic animals.
    • Fig. S4. Example genes from NanoString in Delayed animals.
    • Fig. S5. Interferon response in NanoString CodeSet.
    • Fig. S6. Histogram of deviances from curve fitting.
    • Legends for tables S1 to S3

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Cytokine expression in RNA-seq.
    • Table S2 (Microsoft Excel format). Early expressed genes in the Delayed group.
    • Table S3 (Microsoft Excel format). Normalized NanoString counts.

    [Download Tables S1 to S3]

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