Therapeutics of Ebola Hemorrhagic Fever: Whole-Genome Transcriptional Analysis of Successful Disease Mitigation

Abstract

The mechanisms of Ebola (EBOV) pathogenesis are only partially understood, but the dysregulation of normal host immune responses (including destruction of lymphocytes, increases in circulating cytokine levels, and development of coagulation abnormalities) is thought to play a major role. Accumulating evidence suggests that much of the observed pathology is not the direct result of virus-induced structural damage but rather is due to the release of soluble immune mediators from EBOV-infected cells. It is therefore essential to understand how the candidate therapeutic may be interrupting the disease process and/or targeting the infectious agent. To identify genetic signatures that are correlates of protection, we used a DNA microarray-based approach to compare the host genome-wide responses of EBOV-infected nonhuman primates (NHPs) responding to candidate therapeutics. We observed that, although the overall circulating immune response was similar in the presence and absence of coagulation inhibitors, surviving NHPs clustered together. Noticeable differences in coagulation-associated genes appeared to correlate with survival, which revealed a subset of distinctly differentially expressed genes, including chemokine ligand 8 (CCL8/MCP-2), that may provide possible targets for early-stage diagnostics or future therapeutics. These analyses will assist us in understanding the pathogenic mechanisms of EBOV infection and in identifying improved therapeutic strategies.

Transcriptional Profiling of the Immune Response to Marburg Virus Infection

“…This response is detected within 24 h postexposure, highlighting the rapid response of the circulating immune system following a respiratory challenge. This response is consistent with a strong early innate response seen in models of other virus infections (25,26,29,33,34). The cluster of genes stimulated during early infection (days 1 to 3 p.e.)…”

“…Expression of coagulation response genes following MARV infection is delayed compared to that observed in Ebola virus-infected NHPs, in which expression was observed as early as 4 days p.e. (25,29). Compared with earlier experimental results, our data suggest that the involvement of PBMCs in the thrombotic dysregulation in MARV infection is similar but not identical to that seen in Ebola virus-infected animals (16,29).…”

“…RNA was prepared from the PBMCs, labeled, and hybridized to Agilent two-color microarray chips according to previously described methods (26). All arrays were computationally analyzed using the Limma software package in Bioconductor, a suite of packages in R (25).…”

“…PBMCs were isolated from blood prediluted with saline using Accuspin system Histopaque-1077 tubes (Sigma-Aldrich, St. Louis, MO) as per the manufacturer's recommendations and were subsequently lysed in TRI Reagent LS (Sigma-Aldrich) at USAMRIID. PBMCs were processed for microarray analysis as described earlier (25). Briefly, total RNA was extracted from the TRI Reagent LS samples and then amplified using the low-input Quick Amp labeling kit (Agilent Technologies, Santa Clara, CA).…”

Inhibition of Ebola Virus by Anti-Ebola miRNAs in silico

“…In this case, we downloaded the available miRNAs from miRbase and aligned the most recently sequenced member of the Ebola virus published to date [11]. These sequences were analyzed for homologies in each of the genes of the Ebola genome for 1) seed sequence complementarity: a near-perfect alignment at miRNA seed sequences located at the 3'-untranslated region (UTR) base pair 2 to 8 that signals a successful silencing match [12] and 2) a high degree of complementarity: an 80%-90% degree level of homology of the sequences of miRNAs with each of the Ebola genes was considered as highly significant (p < 0.001) and reported to significantly reduce the "offtarget" silencing of other genes [13]. Figure 2).…”