NEW YORK (GenomeWeb) – A team of researchers at Boston University and the United States Army Medical Research Institute of Infectious Diseases have developed a model for the early detection of Ebola infection, demonstrating that Ebola virus disease (EVD) has a uniform and predictable response to infection, and that the expression of a subset of genes was more efficacious for helping them predict disease development than other host-based indications of infection such as fever.
EVD is caused by the Ebola virus (EBOV), and the illness is characterized by fatality rates of up to 90 percent, the researchers wrote in their study in Science Translational Medicine. Clinicians currently do not have a method to diagnose EVD until symptoms appear in patients, which can strike victims anytime between two and 21 days after exposure to EBOV.
Typical nonhuman primate models of the disease use a large infectious dose and have a fairly uniform disease course, the researchers noted. In this study, however, they infected 12 cynomolgus macaques with 100 plaque-forming units of EBOV and Makona virus through intranasal exposure in order to present a model of varying times to onset of EVD. They then tracked host response over time.
The researchers clustered the macaques into four distinct groups based on disease symptoms, onset of viremia, and time of death. Three animals had an expected disease course, four had a delayed onset, and an additional three had a late onset of EVD. Overall, the population had an 83 percent fatality rate.
Investigating clinical, biochemical, physiological and transcriptomic data, the team aimed to identify biomarkers of disease onset in the group. It applied two approaches for transcriptomic analysis — RNA sequencing and a newly developed NanoString CodeSet — in order to monitor the host response via changes in RNA transcripts over time.
Using RNA-seq, the team saw enriched pathways that included activation of innate immune pathways, acute phase response signaling, and pathways associated with nitric oxide and reactive oxygen species. The top 20 regulators of downstream targets were also consistent with an overactivation of the innate immune response and showed a strongly conserved host response during the symptomatic stage of EVD.
To determine if there was evidence of host-response gene expression changes at early times after exposure, the team looked at the RNA-seq data for indications that any transcripts were induced before the occurrence of visible symptoms or quantifiable viremia. They identified multiple interferon-stimulated genes (ISGs) that were induced at or before the specimen's fever started, indicating that ISG mRNA up-regulation is a presymptomatic indicator of EVD in non-human primates.
Using the Nanostring assays, the team also saw smaller changes in RNA abundance in all groups. The immune responses in hosts mostly contained proinflammatory cytokines and general immune response genes. The cytokine response was similar to what researchers saw in past infections with 100 percent lethality, including cytokines such as IL 6, CCL8, and CXCL10.
The researchers noticed that the cytokine response developed after the interferon response and reached higher maximum levels of expression than ISGs. By modeling the timing of cytokine expression, the team saw that the the up-regulated cytokines were consistent with the cytokine storm often found immune system dysregulation.
In order to find out whether the ISGs upregulated in the primates appeared in clinical patients with EVD, the researchers compared the ISG data to ISGs that showed increased expression in a transcriptional analysis of EVD-infected patients from Guinea. The team found a genetic correlation between the two groups during the acute phase of EVD, suggesting that the genes may be upregulated early in infection in humans. The ISGs the researchers believe could act as early biomarkers include IFI44, IFI44L, IFIT2, IFIT3, IFIT5, ISG15, MX1, and OASL.
By shifting the sampling days to align the different response groups by time to symptom onset, the team observed that the host's response follows a conserved and predictable pattern of gene expression. The team generated mathematical models of gene expression to show that presymptomatic gene expression could be detected up to four days before fevers occur.
In terms of limitations, the authors acknowledged that the small sample size in the study was a byproduct of the challenges linked to primate studies. While they could detect and describe large changes, they noted that a more complete analysis would require further studies.
In addition, they could not claim anything about the infection route and its relationship to the differential onset of EVD, and noted that future primate studies may help identify any potential links between exposure route, survival, and time to onset of the disease.
The study did demonstrate, however, that lethal EVD has a uniform and predictable response to infection regardless of time to onset. While interferon response is not unique to EBOV, the researchers believe that identifying other genes with a similar expression pattern might help to isolate potential early markers of infection.