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Clin Proteomics. 2020 Mar 17;17:11. doi: 10.1186/s12014-020-09273-y. eCollection 2020.

Early detection of Ebola virus proteins in peripheral blood mononuclear cells from infected mice.

Author information

1
1Systems and Stuctural Biology Division, Protein Sciences Branch, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD USA.
2
2Therapeutic Development Center, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD USA.
3
3Applied Biological Sciences, The Johns Hopkins University Applied Physics Laboratory, 11100 Johns Hopkins Road, Laurel, MD USA.

Abstract

Background:

Detection of viral ribo-nucleic acid (RNA) via real-time polymerase chain reaction (RT-PCR) is the gold standard for the detection of Ebola virus (EBOV) during acute infection. However, the earliest window for viral RNA detection in blood samples is 48-72 h post-onset of symptoms. Therefore, efforts to develop additional orthogonal assays using complementary immunological and serological technologies are still needed to provide simplified methodology for field diagnostics. Furthermore, unlike RT-PCR tests, immunoassays that target viral proteins and/or early host responses are less susceptible to sequence erosion due to viral genetic drift. Although virus is shed into the bloodstream from infected cells, the wide dynamic range of proteins in blood plasma makes this a difficult sample matrix for the detection of low-abundant viral proteins. We hypothesized that the isolation of peripheral blood mononuclear cells (PBMCs), which are the first cellular targets of the Ebola virus (EBOV), may provide an enriched source of viral proteins.

Methods:

A mouse infection model that employs a mouse-adapted EBOV (MaEBOV) was chosen as a proof-of-principal experimental paradigm to determine if viral proteins present in PBMCs can help diagnose EBOV infection pre-symptomatically. We employed a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) platform to provide both high sensitivity and specificity for the detection and relative quantitation of viral proteins in PBMCs collected during MaEBOV infection. Blood samples pooled from animals at the post-infection time-points were used to determine the viral load by RT-PCR and purify PBMCs.

Results:

Using quantitative LC-MS/MS, we detected two EBOV proteins (vp40 and nucleoprotein) in samples collected on Day 2 post-infection, which was also the first day of detectable viremia via RT-PCR. These results were confirmed via western blot which was performed on identical PBMC lysates from each post-infection time point.

Conclusions:

While mass spectrometry is not currently amenable to field diagnostics, these results suggest that viral protein enrichment in PBMCs in tandem with highly sensitive immunoassays platforms, could lead to the development of a rapid, high-throughput diagnostic platform for pre-symptomatic detection of EBOV infection.

KEYWORDS:

Diagnostics; Ebola virus; Mouse adapted Ebola virus; Quantitative proteomics

Conflict of interest statement

Competing interestsOpinions, interpretations, conclusions and recommendations are those of the authors and not necessarily endorsed by the U.S. Army. The authors declare that they have no competing interests

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