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

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-ofprincipal 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.

Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein

“…For this reason, targeted MS is gaining popularity as a promising clinical diagnostic tool in a wide variety of disease conditions such as cancer , and cardiovascular disease and can also be used to monitor the levels of proteins that are targets of therapeutic treatment . While preliminary studies have indicated feasibility, , the potential for the application of targeted MS has not been fully explored for infectious disease diagnostics.…”

BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow

“…In this study, we successfully demonstrated the serological diagnosis of HBV infection by combining BAC-DROP and LC-SRM. Interest in molecular diagnosis of viral infections using MS is growing rapidly, − as exemplified by recent attempts to diagnose COVID-19. − The high-throughput approach shown in this study to process clinical samples with BAC-DROP will be applicable not only to HBV but also to other viral infection samples and could further accelerate the MS-based diagnosis of viral and microbial infections.…”

COVID-19: A Global Emergency

“…The 2014-2016 outbreak of EVD in West Africa is the largest recorded EVD outbreak [29]. The detection of viral ribonucleic acid based on polymerase chain reaction (RT-PCR) is a standard for testing EBOVs [30]. According to the CDC, EVD is most often 80% fatal.…”