Abstract
BackgroundDetection 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.MethodsA 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.ResultsUsing 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.ConclusionsWhile 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.
Highlights
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
mouse-adapted EBOV (MaEBOV) infection recapitulates many facets of human Ebola virus disease (EVD) and has been used as a preclinical model to evaluate the efficacy of antiviral therapeutics, vaccines, and to investigate EBOV pathogenesis in the context of the mouse immune system [16, 17]
After purification of Peripheral blood mononuclear cell (PBMC) from each PI time-point, lysates were prepared in RIPA buffer, samples were removed from bio-containment
Summary
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. The earliest window for viral RNA detection in blood samples is 48–72 h post-onset of symptoms. Real-time polymerase chain reaction (RT-PCR) is the gold standard for the detection of EBOV during acute infection in outbreak settings, but the earliest window for viral genomic RNA detection in blood samples is roughly 48–72 h postonset of symptoms [1, 2]. Current commercially available RT-PCR and immunoassay tests for EVD diagnosis are limited for clinical conditions after onset of clinical symptoms [4] where the likelihood of the virus being transmitted is significantly increased and the outcome of current medical interventions are less effective
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
