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. 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. 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. 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.

中文翻译：

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早期检测感染小鼠外周血单个核细胞中的埃博拉病毒蛋白

通过实时聚合酶链反应 (RT-PCR) 检测病毒核糖核酸 (RNA) 是急性感染期间检测埃博拉病毒 (EBOV) 的金标准。然而，血液样本中病毒 RNA 检测的最早窗口是症状发作后 48-72 小时。因此，仍然需要努力使用互补的免疫学和血清学技术开发额外的正交测定，以便为现场诊断提供简化的方法。此外，与 RT-PCR 测试不同，针对病毒蛋白和/或早期宿主反应的免疫测定不太容易受到病毒遗传漂移导致的序列侵蚀。尽管病毒从受感染的细胞中脱落到血液中，但血浆中蛋白质的广泛动态范围使其成为检测低丰度病毒蛋白质的困难样品基质。我们假设作为埃博拉病毒 (EBOV) 的第一个细胞靶标的外周血单个核细胞 (PBMC) 的分离可能提供丰富的病毒蛋白来源。选择采用小鼠适应型 EBOV (MaEBOV) 的小鼠感染模型作为原理验证实验范式，以确定 PBMC 中存在的病毒蛋白是否有助于在症状前诊断 EBOV 感染。我们采用液相色谱与串联质谱 (LC-MS/MS) 平台相结合，为 MaEBOV 感染期间收集的 PBMC 中病毒蛋白的检测和相对定量提供高灵敏度和特异性。在感染后时间点从动物收集的血液样本用于通过 RT-PCR 确定病毒载量并纯化 PBMC。使用定量 LC-MS/MS，我们在感染后第 2 天收集的样本中检测到两种 EBOV 蛋白（vp40 和核蛋白），这也是通过 RT-PCR 检测到病毒血症的第一天。这些结果通过蛋白质印迹得到证实，蛋白质印迹在每个感染后时间点对相同的 PBMC 裂解物进行。虽然质谱法目前不适用于现场诊断，但这些结果表明，PBMC 中的病毒蛋白富集与高度敏感的免疫分析平台相结合，可能会导致开发用于症状前检测 EBOV 感染的快速、高通量诊断平台. 这些结果通过蛋白质印迹得到证实，蛋白质印迹在每个感染后时间点对相同的 PBMC 裂解物进行。虽然质谱法目前不适用于现场诊断，但这些结果表明，PBMC 中的病毒蛋白富集与高度敏感的免疫分析平台相结合，可能会导致开发用于症状前检测 EBOV 感染的快速、高通量诊断平台. 这些结果通过蛋白质印迹得到证实，蛋白质印迹在每个感染后时间点对相同的 PBMC 裂解物进行。虽然质谱法目前不适用于现场诊断，但这些结果表明，PBMC 中的病毒蛋白富集与高度敏感的免疫分析平台相结合，可能会导致开发用于症状前检测 EBOV 感染的快速、高通量诊断平台.