Accelerated and improved quantification of lymphocytic choriomeningitis virus (LCMV) titers by flow cytometry.
Accelerated and improved quantification of lymphocytic choriomeningitis virus (LCMV) titers by flow cytometry.
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DOI：
10.1371/journal.pone.0037337
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Homann D
中科院分区：
文献类型：
--
作者：
Korns Johnson D;Homann D
淋巴细胞性脉络丛脑膜炎病毒（LCMV）是一种天然的小鼠病原体，是沙粒病毒家族的一员，可引起人类非典型脑膜炎，已被广泛用作病毒诱导疾病和免疫反应研究的模型病原体。从历史上看，病毒滴度是通过标准的空斑测定法来量化的，但对于包括LCMV在内的非细胞病变病毒，这需要很长时间的孵育，因此不能快速获得结果。此外，由于菌斑测定的特定技术限制，包括视觉检测格式，它具有主观性因素和有限的灵敏度。在这项研究中，我们描述了一种基于facs的检测方法的发展，该方法利用检测感染细胞中的LCMV核蛋白（NP）表达来确定病毒滴度，并且与标准斑块检测相比，该方法具有几个优势。我们表明，LCMV-NP FACS检测是一种客观且可重复的检测方法，需要更小的样本量，对空斑检测的灵敏度增加~ 20倍，产生结果的速度比空斑检测快3倍。重要的是，当应用于急性和慢性LCMV感染模型时，LCMV- np FACS检测显示，通过斑块检测确定为阴性的样品中存在感染性病毒。因此，该技术代表了一种快速、增强和客观的检测传染性LCMV的替代方法，可以适应其他病毒感染以及高通量诊断平台。
Lymphocytic choriomeningitis virus (LCMV), a natural murine pathogen, is a member of the Arenavirus family, may cause atypical meningitis in humans, and has been utilized extensively as a model pathogen for the study of virus-induced disease and immune responses. Historically, viral titers have been quantified by a standard plaque assay, but for non-cytopathic viruses including LCMV this requires lengthy incubation, so results cannot be obtained rapidly. Additionally, due to specific technical constraints of the plaque assay including the visual detection format, it has an element of subjectivity along with limited sensitivity. In this study, we describe the development of a FACS-based assay that utilizes detection of LCMV nucleoprotein (NP) expression in infected cells to determine viral titers, and that exhibits several advantages over the standard plaque assay. We show that the LCMV-NP FACS assay is an objective and reproducible detection method that requires smaller sample volumes, exhibits a ∼20-fold increase in sensitivity to and produces results three times faster than the plaque assay. Importantly, when applied to models of acute and chronic LCMV infection, the LCMV-NP FACS assay revealed the presence of infectious virus in samples that were determined to be negative by plaque assay. Therefore, this technique represents an accelerated, enhanced and objective alternative method for detection of infectious LCMV that is amenable to adaptation for other viral infections as well as high throughput diagnostic platforms.
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