Mahid Maharah Binte Abdul, Bist Pradeep, Sigmundsson Kristmundur, Mazlan Muhammad Danial Bin Mohd, Watanabe Satoru, Choy Milly M, Vasudevan Subhash G, Chan Kitti Wing Ki
Programme in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore, Singapore.
Programme in Cardiovascular & Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore, Singapore.
Bio Protoc. 2024 Oct 20;14(20):e5084. doi: 10.21769/BioProtoc.5084.
Dengue virus (DENV), a common and prevalent mosquito-borne endemic disease, is caused by four serotypes (DENV-1-4) and has spread rapidly on a global scale over the past decade. A crucial step in the development of antiviral therapeutics requires the utilization of in vitro cell-based techniques, such as plaque assays and focus-forming assays (FFA) for virus quantification. Vero cells have been widely used for FFA and plaque assay; however, there are instances when their efficacy and efficiency in the detection of certain clinical DENV isolates are low. Here, we showed that BHK-21 cells are more sensitive than Vero cells in the detection of all DENV-1-4 plaques and foci. In addition, we developed an improved FFA protocol for the quantification of all four DENV serotypes. Using a pan-flavivirus envelope (E) antibody, we reduce the possibility of false positives by defining a focus to consist of a minimum of eight infected cells. We outlined a protocol using the Operetta® high-content imaging system to automate the digital capture of these infected cells. A pipeline was also designed using the CellProfilerTM automated image analysis software to detect these foci. We then compare the results of the improved FFA with plaque assay. Notably, the improved FFA detected clear foci of the DENV-4 strain that does not form distinct plaques. We subsequently demonstrated the potential application of the improved FFA protocol in antiviral testing, utilizing a nucleoside inhibitor of DENV, NITD008 as a control. The protocol is amenable to a diverse array of applications, including high-throughput compound screening (HTS). Key features • An improved focus-forming assay that has the potential to quantify the DENV-4 strain, which was previously hard to plaque. • Improvements have been made to reduce the possibility of false positives. • Improved workflow using automated digital imaging process and counting of foci. • Applicable to antiviral compounds screening and is amenable to high-throughput screening.
登革病毒（DENV）是一种常见且流行的蚊媒地方性疾病，由四种血清型（DENV-1-4）引起，在过去十年中已在全球范围内迅速传播。抗病毒治疗药物研发的关键步骤需要利用基于体外细胞的技术，如噬斑测定和焦点形成测定（FFA）来进行病毒定量。Vero细胞已被广泛用于FFA和噬斑测定；然而，在某些情况下，它们检测某些临床DENV分离株的效力和效率较低。在这里，我们表明BHK-21细胞在检测所有DENV-1-4噬斑和病灶方面比Vero细胞更敏感。此外，我们开发了一种改进的FFA方案，用于定量所有四种DENV血清型。使用泛黄病毒包膜（E）抗体，我们通过将一个焦点定义为由至少八个感染细胞组成，降低了假阳性的可能性。我们概述了一种使用Operetta®高内涵成像系统自动数字捕获这些感染细胞的方案。还使用CellProfilerTM自动图像分析软件设计了一个管道来检测这些病灶。然后我们将改进的FFA结果与噬斑测定结果进行比较。值得注意的是，改进的FFA检测到了不形成明显噬斑的DENV-4毒株的清晰病灶。随后，我们利用DENV的核苷抑制剂NITD008作为对照，证明了改进的FFA方案在抗病毒测试中的潜在应用。该方案适用于多种应用，包括高通量化合物筛选（HTS）。关键特性 • 一种改进的焦点形成测定，有可能对以前难以形成噬斑的DENV-4毒株进行定量。 • 已进行改进以降低假阳性的可能性。 • 使用自动数字成像过程和病灶计数改进了工作流程。 • 适用于抗病毒化合物筛选，适合高通量筛选。
