Emi Akino, Suzuki Youichi, Odake Chinami, Sakaguchi Shoichi, Wu Hong, Nakano Takashi
Department of Microbiology and Infection Control, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Japan.
Sci Rep. 2025 Apr 11;15(1):12429. doi: 10.1038/s41598-025-97590-3.
Chikungunya virus (CHIKV) induces a massive cytopathic effect (CPE) on various cell types. Therefore, the plaque assay, a CPE-based virus titration method, remains the gold standard for quantifying the infectious units of CHIKV. However, manual plaque counting is often a labor-intensive task, especially in experiments involving multiple samples. In this study, we developed plaQuest, a stand-alone plaque-counting software running on a Windows operating system, for rapid and reliable quantification of CHIKV plaques in a 24-well plate. Our evaluation experiments using the conventional CPE-based plaque assay showed that the CHIKV plaque counts provided by plaQuest strongly correlated with the plaque counts manually determined by four analysts. In addition, the CHIKV inhibition curve of mycophenolic acid (MPA) determined by plaQuest was identical to that determined by manual counting, resulting in a similar 50% inhibitory concentration of MPA. Furthermore, the automated plaque counting by plaQuest was applicable to the evaluation of inhibitors against other RNA viruses using the CPE-based and immunostain-based plaque assay, which is an alternative titration assay for non- (or less) cytopathic viruses. Thus, our study demonstrates that plaQuest is an effective option for quantifying infectious virus titers, reducing the workload of the plaque assay.
基孔肯雅病毒（CHIKV）对多种细胞类型会产生大规模细胞病变效应（CPE）。因此，空斑试验作为一种基于CPE的病毒滴定方法，仍然是量化CHIKV感染单位的金标准。然而，手动计数空斑往往是一项劳动强度大的任务，尤其是在涉及多个样本的实验中。在本研究中，我们开发了plaQuest，这是一款运行在Windows操作系统上的独立空斑计数软件，用于快速、可靠地量化24孔板中的CHIKV空斑。我们使用传统的基于CPE的空斑试验进行的评估实验表明，plaQuest提供的CHIKV空斑计数与四位分析人员手动确定的空斑计数高度相关。此外，plaQuest测定的霉酚酸（MPA）的CHIKV抑制曲线与手动计数测定的曲线相同，导致MPA的50%抑制浓度相似。此外，plaQuest的自动空斑计数适用于使用基于CPE和基于免疫染色的空斑试验评估针对其他RNA病毒的抑制剂，这是一种针对非（或较少）细胞病变病毒的替代滴定试验。因此，我们的研究表明，plaQuest是量化感染性病毒滴度的有效选择，可减少空斑试验的工作量。
