Keiser Patrick T, Anantpadma Manu, Staples Hilary, Carrion Ricardo, Davey Robert A
Department of Microbiology, National Emerging Infectious Disease Laboratories, Boston University, Boston, MA 02215, USA.
Disease Intervention & Prevention, Texas Biomedical Research Institute, San Antonio, TX 78227, USA.
Microorganisms. 2021 Jan 12;9(1):156. doi: 10.3390/microorganisms9010156.
Ongoing efforts to develop effective therapies against filoviruses rely, to different extents, on quantifying the amount of viable virus in samples by plaque, TCID, and focus assays. Unfortunately, these techniques have inherent variance, and laboratory-specific preferences make direct comparison of data difficult. Additionally, human errors such as operator errors and subjective bias can further compound the differences in outcomes. To overcome these biases, we developed a computer-based automated image-processing method for a focus assay based on the open-source CellProfiler software platform, which enables high-throughput screening of many treatment samples at one time. We compared virus titers calculated using this platform to plaque and TCID assays using common stocks of virus for 3 major Filovirus species, , , and with each assay performed by multiple operators on multiple days. We show that plaque assays give comparable findings that differ by less than 3-fold. Focus-forming unit (FFU) and TCID assays differ by 10-fold or less from the plaque assays due a higher (FFU) and lower (TCID) sensitivity. However, reproducibility and accuracy of each assay differs significantly with Neutral Red Agarose Overlay plaque assays and TCID with the lowest reproducibility due to subjective analysis and operator error. Both crystal violet methylcellulose overlay plaque assay and focus assays perform best for accuracy and the focus assay performs best for speed and throughput.
目前针对丝状病毒开发有效疗法的努力在不同程度上依赖于通过蚀斑、组织培养感染剂量（TCID）和病灶测定法来量化样本中活病毒的数量。不幸的是，这些技术存在固有差异，而且实验室特定的偏好使得数据的直接比较变得困难。此外，诸如操作员失误和主观偏差等人为错误会进一步加剧结果的差异。为了克服这些偏差，我们基于开源的CellProfiler软件平台开发了一种用于病灶测定的基于计算机的自动图像处理方法，该方法能够一次对许多治疗样本进行高通量筛选。我们将使用该平台计算的病毒滴度与使用3种主要丝状病毒（ 、 和 ）的普通病毒株进行的蚀斑和TCID测定进行了比较，每种测定由多个操作员在多天内进行。我们发现蚀斑测定法得出的结果相当，差异小于3倍。由于病灶形成单位（FFU）灵敏度较高而TCID灵敏度较低，FFU和TCID测定结果与蚀斑测定结果的差异在10倍或更小。然而，由于主观分析和操作员失误，每种测定方法的可重复性和准确性存在显著差异，中性红琼脂糖覆盖蚀斑测定法和TCID的可重复性最低。结晶紫甲基纤维素覆盖蚀斑测定法和病灶测定法在准确性方面表现最佳，而病灶测定法在速度和通量方面表现最佳。
