PDF(Pubmed)
Abstract：
BACKGROUND: The organism-wide effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection are well studied, but little is known about the dynamics of how the infection spreads in time among or within cells due to the scarcity of suitable high-resolution experimental systems. It has been reported that SARS-CoV-2 infection pathways converge at calcium influx and subcellular calcium distribution changes. Imaging combined with a proper staining technique is an effective tool for studying subcellular calcium-related infection and replication mechanisms at such resolutions.
METHODS: Using two-photon (2P) fluorescence imaging with our novel Ca-selective dye, automated image analysis and clustering analysis were applied to reveal titer and variant effects on SARS-CoV-2-infected Vero E6 cells.
RESULTS: The application of a new calcium sensor molecule is shown, combined with a high-end 2P technique for imaging and identifying the patterns associated with cellular infection damage within cells. Vero E6 cells infected with SARS-CoV-2 variants, D614G or B.1.1.7, exhibit elevated cytosolic calcium levels, allowing infection monitoring by tracking the cellular changes in calcium level by the internalized calcium sensor. The imaging provides valuable information on how the level and intracellular distribution of calcium are perturbed during the infection. Moreover, two-photon calcium sensing allowed the distinction of infections by two studied viral variants via cluster analysis of the image parameters. This approach will facilitate the study of cellular correlates of infection and their quantification depending on viral variants and viral load.
CONCLUSIONS: We propose a new two-photon microscopy-based method combined with a cell-internalized sensor to quantify the level of SARS-CoV-2 infection. We optimized the applied dye concentrations to not interfere with viral fusion and viral replication events. The presented method ensured the proper monitoring of viral infection, replication, and cell fate. It also enabled distinguishing intracellular details of cell damage, such as vacuole and apoptotic body formation. Using clustering analysis, 2P microscopy calcium fluorescence images were suitable to distinguish two different viral variants in cell cultures. Cellular harm levels read out by calcium imaging were quantitatively related to the initial viral multiplicity of infection numbers. Thus, 2P quantitative calcium imaging might be used as a correlate of infection or a correlate of activity in cellular antiviral studies.
METHODS: Using two-photon (2P) fluorescence imaging with our novel Ca-selective dye, automated image analysis and clustering analysis were applied to reveal titer and variant effects on SARS-CoV-2-infected Vero E6 cells.
RESULTS: The application of a new calcium sensor molecule is shown, combined with a high-end 2P technique for imaging and identifying the patterns associated with cellular infection damage within cells. Vero E6 cells infected with SARS-CoV-2 variants, D614G or B.1.1.7, exhibit elevated cytosolic calcium levels, allowing infection monitoring by tracking the cellular changes in calcium level by the internalized calcium sensor. The imaging provides valuable information on how the level and intracellular distribution of calcium are perturbed during the infection. Moreover, two-photon calcium sensing allowed the distinction of infections by two studied viral variants via cluster analysis of the image parameters. This approach will facilitate the study of cellular correlates of infection and their quantification depending on viral variants and viral load.
CONCLUSIONS: We propose a new two-photon microscopy-based method combined with a cell-internalized sensor to quantify the level of SARS-CoV-2 infection. We optimized the applied dye concentrations to not interfere with viral fusion and viral replication events. The presented method ensured the proper monitoring of viral infection, replication, and cell fate. It also enabled distinguishing intracellular details of cell damage, such as vacuole and apoptotic body formation. Using clustering analysis, 2P microscopy calcium fluorescence images were suitable to distinguish two different viral variants in cell cultures. Cellular harm levels read out by calcium imaging were quantitatively related to the initial viral multiplicity of infection numbers. Thus, 2P quantitative calcium imaging might be used as a correlate of infection or a correlate of activity in cellular antiviral studies.
摘要：
背景：对严重急性呼吸道综合症冠状病毒2（SARS-CoV-2）病毒感染的全生物体影响进行了充分研究，但是由于缺乏合适的高分辨率实验系统，对感染如何在细胞之间或细胞内及时传播的动力学知之甚少。据报道，SARS-CoV-2感染途径在钙流入和亚细胞钙分布变化时趋同。成像结合适当的染色技术是以这种分辨率研究亚细胞钙相关感染和复制机制的有效工具。
方法：使用我们的新型Ca选择性染料进行双光子（2P）荧光成像，自动图像分析和聚类分析用于揭示SARS-CoV-2感染的VeroE6细胞的滴度和变异效应。
结果：显示了一种新的钙传感器分子的应用，结合高端2P技术进行成像和识别与细胞内细胞感染损伤相关的模式。VeroE6细胞感染SARS-CoV-2变体，D614G或B.1.1.7，表现出胞浆钙水平升高，允许通过内化钙传感器跟踪钙水平的细胞变化来监测感染。成像提供了关于在感染期间钙的水平和细胞内分布如何被扰乱的有价值的信息。此外，双光子钙感应允许通过图像参数的聚类分析来区分两种研究的病毒变体的感染。这种方法将有助于研究感染的细胞相关性及其取决于病毒变体和病毒载量的定量。
结论：我们提出了一种新的基于双光子显微镜的方法，该方法与细胞内化传感器相结合，以量化SARS-CoV-2感染的水平。我们优化了应用的染料浓度以不干扰病毒融合和病毒复制事件。所提出的方法确保了对病毒感染的正确监测，复制,细胞命运它还能够区分细胞损伤的细胞内细节，如液泡和凋亡体的形成。使用聚类分析，2P显微镜钙荧光图像适用于区分细胞培养物中的两种不同病毒变体。通过钙成像读出的细胞伤害水平与感染数量的初始病毒复数定量相关。因此，2P定量钙成像可用作感染的相关性或细胞抗病毒研究中活性的相关性。
方法：使用我们的新型Ca选择性染料进行双光子（2P）荧光成像，自动图像分析和聚类分析用于揭示SARS-CoV-2感染的VeroE6细胞的滴度和变异效应。
结果：显示了一种新的钙传感器分子的应用，结合高端2P技术进行成像和识别与细胞内细胞感染损伤相关的模式。VeroE6细胞感染SARS-CoV-2变体，D614G或B.1.1.7，表现出胞浆钙水平升高，允许通过内化钙传感器跟踪钙水平的细胞变化来监测感染。成像提供了关于在感染期间钙的水平和细胞内分布如何被扰乱的有价值的信息。此外，双光子钙感应允许通过图像参数的聚类分析来区分两种研究的病毒变体的感染。这种方法将有助于研究感染的细胞相关性及其取决于病毒变体和病毒载量的定量。
结论：我们提出了一种新的基于双光子显微镜的方法，该方法与细胞内化传感器相结合，以量化SARS-CoV-2感染的水平。我们优化了应用的染料浓度以不干扰病毒融合和病毒复制事件。所提出的方法确保了对病毒感染的正确监测，复制,细胞命运它还能够区分细胞损伤的细胞内细节，如液泡和凋亡体的形成。使用聚类分析，2P显微镜钙荧光图像适用于区分细胞培养物中的两种不同病毒变体。通过钙成像读出的细胞伤害水平与感染数量的初始病毒复数定量相关。因此，2P定量钙成像可用作感染的相关性或细胞抗病毒研究中活性的相关性。
