Monitoring correlates of SARS-CoV-2 infection in cell culture using a two-photon-active calcium-sensitive dye

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.

Keywords: Calcium sensors; Fluorescence imaging; SARS-CoV-2; Two-photon microscopy; Viral infections.

Fig. 1

Experimental design and manual analysis of infection in Vero E6 cells using SARS-CoV-2 variants D614G and B.1.1.7. A Fluorescence enhancement of the BEEF-CP dye upon Ca²⁺ binding is attributed to the alleviation of the photoinduced electron transfer (PET) quenching. Its low background fluorescence and high two-photon cross-section (TPCS) along with its advantageous cell internalization make BEEF-CP an appropriate candidate for cytosolic Ca²⁺ imaging with 2P microscopy. B Experimental design with 2P microscopy. A pulsed Ti:Sa laser source was used for excitation at 700 nm wavelength, and laser light was diffracted with a pair of galvo mirrors for scanning the region of interest. A high-numerical-aperture objective (Olympus 20×) was used to obtain subcellular resolution. Samples were prepared in 96-well bioassay plates and transferred directly to the microscope. Samples were moved semi-automatically to locate the different wells in the field of view of the microscope. C Exemplary images from samples where most cells correspond to a particular stage of the infection (see “Methods” for more details on the characterization criteria). D The relative number of cells in different stages of infection 48 h post infection for the two variants at two different dye concentrations at various virus titers. Data from wells with the same virus titer, variant, and dye concentration are averaged. Statistical data for the individual cell categories are shown in Supplementary Fig. 5. E 50th percentile values, i.e., the percentage of cells under the median severity of infection, for the data displayed in D. Statistical analysis of the manual cell sorting shows that (i) the ratio of healthy cells is significantly higher with lower virus titer, regardless of the virus variant or dye concentration; and (ii) for high infection levels (TCID₅₀ > 10^–3 mL⁻¹), the proportion of absent cells is higher for the D614G variant

Fig. 2

Automatic analysis of the 2P microscopy images of infected cells using SARS-CoV-2 variants D614G and B.1.1.7. A Purple contours indicate the areas automatically defined as particles during this analysis. B, C, E, F Spider charts showing the effects of B variant, C virus titer, E dye concentration, and F photomultiplier tube (PMT) relative voltage on the range-normalized values of seven different image parameters obtained from 2P microscopy images. The error bars for the spider charts can be found in Supplementary Fig. 7. D The image parameter called “relative signal area” shows a significant increase at virus titers higher than TCID₅₀ 10^–3 mL⁻¹. G The image parameter called “image mean intensity” for the two studied variants at high virus titer (TCID₅₀ > 10^–3 mL⁻¹) at 0.05 μM and 0.5 μM dye concentration. H t-Distributed stochastic neighbor embedding (t-SNE) 2D plot obtained from all the seven image parameters recorded at 0.5 µM dye concentration shows three clusters depending on the virus variant and titer. Inset shows that the three groups, namely no or low infection, infection with D614G variant, and infection with B.1.1.7 variant are clearly separated along the first dimension. I Classification of the images corresponding to different variants at various virus titer in three clusters by seven-dimensional Gaussian mixture model clustering (error bars show standard deviation; significance levels as *p ≤ 0.1; **p ≤ 0.05; ***p ≤ 0.01.)