Specific intracellular signature of SARS-CoV-2 infection using confocal Raman microscopy

Abstract

SARS-CoV-2 infection remains spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers.

Keywords: Bioanalytical chemistry; Biophysical chemistry; SARS-CoV-2.

Fig. 1. Raman confocal images of SARS-CoV-2 and MeV-infected (24 h pi) and noninfected Vero E6 cells.

A Raman images of noninfected cells Light yellow color corresponds to the highest intensities of lipids and proteins and dark shadow for the lowest intensities. B KMCA results for the noninfected cells of part A. The nucleolus is marked in dark green, the nucleus in turquoise, mitochondria and Golgi are in blue, cytoplasm in pink and lipid droplet in purple. The same color code has been used for all analyses. C SARS-CoV-2-infected Raman C-H images and D relevant KMCA illustrations for the C part. E MeV-infected Raman C-H images and F relevant KMCA illustrations for E part. Scale bars are variable between 6–10 µm as indicated.

Fig. 2. Mean Raman spectra from the cytoplasm, Golgi-mitochondria bodies, and nucleus region shown for noninfected (Control) and infected Vero E6 cells (SARS-CoV-2 and MeV) along with the standard deviation.

A Raman spectra were extracted from the cytoplasm region, B Raman spectra were extracted from Golgi-mitochondria bodies, and C Raman spectra were extracted from the nucleus region. D–F Difference Raman spectra calculated between noninfected (Control) and virus-infected cells (MeV and SARS-CoV-2 taken together) for D cytoplasm, E Golgi-mitochondria, and F nucleus region of the Vero E6 cells. G–I Difference Raman spectra calculated between the SARS-CoV-2-infected cells and MeV-infected cells and for G cytoplasm, H Golgi-mitochondria, and I nucleus region of the Vero E6 cells. The difference Raman spectra have been smoothed using the Savitzky–Golay method with points of window 15 and polynomial order 2. The Raman spectra are shifted on the y-axis for sake of clarity.

Fig. 3. Principal component analysis (PCA) of Raman spectra extracted from intracellular components of SARS-CoV-2 and MeV-infected and noninfected (Control) Vero E6 cells.

3D PCA score plot of Raman spectra from A cells’ cytoplasm and B corresponding PC loadings, C cells’ Golgi-mitochondria bodies, D corresponding PC loadings, E cells’ nucleus, and F corresponding PC loadings. The loading coefficients are shifted on the y-axis for sake of clarity.

Fig. 4. Raman model generated using support vector machine (SVM) algorithm to differentiate.

A noninfected (Control, black) from measles virus (MeV, blue) infected Vero E6 cells, B noninfected (Control, black) from SARS-CoV-2 (red) infected Vero E6 cells, and C MeV and SARS-CoV-2-infected Vero E6 cells. The model was generated using Raman spectra extracted from intracellular components: cytoplasm (Cyto, square), Golgi-mitochondria bodies (Mito, circle), and nucleus (Noy, triangle) of the Vero E6 cells (Total accuracy A 98.89%, B 97.22%, C 97.78%, PCs used 13, tenfold CV other parameters same as below). The difference Raman spectra calculated between D control minus measles virus (MeV), E control minus SARS-CoV-2, and F Measles (MeV) minus SARS-CoV-2.