SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology
NS Ogando, TJ Dalebout, JC Zevenhoven-Dobbe, RWAL Limpens, Y van der Meer, L Caly, J Druce, JJC de Vries, M Kikkert, M Bárcena, I Sidorov, EJ Snijder
Microbiology Society, 22 June 2020; https://doi.org/10.1099/jgv.0.001453
Viruses can be studied with the help of several microscopy techniques. According to the magnification and resolution of the microscope, the observation can be on the tissue level, on the cellular level, or on the virion level (Figure 1). Typically, the virion itself can be resolved only by electron microscopy or super-resolution microscopy. On a cellular level, viruses are mostly observed with the help of advanced widefield fluorescence or confocal microscopy. In a tissue, brightfield microscopy or basic widefield fluorescence microscopy can be sufficient for viral studies. But the differentiation of the microscopy techniques is not done in a strict manner.
Opto-digital image processing tools, such as Computational Clearing, can help to improve the background to signal ratio and reduce the out-of-focus blur. The associated contrast enhancement can reveal additional information in microscopic images.
Amongst sequencing techniques, bioinformatics, and electron microscopy, Ogando et al. analyzed infected cells by fluorescence microscopy: Vero E6 cells were grown on glass cover slips, infected with SARS-CoV-2, and fixed with paraformaldehyde. Then, the cells were incubated with antisera from rabbits or mice which were exposed to SARS-CoV beforehand (Figure 2). The SARS-CoV-originated antibodies, which bind to SARS-CoV-2 structures in Vero E6 cells, were then detected by fluorescently labelled secondary antibodies. In addition, nuclei were stained with Hoechst. Fluorescence imaging was done with a DM6 B upright fluorescence microscope.
Immunofluorescence microscopy revealed cross reactivity of many SARS-CoV antisera in SARS-CoV-2 infected cells (viral proteins nsp3, nsp4, nsp5, nsp8, nsp9, nsp13, nsp15, N, M). This fact means that antisera produced against SARS-CoV also lead to characteristic fluorescent staining in SARS-CoV-2-infected cells (Figure 3). Whereas nsps were found in the perinuclear region of infected cells, the N protein was spread throughout the cytosol. The M protein was detected in the Golgi apparatus.
The cross-reacting antisera described in the study of Ogando et al. will be a useful tool for the characterization of the replication cycle of SARS-CoV-2. This tool, in turn, enables researchers to define potential targets for inhibitors of replication.
A relatively simple experimental setup – immunofluorescence microscopy – is sufficient to draw conclusions of the viral replication cycle. Because the cells are grown on cover slips for this method and mounted on glass slides, an upright fluorescence microscope is a practical solution. Automated versions with a motorized stage in combination with a large field of view (FOV) help users to acquire big overviews quickly. If single snapshots are enough, a mechanical stage is the more reasonable choice.
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