Upright Fluorescence Microscopy for Virus Replication Studies

Communications, Corporate

September 01, 2020

Upright Fluorescence Microscopy for Virus Replication Studies

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and spread over the world quickly. Due to its dramatic impact, researchers are interested in the nature of the virus to finally stop the pandemic. One important aspect is how the virus replicates in host cells. The research of Ogando and co-workers has shed light on replication kinetics, adaption capabilities, and cytopathology of SARS-CoV-2. One of their tools is fluorescence microscopy on SARS-CoV-2 infected Vero E6 cells. By using antibodies which were produced against SARS-CoV (2002) some time before, they discovered strong cross-reactivity with SARS-CoV-2. Thus, they established a tool to study the SARS-CoV-2 replication by immunofluorescence microscopy.

Microscopy for virus observation

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.

Fig. 1: Viruses can be observed on different levels, from a tissue to a single virion. For this approach, a distinct magnification and resolution are necessary. As a result, researchers choose a microscopy technique that fits their needs.

Immunofluorescence of viral proteins

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.

Fig. 2: One of the experiments of Ogando et al. involved the use of immunofluorescence microscopy to image Vero E6 cells. In principle, they explored the potential of antisera which have been produced against SARS-CoV in rabbits (resp. mice), during the outbreak in 2002/2003. They used different rabbit antisera as a 1st set of antibodies. Then, these rabbit antisera were labeled with a 2nd set of antibodies which were fluorescent. Subsequently, fluorescence microscopy was performed with a DM6 B microscope.

SARS-CoV antisera cross-react with SARS-CoV-2

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.

Fig. 3: Immunofluorescence microscopy of SARS-CoV-2 infected Vero E6 cells which were treated with rabbit SARS-CoV antisera. Nsps can be found near the nucleus, while the M protein is located at the Golgi apparatus. Anti-dsRNA antibodies were used to label replicative intermediates of viral RNA synthesis. Nuclei were stained with Hoechst. Scale bar is 25 μm for (a) and (b); 100 μm for (c), (d) and (e). Courtesy of Ogando et al., Journal of General Virology (DOI 10.1099/jgv.0.001453).

Potential of upright fluorescence microscopy for virology research

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.

Read the original publication

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