Analysis of SARS-CoV-2 interactions with the Vero cell lines by scanning electron microscopy

In this study, scanning electron microscopy (SEM) was used to study the cell structure of SARS-CoV-2 infected cells. Our measurements revealed infection remodeling caused by infection, including the emergence of new specialized areas where viral morphogenesis occurs at the cell membrane. Intercellular extensions for viral cell surfing have also been observed. Our results expand knowledge of SARS-CoV-2 interactions with cells, its spread from cell to cell, and their size distribution. Our findings suggest that SEM is a useful microscopic method for intracellular ultrastructure analysis of cells exhibiting specific surface modifications that could also be applied to studying other important biological processes.


Introduction
The SARS-CoV-2 infection, COVID-19, started in December 2019 in China and has since spread throughout the world [1]. In the Czech Republic, the first cases were reported on March 1, 2020, when SARS-CoV-2 was detected in a passenger entering the Czech Republic from Italy. SARS-CoV-2 is a β-coronavirus from Coronaviridae family [1,2]. Coronaviruses are viruses with a single-stranded RNA genome [2], leading generally to a high number of mutations [3,4]. Coronaviruses mainly infect mammals and birds, and can also 1 3 infect humans and cause respiratory and enteric diseases, such as upper and lower respiratory tract infections (bronchitis, pneumonia, severe acute respiratory syndrome -SARS) [5,6]. According to Bakhshandeh et al. [4] SARS-CoV-2 "has a relatively high dynamic mutation rate with respect to other RNA viruses".
The SARS-CoV-2 genome consists of a single-stranded positive-sense RNA molecule of approximately 29,900 nucleotides arranged in 14 open reading frames (ORFs) encoding 31 proteins [7]. SARS-CoV-2 accessory proteins play an important roles in pathogenesis and virulence. The SARS-CoV-2 proteins contain two large polyproteins: ORF1a and ORF1ab; four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), which are common to all coronaviruses and are considered major therapeutic targets for antiviral drug development [7]; and eleven accessory proteins: ORF3a, ORF3b, ORF3c, ORF3d, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c and ORF10 [8][9][10]. Accessory proteins are important virulence factors involved in different mechanisms of pathogenesis during SARS-CoV-2 infection. Although some of them are not essential for replication [11], their role remains unclear in terms of their effect on pathogenesis. In addition, there are other important cellular mechanisms altered by these accessory proteins such as autophagy or apoptosis by ORF3a, mitochondrial function by ORF3d or inflammasome activation by ORF9b. However, there are still many unknowns that need to be explored to better understand not only the current SARS-CoV-2 pandemic but also future public health emergencies that could be caused by a related coronavirus. Additionally, understanding the function of these proteins could lead to the development of new therapies or vaccines against COVID-19 [11].
Understanding virus-cell interactions provides a crucial basis for the development of vaccines, as well as for the treatment and diagnoses of viral diseases. Up to now, most microscopic studies of SARS-CoV-2 have been carried out by using transmission electron microscopy (TEM) [12] , including cryogenic transmission electron microscopy. Pramanick et al. studied SARS-CoV-2 spike glycoprotein conformations by cryogenic TEM [13]. Scanning electron microscopy is a suitable method for studying, among other things, the interaction of viral particles with the cell surface, changes in its morphology, and dynamics of infection, as shown, e.g., in Caldas et al. [14] .
Isolation and rapid sharing of the 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia was described by Caly et al. [12,[15][16][17]. In the presented work, scanning electron microscopy (SEM) was used to study cellular structure of infected cells. We asked the following research questions: What are the possibilities of using scanning electron microscopy in studying SARS-CoV-2? What is the typical size distribution of SARS-CoV-2 virions? The answer to both research questions were sought using well-defined model structures of SARS-CoV-2 infected Vero cells and using standardized procedures for their preparation for SEM imaging.

Cell line and virus
The wild SARS-CoV-2 strain (kindly provided by Assoc. prof. Daniel Růžek, University of South Bohemia, České Budějovice, Czech Republic) was used in the study. The virus was propagated in Vero cell line CCL81 (Monkey African kidney cell line, purchased from Sigma-Aldrich) that was maintained in Dulbecco's Modified and 100 µg/mL Eagle's Medium (DMEM) with high glucose, containing 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin streptomycin (all from Lonza, Swiss). The Vero cell line has been widely distributed among research laboratories and has become one of the most common mammalian immortalized cell lines used in research [18]. These cells are susceptible to a wide variety of viruses, such as simian polyomavirus SV-40, rubella virus, arboviruses, adenoviruses, H5N1 influenza virus, Ebola hemorrhagic fever virus, SARS-CoV, and MERS-CoV [19].
The cells were incubated at 37 °C under 5% CO 2 and were observed every 24 hours until 80-90% of the cells exhibited a cytopathic effect (5-7 days). Afterwards, the stock SARS-CoV-2 virus was harvested, and supernatants were collected, aliquoted, and stored at -80°C. All infectious work was performed under biosafety level 3 (BSL3) conditions in the Laboratory of the Department of Epidemiology at the University of Defence, Czech Republic. The microscopic work on the fixed infected Vero cells was performed at the Department of Physics, University of Hradec Králové. During these experiments, increased biological safety was observed, work surfaces and tools used were decontaminated before and after each measurement. The researchers used protective clothing including respirators.

Infection cycle
The Vero cells were seeded on sterile glass coverslips coated with poly-D-Lysine (Merck, USA) in a 24-well plate and incubated at 37°C in 5% CO 2 until 70% confluency was reached. Subsequently, the glasses were infected with SARS-CoV-2 strain at an MOI (multiplicity of infection -the rate of virus per cell) of 0.5 in a 200 µL addition with shaking to distribute the virus. Then the complete medium was added after an absorption period of 1 h at 37 °C and 5% CO 2 . After that, the plate was incubated under the same conditions for the next 2 days.

Determination of SARS-CoV-2 infectivity by TCID50
The viral titer of SARS-CoV-2 supernatant was determined using an end-point dilution assay and expressed as 50% Tissue Culture Infectious Dose (TCID50)/mL [20] . Briefly, the Vero cells were seeded (20,000 cells per well) onto a 96-well plate (TPP, Swiss) and incubated at 37 °C under 5% CO 2 until the confluent monolayer was observed. The tenfold serial dilution of the viral stocks was prepared from 10 −1 up to 10 −8 . Subsequently, each dilution of the virus was added to the plate in hexa-plicate. Virus-untreated controls were also included. The plate was then incubated for 5 days at 37 °C in 5% CO 2 and the presence of the cytopathic effect was detected under a light microscope. The virus titer was calculated using the method of Spearman and Karber [21].

SARS-CoV-2 virion size analysis
The aim was to determine the size of the virion particles. Based on SEM observations of SARS-CoV-2-infected Vero cells, the sizes of SARS-CoV-2 virions were quantitatively evaluated. For this purpose, three SEM images were used: ImageA.tif (accelerating voltage: 20 kV, magnification: 18,000×, pixel dimensions: 2,560×1,802 px, scale: 2.757 nm/px, 315 virions processed), ImageB.tif (accelerating voltage: 15 kV, magnification: 30,000×, pixel dimensions: 5,120×3,604 px, scale: 0.833 nm/px, 104 virions processed), and ImageC.tif (accelerating voltage: 15 kV, magnification: 15,000×, pixel dimensions: 5,120×3,604 px, scale: 1.665 nm/px, 196 virions processed). Feret's diameters of the virions presented in these images were measured in ImageJ 1.52a software (imagej.nih.gov/ ij/). Each image was initially scaled by assigning the corresponding number of pixels to the given length of its scale bar. To suppress the influence of 3D perspective in the images, only virions located on the upper side of the cells, i.e., horizontally oriented parts of the cell surface, were measured. Feret's diameters were manually marked with the distance measuring tool. In this way, the size distribution of 615 virions coated with a 10 nm thick layer of platinum was obtained.

Results
Scanning electron microscopy showed that the uninfected Vero cells were flat and without distinct surface structures (Fig. 1).
SEM observations of the SARS-CoV-2-infected Vero cells revealed significant changes on their surface induced by the virions (Fig. 2). At the time of their fixation, the individual cells were at different stages of lysis of cell phases of the cycle. A large quantity of extracellular virus was present on the surface of some infected cells. On the other hand, only a few virions were found on other cells, showing an asynchronous infection. Therefore, both damaged cells (Fig. 2C), partially damaged cells (Fig. 2B), and undamaged cells were seen. Furthermore, we observed a large number of vacuoles released during the lysis of virus-infected cells (Fig. 2D).
At higher magnifications, we observed a large number of virus particles (Figs. 3 and 4). The presence of filopodia -protrusions on the cell surface -noticeably increased on the surface of infected cells after infection. The filopodial protrusions on infected cells were also substantially longer and more branched than in the case of uninfected cells. The virus was exported prolifically at the pseudopodia and cell surfaces.
The statistical analysis of SARS-CoV-2 virion sizes revealed that the virion diameters follow a normal (Gaussian) distribution, as shown in Fig. 5. Basic statistical characteristics of the results are given in Table 1.

Discussion
Our measurements revealed remodelling of cells caused by the infection, including the emergence of new specialized regions where viral morphogenesis on the cell membrane occurs. Intercellular extensions intended for virus cell surfing were seen too. Our results extend the knowledge of the SARS-CoV-2 interactions with cells and its cell-to-cell propagation. The results demonstrate the possibility of using SEM with a hot cathode (a tungsten thermionicemission gun) to study different phases of cell infection by the SARS-CoV-2 virus, visualize individual SARS-CoV-2 virions, and determine the number of SARS-CoV-2 virions present The results presented in Fig. 5 and Table 1, obtained from the SEM images ImageA, ImageB, and ImageC, are mutually similar, indicating the reproducibility of the virion size measurements. The lowest standard error was obtained in ImageA, which is probably due to the highest number of evaluated virions in this image. Slight differences in the mean virion diameters between the individual images may be caused by small variations in the focus of the images. The fact that the means are almost equal to the medians gives good evidence of a high symmetry of the measured size distributions.
The determined virion size is in good agreement with other studies: Caldas et al. [14] reported the diameter of SARS-CoV-2 viruses to be around 70 -85 nm, in Zhu et al. [17] the diameters varied from about 60 to 140 nm, in Kim et al. [11] they ranged from 70 to 90 nm, and in Prasad et al. [24] the virion size was around 70 -80 nm. Bakhshandeh et al. [4] report a wider range of SARS-CoV-2 virion diameters between 60 and 140 nm.
Unlike us, the cited studies provide only approximate ranges of virion sizes without specifying the distributions of these values. Therefore, it is not possible to directly compare the mean values or medians.

Conclusion
Our data obtained by scanning electron microscopy can provide new insights into the basic SARS-CoV-2/Vero cell interactions and facilitate the identification of targets for the development of preventive and therapeutic strategies to combat infection caused by these viruses. The observations presented should be confirmed and extended by other appropriate biological experiments. However, our findings suggest that SEM is a useful microscopic method for intracellular ultrastructure analysis of cells exhibiting specific surface modifications that could also be applied to studying other important biological processes.