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 cells were incubated at 37°C under 5% CO2 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.
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% CO2 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% CO2. 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)/mL13. 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% CO2 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% CO2 and the presence of the cytopathic effect was detected under a light microscope. The virus titer was calculated using the method of Spearman and Karber14.
Scanning electron microscopy
After 48 h post-infection, the coverslips were fixed with 2.5% glutaraldehyde (pH 7.2) for 1 h, washed with phosphate-buffers saline (PBS), and post-fixed in 1% osmium tetroxide (OsO4) for 40 minutes. After further wash cycles, the samples were dehydrated through an ethanol series (30%, 50%, 70%, 80% 90%, 95%, and 100%, 10 minutes each step). Subsequently, the Vero cells were dried in a critical point dryer CPD300 (Leica, Germany) in liquid CO2. The dry cells were sputter-coated with a 10 nm thick platinum layer using a sputter coater EM ACE200 (Leica, Germany). Imaging of the samples was conducted by a scanning electron microscope FlexSEM 1000 (Hitachi, Japan) operated in secondary electrons mode at accelerating voltages of 10 kV, 15 kV, and 20 kV.
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.