Monitoring SARS-CoV-2 Surrogate TGEV Individual Virions Structure Survival under Harsh Physicochemical Environments

Abstract

Effective airborne transmission of coronaviruses via liquid microdroplets requires a virion structure that must withstand harsh environmental conditions. Due to the demanding biosafety requirements for the study of human respiratory viruses, it is important to develop surrogate models to facilitate their investigation. Here we explore the mechanical properties and nanostructure of transmissible gastroenteritis virus (TGEV) virions in liquid milieu and their response to different chemical agents commonly used as biocides. Our data provide two-fold results on virus stability: First, while particles with larger size and lower packing fraction kept their morphology intact after successive mechanical aggressions, smaller viruses with higher packing fraction showed conspicuous evidence of structural damage and content release. Second, monitoring the structure of single TGEV particles in the presence of detergent and alcohol in real time revealed the stages of gradual degradation of the virus structure in situ. These data suggest that detergent is three orders of magnitude more efficient than alcohol in destabilizing TGEV virus particles, paving the way for optimizing hygienic protocols for viruses with similar structure, such as SARS-CoV-2.

Keywords: coronavirus; disinfection; mechanical properties; physical virology; uncoating.

Figure 1

Structural characterization of TGEV virions (A) Cryo-electron tomogram presenting a section of a TGEV particle showing the spike proteins (blue arrowheads), the double layer membrane (green arrowheads) and the RNP wrapped inside (red arrowheads). Video S1 shows sections of a reconstructed cryotomogram of a group of purified TGEV virions (B) Intact virions visualized by AFM in a buffered solution. Blue arrowheads point to possible spike debris. The color bar shows the arbitrary color palette used for AFM image visualization, from 0 nm (blue) to 60 nm (white). Maximum color is set to the maximum height in each image (C) Dehydrated virion imaged in air conditions (D) Rehydrated virion imaged in liquid conditions (E) PFA-fixed virion imaged in liquid (F) Dehydrated PFA-fixed virion imaged in air (G) Height chart comparing the five populations with the corresponding AFM images labels. The native and fixed-hydrated populations had the same mean height, although the fixed particles had a narrower height distribution. Dehydration resulted in an average decrease in height of about 50 nm, although rehydration restored the height of native particles. Scale bars represent 50 nm unless otherwise indicated.

Figure 2

Mechanical characterization of TGEV particles. (A,B) Topographical images of TGEV virions after consecutive indentations. Particles can be classified in two groups: indentation resistive ((A), Video S2) and indentation sensitive ((B), Video S3) as shown in the topo images. Frame number indicates how many indentations were performed so far (C). Chart evolution of Δheight as a function of the indentation number (FDC#). The values of height are taken from the indented region (D). Height plot distribution of particles before the indentation experiments, which agrees with the scale bars of (A,B,E,F). The heat map displays all the density of pixels for all the force-distance curves (FDC) of all particles as a function of the strain (Figure S2) for the indentation-resistive (E) and the indentation-sensitive (F) particles. Reddish colors mean more density of points which points to the coincidence of many experiments.

Figure 3

Treatment of TGEV with IGEPAL 0.2% (A). Topographical images before (left) and after (right) IGEPAL treatment (B). Profiles traced over the particles before (black) and after (blue) the treatment. The time interval between images was ~30 s (C). Height distribution of TGEV particles before (black) and after (blue) treatment (n = 103). Counts taken from the distribution curve were normalized for comparison. The peak shifts from the value of the intact particle height to the height of the cores.

Figure 4

Effect of IGEPAL gradient on TGEV virions (A). Topographical images were taken while the amount IGEPAL was increased (Video S4). The frame number is shown in the upper left corner and the concentration of IGEPAL in the right (B). Height loss during the time course assay. Experimental curves are shown in grey and the average curve of 8 observations is shown in black. As a control, the height of 3 particles (Video S5) was tracked in buffer without detergent (blue).

Figure 5

Effect of ethanol gradient on TGEV virions (A). Comparison of virion height distribution after treatment with 60% ethanol, 0.2% IGEPAL or dehydration (B). Consecutive imaging of TGEV virions during the increase in ethanol concentration. The concentration of ethanol is shown in the upper left corner and frame is shown in the lower left corner (Video S6) (C). Track of the height loss during the ethanol time course. Experimental data is shown in grey, the average curve of 23 observations is shown in black and the average of 5 viruses without ethanol is shown in blue (Video S5).

Figure 6

Proposed effects for the different environmental conditions. Paraformaldehyde (PFA) treatment, which fixes the RNPs and membrane proteins to the capsid layer. Desiccation of the viral particle reduces the virion size. IGEPAL solubilizes the membrane lipids while uncovering the core and allows some RNPs to spread out around the virion. Alcohol induces both dehydration and loss of material.