key: cord-0703238-pf9c9tnz
authors: Lyonnais, Sébastien; Hénaut, Mathilde; Neyret, Aymeric; Merida, Peggy; Cazevieille, Chantal; Gros, Nathalie; Chable-Bessia, Christine; Muriaux, Delphine
title: Atomic force microscopy analysis of native infectious and inactivated SARS-CoV-2 virions
date: 2021-06-04
journal: Sci Rep
DOI: 10.1038/s41598-021-91371-4
sha: 59c4f474d8076770bbd3cb8068074b78e11ddf88
doc_id: 703238
cord_uid: pf9c9tnz

SARS-CoV-2 is an enveloped virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Here, single viruses were analyzed by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess at the nanoscale level and in 3D infectious virus morphology in its native conformation, or upon inactivation treatments. AFM imaging reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol combining AFM and plaque assays allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-AFM is a remarkable toolbox for rapid and direct virus analysis based on nanoscale morphology.

We first controlled that the virus isolate was SARS-CoV-2 by revealing the presence of the M, N, and S viral proteins in infected cell lysates using immunoblots, by quantitative RT-PCR targeting the E gene overtime on the cell culture supernatant, and by imaging fixed infected cells by Transmission Electron Microscopy (TEM) (Supplementary Figure S1 ). One can see that the infected cells are producing SARS-CoV-2. Native infectious SARS-CoV-2 from the clarified infected cell supernatant were then directly visualized by AFM using a forcecurve based imaging mode, after smooth adsorption of the sample on an appropriate AFM surface (mica or glass coverslip). Viruses did not adsorb on freshly cleaved mica or glass coverslip (not shown), but were found to bind very quickly (2-3 min) on poly-l-lysine coated mica or glass coverslips coated with an alkyl silane to make them hydrophobic 20 . Virus adsorption on poly-l-lysine coated mica surface showed SARS-CoV-2 virions as roughly spherical or ellipsoidal particles (Fig. 1) , consistent with previous electron microscopy and AFM studies. Surprisingly, a fraction of the viral particles appeared embedded in a network of thin filamentous structures adhering to the surface ( Fig. 1a and Supplementary Figure S2 ), in addition to free particles, suggesting that SARS-CoV-2 particles could be released from the infected culture cells as viral "packages" containing tethered particles. As seen by Kiss et al., the AFM images showed "bald" native viral particles with a blurred, smooth surface, without clear S trimers protruding from the viral surface, even from viruses directly imaged from cell supernatant. This has been attributed to the high mobility of spikes and their rapid motion on virus surface, which thus evade the AFM cantilever tip 18 . We also consider that virion movements on the mica surface during adsorption could break the long spike proteins. Virions adsorbed on the hydrophobic surface showed equivalent topographical structure and the method did not help to resolve S trimers (Fig. 1c) . On this surface, however, images based on the slope of the force-distance curve for an applied force of 1 nN showed particles with a donut-shape, hollowed out in the center and displaying a crown of increased stiffness in the range of 10-20 pN/nm (Fig. 1c) , similar to the stiffness measured by indentation 18 . This supports the hypothesis that SARS-CoV-2 is easily deformable and that the ribonucleoprotein complex, made of N and the viral RNA, contributes little to the viral mechanics. TEM observation of the virus-producing cells confirmed unambiguously the typical structure of coronavirus particles, containing granular densities corresponding to the viral nucleoprotein and showing protruding spikes S proteins incorporated into the viral lipid envelope 22 (Fig. 1d) . The mean central height of the virions (Fig. 1e ) ranged from 45 to 140 nm on both surfaces, with a mean height of 89 nm ± 19 nm (Fig. 1f ), consistent with viral particle diameters in the literature measured by AFM 18 and very close from the 91 ± 11 nm value obtained by cryo-electron tomography for SARS-CoV-2 6 and for other coronaviruses 21 , which are ranging from 50 to 150 nm. By TEM, dehydrated particles show diameters distribution in the range from 40 to 95 nm (mean 61 nm ± 10 nm) , in good accordance with AFM measurement of on SARS-CoV-2 fixed with 5% glutaraldehyde 18 and the known ≈ 25% shrinkage of samples with the dehydration procedure used here for TEM sample preparation 23 .

We then examined and compared virus inactivation using FA and heat, to identify a condition keeping structurally intact SARS-CoV-2 particles. Upon heat or FA treatments, followed by ultra-filtration, viral particles were analyzed directly by AFM for nanoscale morphology (compare Fig. 2a for WT with no treatment with Fig. 2b with treatments) and by plaque assays for infectivity (Table 1 , Supplementary Figure S3 ). SARS-CoV-2 incubated at 58 °C for 30 min was successfully inactivated (Table 1) , as described for other coronavirus 10, 11 . AFM analysis of heated viruses showed severely damaged particles that have lost their spherical shapes (Fig. 2b) . On the other hand, we observed complete inactivation of SARS-CoV-2 after incubation at 20 °C for 30 min with 0.5%, 1%, 2%, or 3.6% FA (see Table 1 ) evaluating infectivity by plaque assays (Supplementary Figure S3 ). AFM analysis of FA-treated viruses showed unaltered particles with a shape and height similar to the untreated control upon 0.5% or 1% FA treatment (Fig. 2b) , while the higher percentage dramatically damaged viral particles, whose morphology is then similar to the samples heated to 58 °C, i.e. a loss of spherical shape and an altered average height ( Fig. 2c -e). FA-fixation did not reveal the S glycoprotein as seen by others and the protrusions of the FA-treated virions did not correspond to S spikes 18 . As shown previously 10, 13 , the FA inactivation effect was temperature-dependent and was strongly reduced at 4 °C. Indeed, SARS-CoV-2 was still infectious for a lower concentration of 0.1% FA with incubation at 4 °C (Table 1 , Supplementary Figure S3 ). Altogether, these results show that SARS-CoV-2 at a concentration of 1-2 × 10 6 PFU/ml is inactivated at 20 °C using 0.5% or 1% FA for 30 min at RT, without major loss of virus physical integrity. In contrary, keeping the virus at 4 °C in buffer, or up to 48 h at 20 °C, has no incidence on its infectious titer (Table 1) indicating a strong stability of SARS-CoV2 particles in buffer ( Fig. 1a and Supplementary Figure S2 ).

In conclusion, in front of the urgent need to perform researches on SARS-CoV-2 to fight COVID-19, the inactivation methods described here can contribute to transfer the virion from the confined laboratory to the lower biosafety class laboratory in an inactivated, non-infectious, form preserving viral morphology. We also show that a low percentage of FA treatment allows retaining the physical integrity of the particles albeit non-infectious. High-resolution AFM analysis of SARS-CoV-2 in buffer also demonstrates its ability to provide direct and fast qualitative information on infectious virus morphology and proves to be a method of choice for analyzing viral preparations with exceptional precision and rapidity.

Virus and cell culture. VeroE6 cells (African green monkey kidney cells) were obtained from ECACC and maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37 °C with 5% CO 2 . The strain BetaCoV/France/IDF0372/2020, was supplied by the National Reference Center for Respiratory Viruses hosted by Institut Pasteur (Paris, France) and headed by Pr. Sylvie van der Werf. The human sample from which strain BetaCoV/France/IDF0372/2020 was isolated has been provided by Dr. X. Lescure 

were washed twice in PBS, detached with versen, pelleted at 250 × g for 6 min, and lysed in RIPA buffer. Total protein concentration was calculated using a Bradford protein assay kit. 20 µg of total cell lysates were diluted in Laemmli buffer and proteins were separated by SDS-PAGE on 8% (for S-protein) and 10% (for M-and N-protein) acrylamide gels. Gels were transferred to PVDF membrane using wet transfer with Tris-glycine-methanol buffer. Membranes were washed in TBS, blocked with 5% milk in TBS-T for 30 min and incubated overnight at 4 °C with primary antibody specific for spike (Gentex, cat# GTX632604), N-protein (Gentex, cat# GTX632269) or M-protein (Tebu, cat# 039100-401-A55), all three diluted at 1:1000 in TBS-T. After washing with 5% milk in TBS-T, the membranes were incubated with HRP conjugated anti-mouse antibodies for N and S protein, and with HRP conjugated anti-rabbit antibody for M protein for 2 h at room temperature, washed again in TBS-T, incubated with ECL reagent (Amersham cat#RPN2236) and imaged using a Chemidoc Imager (Biorad).

All inactivation conditions were performed with a starting viral stock of 1-2 × 10 6 plaque-forming units/ml (PFU/ml). Heat inactivation was performed by incubating 100 μl of SARS-CoV-2 stock at 58 °C for 30 min. Formaldehyde inactivation was performed by incubating 90 µl of virus supplied by 10 × FA-Hepes (0.5 M) at 4 °C or RT (20 °C) for 30 min, 2 h or up to overnight at 4 °C depending on the conditions described in Table 1 Atomic force microcopy. Freshly cleaved muscovite mica sheets (V1 grade, Ted Pella, Inc.) were glued on a glass slide, coated for 10 min at 20 °C with 0.1% poly-L-lysine (Sigma), rinsed with 3 ml of buffer A (10 mM Tris-HCl pH 7.5 and NaCl 100 mM) and dried with a N 2 flux. A plastic O-ring (JPK Instruments) was then glued on the glass slide to assemble a small liquid cell. Virus samples were diluted four-fold in buffer A and 200 µL were deposited on the mica to allow passive virus adsorption. The liquid cell was completed with 200 µl of buffer A before imaging. Glass coverslips coated with hexamethyldisilazane (HDMS, Sigma) were prepared according to 20 and AFM imaging was performed using a coverslip holder adapted to the AFM (Bruker) using the same procedure as for poly-l-Lysine coated mica. AFM imaging was performed at room temperature on a NanoWizard IV atomic force microscope (JPK Instruments-Bruker, Berlin, Germany) mounted on an inverted optical microscope (Nikon Instruments, Japan) and operating in a BSL3 laboratory. The modifications of the Bio-AFM included an additional sealing of the AFM scanner head, protection of electronic parts, and a specific machining (smoothing) of the metallic elements (object holder, cantilever) avoiding sharp edges. Topographic Table 1 . Efficiency of SARS-CoV-2 inactivation by FA in relation to temperature and incubation time. Virus titer was determined by plaque assays (see Supplementary Figure S3 ). www.nature.com/scientificreports/ imaging was performed in quantitative imaging (QI) mode, which is a force-curve-based imaging mode, using BL-AC40TS-C2 cantilevers (mean cantilever spring constant k cant = 0.09 N/m, Olympus). Before each experiment, the sensitivity and spring constant (thermal noise method) of the cantilever were calibrated. The applied force was kept at 150 pN and a constant approach/retract speed of 10 µm/s (z range of 100 nm). For Fig. 1c , the applied force was raised to 1 nN and a constant approach/retract speed of 1 µm/s. Images were flattened with a polynomial/histogram line-fit with the JPK data processing software. Low-pass Gaussian and/or median filtering was applied to remove minor noise from the images. The Z-color scale in all images is given as relative after processing, with the offset being kept the same within each of the figures to emphasize the structural features. Particle height analysis was carried out using the height (measured) channel of the QI mode, which corresponds to the height at 80% of the setpoint force determined on the reference F-D curve. Height was calculated as the topographical maximal central height on each virion using the section tool of the analysis software, and the histogram tool of the z channel values using a 300 × 300 nm area for each particle.

Transmission electron microcopy. VeroE6 cells were infected with 1 × 10 6 PFU of SARS-CoV-2 for 24 h. Cells were fixed with 2.5% (v/v) glutaraldehyde in PHEM buffer and post-fixed in osmium tetroxide 1% / K 4 Fe(CN) 6 

A new coronavirus associated with human respiratory disease in China

Characteristics of SARS-CoV-2 and COVID-19

Molecular architecture of the SARS-CoV-2 virus

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Structures and distributions of SARS-CoV-2 spike proteins on intact virions

In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges

Methods of Inactivation of SARS-CoV-2 for downstream biological assays

Rapid and complete inactivation of SARS-CoV-2 by ultraviolet-C irradiation

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV

Stability and inactivation of SARS coronavirus

Formaldehyde fixation

Evaluation of virus inactivation by formaldehyde to enhance biosafety of diagnostic electron microscopy

Formaldehyde inactivation of foot-and-mouth disease virus. Conditions for the preparation of safe vaccine

A review of theoretical, experimental, and practical considerations in the use of formaldehyde for the inactivation of poliovirus

Atomic force microscopy in imaging of viruses and virus-infected cells. Microbiol

Single-particle virology

Topography, spike dynamics, and nanomechanics of individual native SARS-CoV-2 virions

Interpretation of SARS-CoV-2 behaviour on different substrates and denaturation of virions using ethanol: An atomic force microscopy study

How to perform a nanoindentation experiment on a virus

Supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy

Chapter one-Supramolecular architecture of the coronavirus particle

Artifacts caused by dehydration and epoxy embedding in transmission electron microscopy

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

/scientificreports/ University through a Montpellier Université d'Excellence (MUSE) support. The BSL3-AFM was funded by the REDSAIM project

We thank Dr Olivier Moncorgé, Dr Caroline Goujon and Dr Raphaël Gaudin from The Institut de Recherche en Infectiologie de Montpellier (IRIM) for initial advices in virus production and plaque assay setup. We also thank Torsten Müller from Bruker-JPK for his support. We are grateful to Dr Edouard Tuaillon and Dr Vincent Foulongne for provision of the SARS-CoV-2 strain from the Centre de Ressources Biologiques collection of the University Hospital of Montpellier, France, and to Dr Monsef Benkirane (IGF, Montpellier, France) for providing the first amplification of this virus on VeroE6 cells. This study was supported by the CNRS and Montpellier

The authors declare no competing interests.

The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598-021-91371-4.Correspondence and requests for materials should be addressed to S.L. or D.M.Reprints and permissions information is available at www.nature.com/reprints.Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.