Atomic force microscopy analysis of native infectious and inactivated SARS-CoV-2 virions

Lyonnais, Sébastien; Hénaut, Mathilde; Neyret, Aymeric; Mérida, Peggy; Cazevieille, Chantal; Gros, Nathalie; Chable‐Bessia, Christine; Muriaux, Delphine

doi:10.1038/s41598-021-91371-4

Cited by 41 publications

(62 citation statements)

References 24 publications

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“…These data are quite desirable to promote the evaluation of new products, especially for disinfection purposes. The nanoindentation analysis also corroborated the suggestion made by Lyonnais et al, 23 confirming the deformable feature of the SARS-CoV-2. Finally, the most innovative result is that the SARS-CoV-2 virus may self-assemble after mechanical stress.…”

Section: Resultssupporting

confidence: 87%

“…Their findings suggest that the spikes interspacing is ∼15 nm and they reported other densities spaced 5 to 8 nm apart, connecting the RNP (ribonucleoprotein complex: composed by the N protein and the genome) to the membrane region, but could not confidently affirm if these densities were related to M or N proteins. For the SARS-CoV-2, Kiss et al 22 and Lyonnais et al 23 reported a “bald” viral particle showing a smooth surface, without clear smaller structures. Opposingly, our data show that the viral particle membrane is crowded with smaller structures, suggesting the structural description of the proteins on the membrane surface.…”

Section: Resultsmentioning

confidence: 99%

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SARS-CoV-2 Unrevealed: Ultrastructural and Nanomechanical Analysis

et al. 2021

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The ongoing outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) started in late 2019 and spread across the world, infecting millions of people, with over 3.3 million deaths worldwide. To fight back the virus, it is necessary to understand how the main structures work, especially those responsible for the virus infectivity pathogenicity. Here, using the most advanced atomic force microscopy techniques, SARS-CoV-2 viral particles were analyzed, with a special focus on their ultrastructure, adsorption conformation, and nanomechanical behavior. The results uncovered the aspects of the organization and the spatial distribution of the proteins on the surface of the viral particles. It also showed the compliant behavior of the membrane and ability to recover from mechanical injuries. At least three layers composing the membrane and their thickness were measured, protecting the virus from external stress. This study provides new insight into the ultrastructure of SARS-CoV-2 particles at the nanoscale, offering new prospects that could be employed for mapping viral surfaces. The understanding of the viruses’ capacity to survive mechanical disruptions at any level and their ability to recover from such injuries can shed a light on the structure–function relationship and help us to find targets for drug action, especially for this virus that, to this day, has no course of treatment approved.

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

SARS-CoV-2 Unrevealed: Ultrastructural and Nanomechanical Analysis

et al. 2021

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“…Using FCS and PALM, we found a 120-140nm particle size range (Figure 2D,E) : 100-110 nm for MNE VLPs, and 130nm for MNES VLPs, which is consistent with AFM results (Figure 1D ). It is to notice that VLPs size are slightly bigger than wild-type virus particles as it has been described using AFM [19], TEM [19] or cryoET [27] [28] [29].…”

Section: Discussionmentioning

confidence: 99%

“…The purified MNE and MNES VLPs were next imaged by AFM in buffer for size and shape characterization (Figure 1D), in the conditions previously used for the wild-type virus [19]. We then determined the M(GFP)NES and M(GFP)NE VLPs size and approximate concentration using Fluorescence Correlative Microscopy (FCS) (Figure 2D).…”

Section: -Optimized Virus-like-particles Production By Mimicking Sars...mentioning

confidence: 99%

Optimized production and fluorescent labelling of SARS-CoV-2 Virus-Like-Particles to study virus assembly and entry

Muriaux

Gourdelier

Swain

et al. 2022

Preprint

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SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemic that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the virus RNA ratio allowing SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and S. Furthermore, monochrome, dual-color fluorescent or photoconvertible VLPs were produced. Thanks to live fluorescence and super-resolution microscopy, we quantified VLPs size and concentration. It shows a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a minimum concentration of 10e12 VLP/ml. SARS-CoV-2 VLPs could tolerate the integration of fluorescent N and M tagged proteins without impairing particle assembly. In this condition, we were able to establish incorporation of the mature Spike in fluorescent VLPs. The Spike functionality was then shown by monitoring fluorescent MNES-VLPs docking and endocytosis in human pulmonary cells expressing the receptor hACE2. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.

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“…Ethical approval was obtained for the use of anonymized oropharyngeal specimens from patients for the diagnosis of SARS-CoV-2 (North West-Greater Manchester South Research Ethics Committee [REC], REC Ref:19/NW/0730). Specimens were maintained in Copan Universal Transport Medium (UTM) before being inactivated in a 1% final concentration of formaldehyde (Pierce) for 5 min at room temperature 51 , followed by clarification at 13,000× g for 5–10 min and diluted 1:10 in viral resuspension buffer (10 mM Tris HCl pH8, 100 mM NaCl) prior to use.…”

Section: Methodsmentioning

confidence: 99%

Viral detection and identification in 20 min by rapid single-particle fluorescence in-situ hybridization of viral RNA

Hepp

Shiaelis

Robb

et al. 2021

Sci Rep

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The increasing risk from viral outbreaks such as the ongoing COVID-19 pandemic exacerbates the need for rapid, affordable and sensitive methods for virus detection, identification and quantification; however, existing methods for detecting virus particles in biological samples usually depend on multistep protocols that take considerable time to yield a result. Here, we introduce a rapid fluorescence in situ hybridization (FISH) protocol capable of detecting influenza virus, avian infectious bronchitis virus and SARS-CoV-2 specifically and quantitatively in approximately 20 min, in virus cultures, combined nasal and throat swabs with added virus and likely patient samples without previous purification. This fast and facile workflow can be adapted both as a lab technique and a future diagnostic tool in enveloped viruses with an accessible genome.

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Atomic force microscopy analysis of native infectious and inactivated SARS-CoV-2 virions

Cited by 41 publications

References 24 publications

SARS-CoV-2 Unrevealed: Ultrastructural and Nanomechanical Analysis

SARS-CoV-2 Unrevealed: Ultrastructural and Nanomechanical Analysis

Optimized production and fluorescent labelling of SARS-CoV-2 Virus-Like-Particles to study virus assembly and entry

Viral detection and identification in 20 min by rapid single-particle fluorescence in-situ hybridization of viral RNA

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