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. 2020 Aug 19:11:2014.
doi: 10.3389/fmicb.2020.02014. eCollection 2020.

The Strengths of Scanning Electron Microscopy in Deciphering SARS-CoV-2 Infectious Cycle

Djamal Brahim Belhaouari  1 , Anthony Fontanini  2 , Jean-Pierre Baudoin  1 , Gabriel Haddad  1 , Marion Le Bideau  1 , Jacques Yaacoub Bou Khalil  2 , Didier Raoult  1 , Bernard La Scola  1   2
Affiliations

The Strengths of Scanning Electron Microscopy in Deciphering SARS-CoV-2 Infectious Cycle

Djamal Brahim Belhaouari et al. Front Microbiol. .

Abstract

Electron microscopy is a powerful tool in the field of microbiology. It has played a key role in the rapid diagnosis of viruses in patient samples and has contributed significantly to the clarification of virus structure and function, helping to guide the public health response to emerging viral infections. In the present study, we used scanning electron microscopy (SEM) to study the infectious cycle of SARS-CoV-2 in Vero E6 cells and we controlled some key findings by classical transmission electronic microscopy (TEM). The replication cycle of the virus was followed from 1 to 36 h post-infection. Our results revealed that SARS-CoV-2 infected the cells through membrane fusion. Particles are formed in the peri-nuclear region from a budding of the endoplasmic reticulum-Golgi apparatus complex into morphogenesis matrix vesicae. New SARS-CoV-2 particles were expelled from the cells, through cell lysis or by fusion of virus containing vacuoles with the cell plasma membrane. Overall, this cycle is highly comparable to that of SARS-CoV. By providing a detailed and complete SARS-CoV-2 infectious cycle, SEM proves to be a very rapid and efficient tool compared to classical TEM.

Keywords: Coronavirus; SARS-CoV-2; Vero E6 cells; infectious cycle; scanning electron microscopy.

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Figures

FIGURE 1
FIGURE 1
SARS-CoV-2 infected Vero E6 cells At early post-infection time-point with virus (A,B) at the periphery of Vero E6 cells (arrows). (C,D) SEM (C) and TEM (D) views of the same cellular region with a SARS-CoV-2 particle (arrow) attached to the plasma membrane, the corona spikes of which are located between the particle and the cell plasma membrane. (E,F) SEM (E) and TEM (F) views of the same cellular region showing SARS-CoV-2 virus particles attached to the cell plasma membrane; one particle (arrow in E,F) is glued to the plasma membrane, fusing with the cell plasma membrane.
FIGURE 2
FIGURE 2
SARS-CoV-2 infected Vero E6 cells with endocytic vesicles in the cytoplasm. (A–F) SEM (A,C,E,G) and TEM (B,D,F,H) views of the same cellular regions with clathrin-coated vesicles (arrows) containing rod-like amorphous material. Crescent-like electron-dense structures (arrowhead in A) were often depicted in infected cells cytoplasm. Solid arrows (E–H) point to glued SARS-CoV-2 particles on cells plasma membrane.
FIGURE 3
FIGURE 3
SARS-CoV-2 infected Vero E6 cells at 12 h post-infection. (A) Cell in lytic process with vacuole filled with nascent particles (arrows). (B) producing-virus cell adjacent to lysed cell. (C,D) zooming on virus-producing cell shows mature SARS-CoV-2 particles with their corona spikes (arrows) in small cytoplasmic vacuoles between the nucleus and the cell periphery, observed by SEM (C) or TEM (D) in a cell. Extracellular particles attached to the plasma membrane (arrows).
FIGURE 4
FIGURE 4
SARS-CoV-2 infected Vero E6 cells at 12–18 h post-infection. (A,B) SEM (A) and TEM (B) views of the same cell region showing extreme Golgi apparatus budding as well as a vacuole filled with nascent particles close to the Golgi apparatus. (C,D) Thick and distorted endoplasmic reticulum (ER) tubules were observed by SEM at peri-nuclear location and at the cells periphery. (E) SEM view of the extensive enlargement and budding of the ER and Golgi apparatus, as well as a myelin-like membranes whorl (mw) close to the Golgi apparatus and mitochondria (mito.) in the perinuclear region. (F) SEM low-magnification view of Golgi budding between a nucleus and the peripheral plasma membrane. (G) SEM image of zippered endoplasmic reticulum (ER) at apical location (arrow).
FIGURE 5
FIGURE 5
SARS-CoV-2 infected Vero E6 cells at 36 h post-infection. (A,B) SEM views of doughnut-like particles (arrow) with a pronounced electron-opaque edge observed at peri-nuclear locations dispersed into the cytoplasm (solid arrowhead), or within vacuoles, which seemed derived from the ER, forming virus morphogenesis matrix vesicae (VMMV). VMMV were observed as opened sacs (asterisk in B), assembling next to the nucleus (A,B). Protrusions of the doughnut-like particles could be observed in the VMMV (arrowhead in B). (C,D) VMMV observed as a closed sac (asterisk in D) in a larger vacuole in the cytoplasm. (A–D) SEM images.
FIGURE 6
FIGURE 6
SARS-CoV-2 infected Vero cells at 36 h post-infection (A) SEM depiction of an extensive peri-nuclear membrane whorls (mw) network, with larger inner-membranous distances as infection progresses. (B) Nascent virions particles (≈70 nm) observed by SEM mixed with membranes whorls in large bags. (C–F) SARS-CoV-2-morphogenesis matrix vesicae (asterisk) with different appearances, with nascent particles (≈70 nm) located in more or less large vacuoles, containing more or less electron-dense materials (asterisk). (F) Translucent VMMV/vacuoles with well-individualized virions particles (white arrows).
FIGURE 7
FIGURE 7
SEM of SARS-CoV-2 mature particles at 18–36 h post-infection. observed at intra- and extra- cellular locations. (A–C) Viral mature particles lying between cellular microvilli. (D,E) Virus particles within smooth vesicles at the cell periphery and eventually fused with the cell membrane (arrowhead) to release the virus (arrow). (F,G) Viral particles in translucent vacuoles or attached to the plasma membrane of a lysed cell. Small arrow points to a SARS-CoV-2 corona spike.
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