Cargando…

Innovative Approach to Fast Electron Microscopy Using the Example of a Culture of Virus-Infected Cells: An Application to SARS-CoV-2

Despite the development of new diagnostic methods, co-culture, based on sample inoculation of cell monolayers coupled with electron microscopy (EM) observation, remains the gold standard in virology. Indeed, co-culture allows for the study of cell morphology (infected and not infected), the ultrastr...

Descripción completa

Detalles Bibliográficos
Autores principales: Le Bideau, Marion, Wurtz, Nathalie, Baudoin, Jean-Pierre, La Scola, Bernard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228702/
https://www.ncbi.nlm.nih.gov/pubmed/34073053
http://dx.doi.org/10.3390/microorganisms9061194
_version_ 1783712804177444864
author Le Bideau, Marion
Wurtz, Nathalie
Baudoin, Jean-Pierre
La Scola, Bernard
author_facet Le Bideau, Marion
Wurtz, Nathalie
Baudoin, Jean-Pierre
La Scola, Bernard
author_sort Le Bideau, Marion
collection PubMed
description Despite the development of new diagnostic methods, co-culture, based on sample inoculation of cell monolayers coupled with electron microscopy (EM) observation, remains the gold standard in virology. Indeed, co-culture allows for the study of cell morphology (infected and not infected), the ultrastructure of the inoculated virus, and the different steps of the virus infectious cycle. Most EM methods for studying virus cycles are applied after infected cells are produced in large quantities and detached to obtain a pellet. Here, cell culture was performed in sterilized, collagen-coated single-break strip wells. After one day in culture, cells were infected with SARS-CoV-2. Wells of interest were fixed at different time points, from 2 to 36 h post-infection. Microwave-assisted resin embedding was accomplished directly in the wells in 4 h. Finally, ultra-thin sections were cut directly through the infected-cell monolayers. Our methodology requires, in total, less than four days for preparing and observing cells. Furthermore, by observing undetached infected cell monolayers, we were able to observe new ultrastructural findings, such as cell–cell interactions and baso-apical cellular organization related to the virus infectious cycle. Our innovative methodology thus not only saves time for preparation but also adds precision and new knowledge about viral infection, as shown here for SARS-CoV-2.
format Online
Article
Text
id pubmed-8228702
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-82287022021-06-26 Innovative Approach to Fast Electron Microscopy Using the Example of a Culture of Virus-Infected Cells: An Application to SARS-CoV-2 Le Bideau, Marion Wurtz, Nathalie Baudoin, Jean-Pierre La Scola, Bernard Microorganisms Article Despite the development of new diagnostic methods, co-culture, based on sample inoculation of cell monolayers coupled with electron microscopy (EM) observation, remains the gold standard in virology. Indeed, co-culture allows for the study of cell morphology (infected and not infected), the ultrastructure of the inoculated virus, and the different steps of the virus infectious cycle. Most EM methods for studying virus cycles are applied after infected cells are produced in large quantities and detached to obtain a pellet. Here, cell culture was performed in sterilized, collagen-coated single-break strip wells. After one day in culture, cells were infected with SARS-CoV-2. Wells of interest were fixed at different time points, from 2 to 36 h post-infection. Microwave-assisted resin embedding was accomplished directly in the wells in 4 h. Finally, ultra-thin sections were cut directly through the infected-cell monolayers. Our methodology requires, in total, less than four days for preparing and observing cells. Furthermore, by observing undetached infected cell monolayers, we were able to observe new ultrastructural findings, such as cell–cell interactions and baso-apical cellular organization related to the virus infectious cycle. Our innovative methodology thus not only saves time for preparation but also adds precision and new knowledge about viral infection, as shown here for SARS-CoV-2. MDPI 2021-05-31 /pmc/articles/PMC8228702/ /pubmed/34073053 http://dx.doi.org/10.3390/microorganisms9061194 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Le Bideau, Marion
Wurtz, Nathalie
Baudoin, Jean-Pierre
La Scola, Bernard
Innovative Approach to Fast Electron Microscopy Using the Example of a Culture of Virus-Infected Cells: An Application to SARS-CoV-2
title Innovative Approach to Fast Electron Microscopy Using the Example of a Culture of Virus-Infected Cells: An Application to SARS-CoV-2
title_full Innovative Approach to Fast Electron Microscopy Using the Example of a Culture of Virus-Infected Cells: An Application to SARS-CoV-2
title_fullStr Innovative Approach to Fast Electron Microscopy Using the Example of a Culture of Virus-Infected Cells: An Application to SARS-CoV-2
title_full_unstemmed Innovative Approach to Fast Electron Microscopy Using the Example of a Culture of Virus-Infected Cells: An Application to SARS-CoV-2
title_short Innovative Approach to Fast Electron Microscopy Using the Example of a Culture of Virus-Infected Cells: An Application to SARS-CoV-2
title_sort innovative approach to fast electron microscopy using the example of a culture of virus-infected cells: an application to sars-cov-2
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228702/
https://www.ncbi.nlm.nih.gov/pubmed/34073053
http://dx.doi.org/10.3390/microorganisms9061194
work_keys_str_mv AT lebideaumarion innovativeapproachtofastelectronmicroscopyusingtheexampleofacultureofvirusinfectedcellsanapplicationtosarscov2
AT wurtznathalie innovativeapproachtofastelectronmicroscopyusingtheexampleofacultureofvirusinfectedcellsanapplicationtosarscov2
AT baudoinjeanpierre innovativeapproachtofastelectronmicroscopyusingtheexampleofacultureofvirusinfectedcellsanapplicationtosarscov2
AT lascolabernard innovativeapproachtofastelectronmicroscopyusingtheexampleofacultureofvirusinfectedcellsanapplicationtosarscov2