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

2021 ◽  
Vol 9 (6) ◽  
pp. 1194
Author(s):  
Marion Le Bideau ◽  
Nathalie Wurtz ◽  
Jean-Pierre Baudoin ◽  
Bernard La Scola
Keyword(s):  
Electron Microscopy ◽  
Infected Cell ◽  
Diagnostic Methods ◽  
Thin Sections ◽  
Cell Monolayers ◽  
Culture Cells ◽  
Innovative Methodology ◽  
Infected Cells ◽  
Infectious Cycle ◽  
Ultrastructural Findings

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.

Studies of a Defective Measles Virus

1968 ◽  
Vol 26 ◽  
pp. 208-209
Author(s):  
R. M. McCombs ◽  
M. Benyesh-Melnick ◽  
J. P. Brunschwig
Keyword(s):  
Inclusion Bodies ◽  
Measles Virus ◽  
Giant Cells ◽  
Helical Structures ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Culture Cells ◽  
Infected Cells ◽  
Eosinophilic Inclusions

Measles virus is an agent that is capable of replicating in a number of different culture cells and generally causes the formation of multinucleated giant cells. As a result of infection, virus is released from the cells into the culture fluids and reinfection can be initiated by this cell-free virus. The extracellular virus has been examined by negative staining with phosphotungstic acid and has been shown to be a rather pleomorphic particle with a diameter of about 140 mμ. However, no such virus particles have been detected in thin sections of the infected cells. Rather, the only virus-induced structures present in the giant cells are eosinophilic inclusions (intracytoplasmic or intranuclear). These inclusion bodies have been shown to contain helical structures, resembling the nucleocapsid observed in negatively stained preparations.

Electron Microscopic Study of the Morphogenesis of Echovirus 23

1973 ◽  
Vol 31 ◽  
pp. 590-591
Author(s):  
C. M. Trant ◽  
R. M. Jamison
Keyword(s):  
Electron Microscopy ◽  
Microscopic Study ◽  
Electron Microscopic Study ◽  
Post Infection ◽  
Glass Surface ◽  
Kidney Cells ◽  
Electron Microscopic ◽  
Monolayer Cultures ◽  
Infected Cells ◽  
Infectious Cycle

The cytopathology which accompanies the replication of Echovirus 23 in monkey kidney cells has been studied. Cells grown in monolayer cultures in glass bottles were infected with Echovirus 23 and incubated at 37°C. At varying intervals, cells were harvested for electron microscopy. Multiple embeddings of specimens at each interval post-infection were performed to confirm and extend the observations. It was noted that as the interval postinfection lengthened, individual infected cells rounded and detached from the surface of the monolayer. As the infectious cycle continued, virtually all cells in the bottle became detached. Accordingly, each infected bottle provided two specimens for electron microscopy; a) those free in the supernatent. fluid and b) those remaining on.the glass surface.

scholarly journals Bromodeoxyuridine Labelling to Determine Viral DNA Localization in Fluorescence and Electron Microscopy: The Case of Adenovirus

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1863 ◽  
Author(s):  
Gabriela N. Condezo ◽  
Carmen San Martín
Keyword(s):  
Electron Microscopy ◽  
Nucleic Acids ◽  
Cell Metabolism ◽  
Viral Dna ◽  
Nucleotide Analogs ◽  
Viral Genomes ◽  
Fluorescence And Electron Microscopy ◽  
Brdu Labeling ◽  
Infected Cells ◽  
Infectious Cycle

The localization of viral nucleic acids in the cell is essential for understanding the infectious cycle. One of the strategies developed for this purpose is the use of nucleotide analogs such as bromodeoxyuridine (BrdU, analog to thymine) or bromouridine (BrU, analog of uridine), which are incorporated into the nucleic acids during replication or transcription. In adenovirus infections, BrdU has been used to localize newly synthesized viral genomes in the nucleus, where it is key to distinguish between host and viral DNA. Here, we describe our experience with methodological variations of BrdU labeling to localize adenovirus genomes in fluorescence and electron microscopy. We illustrate the need to define conditions in which most of the newly synthesized DNA corresponds to the virus and not the host, and the amount of BrdU provided is enough to incorporate to the new DNA molecules without hampering the cell metabolism. We hope that our discussion of problems encountered and solutions implemented will help other researches interested in viral genome localization in infected cells.

Freeze-Etching of Intranuclear Adenovirus

1971 ◽  
Vol 29 ◽  
pp. 384-385
Author(s):  
Dennis T. Brown ◽  
Byron T. Burlingham
Keyword(s):  
Osmium Tetroxide ◽  
Adenovirus Type ◽  
Etching Time ◽  
Human Carcinoma ◽  
Thin Sections ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Infected Cells ◽  
Freeze Etching

Adenoviruses are revealed by negative staining to be icosahedral in structure and composed of 252 subunits (insert Fig. 1). The vertices have a fiber-like projection which has been implicated in the adsorbtion of the virus to the host cell. Ultra thin sections of adenovirus-infected cells has revealed that virus morphogenesis occurs in the nucleus.Human carcinoma (KB) tissue culture cells were productively infected with adenovirus type 2 at a multiplicity of 10 and incubated for 40 hours at 37°C. The infected monolayers were fixed in place with 1%, gluteraldehyde buffered in Millonigs phosphate buffer. The fixed cells were suspended in 30%, glycerol and processed according to the procedure of Moor in a Balzers 360 M freeze etch unit with an etching time of 70 seconds at -100°C. A second portion of the fixed cells in glycerol was post-fixed with 1% osmium tetroxide and embeded in epon 812 for ultrathin sectioning.

Enzyme cytochemistry and autoradiography of adenovirus-infected cells as determined with the electron microscope

1971 ◽  
Vol 17 (2) ◽  
pp. 249-256 ◽  
Author(s):  
T. Yamamoto ◽  
M. S. Shahrabadi
Keyword(s):  
Electron Microscope ◽  
Infected Cell ◽  
Deoxyribonucleic Acid ◽  
Enzyme Digestion ◽  
Specific Enzyme ◽  
Enzyme Cytochemistry ◽  
Thin Sections ◽  
Ring Form ◽  
Infected Cells ◽  
Eosinophilic Inclusions

The biochemical nature of adenovirus-induced inclusions was determined by specific enzyme digestion of thin sections combined with autoradiography using tritiated precursors.The early basophilic inclusions and the ring form inclusions were found to contain newly synthesized deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein. The later appearing eosinophilic inclusions, both the electron light appearing ones and the electron dark appearing ones, consisted of protein.The total RNA synthesized by the infected cell was slightly increased; part of the increase was due to continuing synthesis of RNA by the nucleolus. The host DNA, after infection, was marginated along the nuclear membrane and was not subsequently incorporated into virus DNA.

The Effect of Temperature on Coronavirus Replication

1975 ◽  
Vol 33 ◽  
pp. 650-651
Author(s):  
R. M. Jamison ◽  
L. H. Cowley
Keyword(s):  
Electron Microscopy ◽  
Cell Monolayer ◽  
Uranyl Acetate ◽  
Effect Of Temperature ◽  
Post Inoculation ◽  
Minimal Essential Medium ◽  
Thin Sections ◽  
Cell Monolayers ◽  
Input Multiplicity ◽  
Ethanol Series

Monolayers of human foreskin fibroblast cells were infected with a high input multiplicity of coronavirus 229-E. Virus was allowed to adsorb to the cells for 2 hours at 37°C. After this interval, one series of cell monolayers was covered with Eagle's minimal essential medium with 2% bovine serum warmed to 33°C and incubated in an atmosphere of 5% CO2 at 33°C. Another cell monolayer series was covered with the same medium at 37°C and incubated in an atmosphere of 5% CO2 at 37°C. At appropriate intervals, cultures were switched from 33°C to 37°C and incubated for a total of 24 hours post-inoculation. After incubation, all cultures were harvested for titration of infectious virus or electron microscopy.The monolayers for electron microscopy were fixed in 6% glutaraldehyde, post-fixed in 2% OsO4 in Clark's buffer, dehydrated in an ethanol series, and embedded in Epon 812. Ultra-thin sections were prepared and double stained with lead citrate and uranyl acetate.

Two- or three-way observations of the identical cell elements within the same sections by LM, TEM and/or SEM

1978 ◽  
Vol 36 (2) ◽  
pp. 82-83 ◽  
Author(s):  
Nakazo Watari ◽  
Yasuaki Hotta ◽  
Yoshio Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Fine Structure ◽  
Light Microscopy ◽  
Thin Sections ◽  
Transmission Electron ◽  
Identical Cell ◽  
Electron Microscopes ◽  
Scanning Electron

It is very useful if we can observe the identical cell elements within the same sections by light microscopy (LM), transmission electron microscopy (TEM) and/or scanning electron microscopy (SEM) sequentially, because, the cell fine structure can not be indicated by LM, while the color is; on the other hand, the cell fine structure can be very easily observed by EM, although its color properties may not. However, there is one problem in that LM requires thick sections of over 1 μm, while EM needs very thin sections of under 100 nm. Recently, we have developed a new method to observe the same cell elements within the same plastic sections using both light and transmission (conventional or high-voltage) electron microscopes.In this paper, we have developed two new observation methods for the identical cell elements within the same sections, both plastic-embedded and paraffin-embedded, using light microscopy, transmission electron microscopy and/or scanning electron microscopy (Fig. 1).

Lead aspartate: a new en bloc stain for electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 34-35
Author(s):  
J.R. Walton
Keyword(s):  
Electron Microscopy ◽  
Calcium Ion ◽  
Enzyme Reaction ◽  
Lead Citrate ◽  
Reaction Products ◽  
En Bloc ◽  
Thin Sections ◽  
Lead Salts ◽  
Thin Sectioning ◽  
High Alkalinity

In electron microscopy, lead is the metal most widely used for enhancing specimen contrast. Lead citrate requires a pH of 12 to stain thin sections of epoxy-embedded material rapidly and intensively. However, this high alkalinity tends to leach out enzyme reaction products, making lead citrate unsuitable for many cytochemical studies. Substitution of the chelator aspartate for citrate allows staining to be carried out at pH 6 or 7 without apparent effect on cytochemical products. Moreover, due to the low, controlled level of free lead ions, contamination-free staining can be carried out en bloc, prior to dehydration and embedding. En bloc use of lead aspartate permits the grid-staining step to be bypassed, allowing samples to be examined immediately after thin-sectioning.Procedures. To prevent precipitation of lead salts, double- or glass-distilled H20 used in the stain and rinses should be boiled to drive off carbon dioxide and glassware should be carefully rinsed to remove any persisting traces of calcium ion.

Further Observations on the Virus of Epizootic Diarrhea of Infant Mice. An Electron Microscopic Study

1967 ◽  
Vol 25 ◽  
pp. 110-111
Author(s):  
W. G. Banfield ◽  
G. Kasnic ◽  
J. H. Blackwell
Keyword(s):  
Electron Microscope ◽  
Infected Cell ◽  
Microscopic Study ◽  
Intestinal Epithelium ◽  
Ultrastructural Study ◽  
Osmium Tetroxide ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Virus Particles ◽  
Infected Cells

An ultrastructural study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (EDIM virus) was first performed by Adams and Kraft. We have extended their observations and have found developmental forms of the virus and associated structures not reported by them.Three-day-old NLM strain mice were infected with EDIM virus and killed 48 to 168 hours later. Specimens of bowel were fixed in glutaraldehyde, post fixed in osmium tetroxide and embedded in epon. Sections were stained with uranyl magnesium acetate followed by lead citrate and examined in an updated RCA EMU-3F electron microscope.The cells containing virus particles (infected) are at the tips of the villi and occur throughout the intestine from duodenum through colon. All developmental forms of the virus are present from 48 to 168 hours after infection. Figure 1 is of cells without virus particles and figure 2 is of an infected cell. The nucleus and cytoplasm of the infected cells appear clearer than the cells without virus particles.

Ectodesmata as Revealed By Freeze-Etch Preparations of Plant Epidermal Cell Walls

1973 ◽  
Vol 31 ◽  
pp. 468-469
Author(s):  
N.C. Lyon ◽  
W. C. Mueller
Keyword(s):  
Electron Microscopy ◽  
Acetic Acid ◽  
Nitric Acid ◽  
Oxalic Acid ◽  
Light Microscopy ◽  
Epidermal Cell ◽  
Cell Walls ◽  
Plant Viruses ◽  
Plant Leaves ◽  
Thin Sections

Schumacher and Halbsguth first demonstrated ectodesmata as pores or channels in the epidermal cell walls in haustoria of Cuscuta odorata L. by light microscopy in tissues fixed in a sublimate fixative (30% ethyl alcohol, 30 ml:glacial acetic acid, 10 ml: 65% nitric acid, 1 ml: 40% formaldehyde, 5 ml: oxalic acid, 2 g: mecuric chloride to saturation 2-3 g). Other workers have published electron micrographs of structures transversing the outer epidermal cell in thin sections of plant leaves that have been interpreted as ectodesmata. Such structures are evident following treatment with Hg++ or Ag+ salts and are only rarely observed by electron microscopy. If ectodesmata exist without such treatment, and are not artefacts, they would afford natural pathways of entry for applied foliar solutions and plant viruses.

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