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1.
Viruses ; 13(10)2021 09 29.
Article in English | MEDLINE | ID: covidwho-1688586

ABSTRACT

Recent advances in light and electron microscopy are uncovering viral lifecycle events with a level of detail never before seen [...].


Subject(s)
Host Microbial Interactions , Image Interpretation, Computer-Assisted/methods , Single Molecule Imaging/methods , Humans , Image Interpretation, Computer-Assisted/instrumentation , Microscopy, Electron/methods , Single Molecule Imaging/instrumentation , Virus Replication
2.
Viruses ; 14(2)2022 01 20.
Article in English | MEDLINE | ID: covidwho-1650717

ABSTRACT

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted public health and the world economy and fueled a worldwide race to approve therapeutic and prophylactic agents, but so far there are no specific antiviral drugs. Understanding the biology of the virus is the first step in structuring strategies to combat it, and in this context several studies have been conducted with the aim of understanding the replication mechanism of SARS-CoV-2 in vitro systems. In this work, studies using transmission and scanning electron microscopy and 3D electron microscopy modeling were performed with the goal of characterizing the morphogenesis of SARS-CoV-2 in Vero-E6 cells. Several ultrastructural changes were observed-such as syncytia formation, cytoplasmic membrane projections, lipid droplets accumulation, proliferation of double-membrane vesicles derived from the rough endoplasmic reticulum, and alteration of mitochondria. The entry of the virus into cells occurred through endocytosis. Viral particles were observed attached to the cell membrane and in various cellular compartments, and extrusion of viral progeny took place by exocytosis. These findings allow us to infer that Vero-E6 cells are highly susceptible to SARS-CoV-2 infection as described in the literature and their replication cycle is similar to that described with SARS-CoV and MERS-CoV in vitro models.


Subject(s)
Microscopy, Electron, Transmission/methods , Microscopy, Electron/methods , SARS-CoV-2/metabolism , SARS-CoV-2/ultrastructure , Animals , Cell Line , Chlorocebus aethiops , SARS-CoV-2/chemistry , Vero Cells , Virus Internalization , Virus Replication
3.
Life Sci Alliance ; 5(4)2022 04.
Article in English | MEDLINE | ID: covidwho-1614505

ABSTRACT

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.


Subject(s)
COVID-19/diagnosis , In Situ Hybridization, Fluorescence/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Virus Replication/genetics , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/virology , Caco-2 Cells , Cell Line, Tumor , Chlorocebus aethiops , Humans , In Situ Hybridization/methods , Microscopy, Electron/methods , RNA, Viral/ultrastructure , Reproducibility of Results , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Sensitivity and Specificity , Vero Cells , Virus Release/drug effects , Virus Release/genetics , Virus Release/physiology , Virus Replication/drug effects , Virus Replication/physiology
4.
J Cutan Pathol ; 49(1): 17-28, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1349942

ABSTRACT

BACKGROUND: The abundance of publications of COVID-19-induced chilblains has resulted in a confusing situation. METHODS: This is a prospective single-institution study from 15 March to 13 May 2020. Thirty-two patients received PCR nasopharyngeal swabs. Of these, 28 patients had a thoracic CT-scan, 31 patients had blood and urine examinations, 24 patients had skin biopsies including immunohistochemical and direct immunofluorescence studies, and four patients had electron microscopy. RESULTS: COVID-19-induced chilblains are clinically and histopathologically identical to chilblains from other causes. Although intravascular thrombi are sometimes observed, no patient had a systemic coagulopathy or severe clinical course. The exhaustive clinical, radiological, and laboratory work-up in this study ruled-out other primary and secondary causes. Electron microscopy revealed rare, probable viral particles whose core and spikes measured from 120 to 133 nm within endothelium and eccrine glands in two cases. CONCLUSION: This study provides further clinicopathologic evidence of COVID-19-related chilblains. Negative PCR and antibody tests do not rule-out infection. Chilblains represent a good prognosis, occurring later in the disease course. No systemic coagulopathy was identified in any patient. Patients presenting with acral lesions should be isolated, and chilblains should be distinguished from thrombotic lesions (livedo racemosa, retiform purpura, or ischemic acral necrosis).


Subject(s)
COVID-19/complications , COVID-19/diagnosis , Chilblains/etiology , Chilblains/pathology , Toes/pathology , Adolescent , Adult , Aged , Biopsy/methods , COVID-19/metabolism , COVID-19/virology , Chilblains/diagnosis , Chilblains/virology , Child , Diagnosis, Differential , Eccrine Glands/pathology , Eccrine Glands/ultrastructure , Eccrine Glands/virology , Endothelium/pathology , Endothelium/ultrastructure , Endothelium/virology , Female , Humans , Livedo Reticularis/pathology , Male , Microscopy, Electron/methods , Middle Aged , Prognosis , Prospective Studies , Purpura/pathology , SARS-CoV-2/genetics , Skin/pathology , Toes/virology , Young Adult
5.
Int J Mol Med ; 47(6)2021 06.
Article in English | MEDLINE | ID: covidwho-1181666

ABSTRACT

The Coronavirus Disease 2019 (COVID­19) pandemic has forced the scientific community to rapidly develop highly reliable diagnostic methods in order to effectively and accurately diagnose this pathology, thus limiting the spread of infection. Although the structural and molecular characteristics of the severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) were initially unknown, various diagnostic strategies useful for making a correct diagnosis of COVID­19 have been rapidly developed by private research laboratories and biomedical companies. At present, rapid antigen or antibody tests, immunoenzymatic serological tests and molecular tests based on RT­PCR are the most widely used and validated techniques worldwide. Apart from these conventional methods, other techniques, including isothermal nucleic acid amplification techniques, clusters of regularly interspaced short palindromic repeats/Cas (CRISPR/Cas)­based approaches or digital PCR methods are currently used in research contexts or are awaiting approval for diagnostic use by competent authorities. In order to provide guidance for the correct use of COVID­19 diagnostic tests, the present review describes the diagnostic strategies available which may be used for the diagnosis of COVID­19 infection in both clinical and research settings. In particular, the technical and instrumental characteristics of the diagnostic methods used are described herein. In addition, updated and detailed information about the type of sample, the modality and the timing of use of specific tests are also discussed.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Animals , Biosensing Techniques/methods , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Inventions , Microscopy, Electron/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Virus Cultivation/methods
6.
Emerg Infect Dis ; 27(4): 1023-1031, 2021 04.
Article in English | MEDLINE | ID: covidwho-1088897

ABSTRACT

Efforts to combat the coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have placed a renewed focus on the use of transmission electron microscopy for identifying coronavirus in tissues. In attempts to attribute pathology of COVID-19 patients directly to tissue damage caused by SARS-CoV-2, investigators have inaccurately reported subcellular structures, including coated vesicles, multivesicular bodies, and vesiculating rough endoplasmic reticulum, as coronavirus particles. We describe morphologic features of coronavirus that distinguish it from subcellular structures, including particle size range (60-140 nm), intracellular particle location within membrane-bound vacuoles, and a nucleocapsid appearing in cross section as dense dots (6-12 nm) within the particles. In addition, although the characteristic spikes of coronaviruses may be visible on the virus surface, especially on extracellular particles, they are less evident in thin sections than in negative stain preparations.


Subject(s)
COVID-19 , Cellular Structures , SARS-CoV-2 , Biopsy/methods , COVID-19/pathology , COVID-19/virology , Cellular Structures/classification , Cellular Structures/ultrastructure , Humans , Microscopy, Electron/methods , SARS-CoV-2/isolation & purification , SARS-CoV-2/ultrastructure
7.
Am J Pathol ; 191(2): 222-227, 2021 02.
Article in English | MEDLINE | ID: covidwho-1025412

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 pandemic has infected millions of individuals in the United States and caused hundreds of thousands of deaths. Direct infection of extrapulmonary tissues has been postulated, and using sensitive techniques, viral RNA has been detected in multiple organs in the body, including the kidney. However, direct infection of tissues outside of the lung has been more challenging to demonstrate. This has been in part due to misinterpretation of electron microscopy studies. In this perspective, we will discuss what is known about coronavirus infection, some of the basic ultrastructural cell biology that has been confused for coronavirus infection of cells, and rigorous criteria that should be used when identifying pathogens by electron microscopy.


Subject(s)
COVID-19 , Coronavirus Infections/virology , Microscopy, Electron , SARS-CoV-2/pathogenicity , COVID-19/epidemiology , COVID-19/virology , Humans , Lung/ultrastructure , Lung/virology , Microscopy, Electron/methods , United States , Virus Diseases
10.
Nat Struct Mol Biol ; 27(10): 925-933, 2020 10.
Article in English | MEDLINE | ID: covidwho-662441

ABSTRACT

The coronavirus (CoV) spike (S) protein, involved in viral-host cell fusion, is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. Here, to better understand S-protein mobility, we implemented a structure-based vector analysis of available ß-CoV S-protein structures. Despite an overall similarity in domain organization, we found that S-proteins from different ß-CoVs display distinct configurations. Based on this analysis, we developed two soluble ectodomain constructs for the SARS-CoV-2 S-protein, in which the highly immunogenic and mobile receptor binding domain (RBD) is either locked in the all-RBDs 'down' position or adopts 'up' state conformations more readily than the wild-type S-protein. These results demonstrate that the conformation of the S-protein can be controlled via rational design and can provide a framework for the development of engineered CoV S-proteins for vaccine applications.


Subject(s)
Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Binding Sites , Cryoelectron Microscopy , Microscopy, Electron/methods , Models, Molecular , Mutation , Protein Conformation , Protein Domains , Protein Subunits/chemistry , Spike Glycoprotein, Coronavirus/genetics
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