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1.
Viruses ; 13(9)2021 09 09.
Article in English | MEDLINE | ID: covidwho-1411084

ABSTRACT

A variety of immunolabeling procedures for both light and electron microscopy were used to examine the cellular origins of the host membranes supporting the SARS-CoV-2 replication complex. The endoplasmic reticulum has long been implicated as a source of membrane for the coronavirus replication organelle. Using dsRNA as a marker for sites of viral RNA synthesis, we provide additional evidence supporting ER as a prominent source of membrane. In addition, we observed a rapid fragmentation of the Golgi apparatus which is visible by 6 h and complete by 12 h post-infection. Golgi derived lipid appears to be incorporated into the replication organelle although protein markers are dispersed throughout the infected cell. The mechanism of Golgi disruption is undefined, but chemical disruption of the Golgi apparatus by brefeldin A is inhibitory to viral replication. A search for an individual SARS-CoV-2 protein responsible for this activity identified at least five viral proteins, M, S, E, Orf6, and nsp3, that induced Golgi fragmentation when expressed in eukaryotic cells. Each of these proteins, as well as nsp4, also caused visible changes to ER structure as shown by correlative light and electron microscopy (CLEM). Collectively, these results imply that specific disruption of the Golgi apparatus is a critical component of coronavirus replication.


Subject(s)
Endoplasmic Reticulum/virology , Golgi Apparatus/virology , SARS-CoV-2/physiology , Virus Replication , Animals , Chlorocebus aethiops , Coronavirus M Proteins/physiology , Coronavirus M Proteins/ultrastructure , Endoplasmic Reticulum/ultrastructure , Golgi Apparatus/ultrastructure , Humans , Intracellular Membranes/ultrastructure , Intracellular Membranes/virology , Microscopy, Electron , SARS-CoV-2/ultrastructure , Vero Cells , Viral Structural Proteins/physiology , Viral Structural Proteins/ultrastructure
2.
Cardiovasc Pathol ; 52: 107338, 2021.
Article in English | MEDLINE | ID: covidwho-1385193
3.
Cardiovasc Pathol ; 52: 107337, 2021.
Article in English | MEDLINE | ID: covidwho-1385192
6.
Cell Host Microbe ; 28(6): 853-866.e5, 2020 12 09.
Article in English | MEDLINE | ID: covidwho-1385263

ABSTRACT

Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2-infected human lung epithelial cells. We report 3D electron microscopy reconstructions of whole cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodeling of cytoskeleton elements. Pharmacological inhibition of their dynamics suppresses SARS-CoV-2 replication. We thus report insights into virus-induced cytopathic effects and provide alongside a comprehensive publicly available repository of 3D datasets of SARS-CoV-2-infected cells for download and smooth online visualization.


Subject(s)
COVID-19/genetics , Endoplasmic Reticulum/ultrastructure , SARS-CoV-2/ultrastructure , Viral Replication Compartments/ultrastructure , COVID-19/diagnostic imaging , COVID-19/pathology , COVID-19/virology , Cell Death/genetics , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/virology , Humans , Microscopy, Electron , Pandemics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Viral Replication Compartments/metabolism , Virus Replication/genetics
7.
Pediatr Nephrol ; 36(11): 3789-3793, 2021 11.
Article in English | MEDLINE | ID: covidwho-1361293

ABSTRACT

BACKGROUND: Histological findings of kidney involvement have been rarely reported in pediatric patients with SARS-CoV-2 infection. Here, we describe clinical, laboratory, and histological findings of two pediatric cases with almost exclusive kidney involvement by SARS-CoV-2. RESULTS: A 10-year-old girl with IgA vasculitis nephritis underwent kidney biopsy, showing diffuse and segmental mesangial-proliferative glomerulonephritis, and steroid therapy was initiated. After the worsening of the clinical picture, including an atypical skin rash, she was diagnosed with SARS-CoV-2. The re-evaluation of initial biopsy showed cytoplasmatic blebs and virus-like particles in tubular cells at electron microscopy. Despite SARS-CoV-2 clearance and the intensification of immunosuppression, no improvement was observed. A second kidney biopsy showed a crescentic glomerulonephritis with sclerosis, while virus-like particles were no longer evident. The second patient was a 12-year-old girl with a 3-week history of weakness and weight loss. Rhinitis was reported the month before. No medications were being taken. Blood and urine analysis revealed elevated serum creatinine, hypouricemia, low molecular weight proteinuria, and glycosuria. A high SARS-CoV-2-IgG titre was detected. Kidney biopsy showed acute tubular-interstitial nephritis. Steroid therapy was started with a complete resolution of kidney involvement. CONCLUSION: We can speculate that in both cases SARS-CoV-2 played a major role as inflammatory trigger of the kidney damage. Therefore, we suggest investigating the potential kidney damage by SARS-CoV-2 in children. Moreover, SARS-CoV-2 can be included among infectious agents responsible for pediatric acute tubular interstitial nephritis.


Subject(s)
COVID-19/complications , Glomerulonephritis, IGA/immunology , Kidney/pathology , Nephritis, Interstitial/immunology , SARS-CoV-2/immunology , Biopsy , COVID-19/immunology , COVID-19/virology , Child , Female , Glomerulonephritis, IGA/diagnosis , Glomerulonephritis, IGA/pathology , Glomerulonephritis, IGA/virology , Humans , Kidney/immunology , Kidney/ultrastructure , Kidney/virology , Microscopy, Electron , Nephritis, Interstitial/diagnosis , Nephritis, Interstitial/pathology , Nephritis, Interstitial/virology , SARS-CoV-2/isolation & purification
8.
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
10.
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
11.
Viruses ; 13(2)2021 01 28.
Article in English | MEDLINE | ID: covidwho-1058918

ABSTRACT

During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in the cytoplasm of the infected cell. These viral replication organelles (ROs) play a key role in the replicative cycle of important human pathogens like coronaviruses, enteroviruses, or flaviviruses. From their discovery to date, progress in our understanding of viral ROs has closely followed new developments in electron microscopy (EM). This review gives a chronological account of this progress and an introduction to the different EM techniques that enabled it. With an ample repertoire of imaging modalities, EM is nowadays a versatile technique that provides structural and functional information at a wide range of scales. Together with well-established approaches like electron tomography or labeling methods, we examine more recent developments, such as volume scanning electron microscopy (SEM) and in situ cryotomography, which are only beginning to be applied to the study of viral ROs. We also highlight the first cryotomography analyses of viral ROs, which have led to the discovery of macromolecular complexes that may serve as RO channels that control the export of newly-made viral RNA. These studies are key first steps towards elucidating the macromolecular complexity of viral ROs.


Subject(s)
Microscopy, Electron , RNA Viruses/physiology , Viral Replication Compartments/ultrastructure , Virus Replication , Cryoelectron Microscopy , Electron Microscope Tomography , Image Processing, Computer-Assisted , Intracellular Membranes/ultrastructure , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Microscopy, Immunoelectron , RNA, Viral/biosynthesis , Viral Nonstructural Proteins/analysis , Viral Nonstructural Proteins/metabolism , Viral Replication Compartments/chemistry
13.
Nat Commun ; 12(1): 1346, 2021 03 01.
Article in English | MEDLINE | ID: covidwho-1111984

ABSTRACT

SARS-CoV-2 is the underlying cause for the COVID-19 pandemic. Like most enveloped RNA viruses, SARS-CoV-2 uses a homotrimeric surface antigen to gain entry into host cells. Here we describe S-Trimer, a native-like trimeric subunit vaccine candidate for COVID-19 based on Trimer-Tag technology. Immunization of S-Trimer with either AS03 (oil-in-water emulsion) or CpG 1018 (TLR9 agonist) plus alum adjuvants induced high-level of neutralizing antibodies and Th1-biased cellular immune responses in animal models. Moreover, rhesus macaques immunized with adjuvanted S-Trimer were protected from SARS-CoV-2 challenge compared to vehicle controls, based on clinical observations and reduction of viral loads in lungs. Trimer-Tag may be an important platform technology for scalable production and rapid development of safe and effective subunit vaccines against current and future emerging RNA viruses.


Subject(s)
COVID-19 Vaccines/therapeutic use , COVID-19/immunology , COVID-19/prevention & control , SARS-CoV-2/pathogenicity , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Blotting, Western , COVID-19/therapy , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunity, Cellular/physiology , Immunization, Passive , Immunohistochemistry , Macaca mulatta , Mice , Mice, Inbred BALB C , Microscopy, Electron , SARS-CoV-2/immunology
14.
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
15.
Cell Microbiol ; 23(7): e13319, 2021 07.
Article in English | MEDLINE | ID: covidwho-1087999

ABSTRACT

The ongoing SARS-CoV-2 pandemic with over 80 million infections and more than a million deaths worldwide represents the worst global health crisis of the 21th century. Beyond the health crisis, the disruptions caused by the COVID-19 pandemic have serious global socio-economic consequences. It has also placed a significant pressure on the scientific community to understand the virus and its pathophysiology and rapidly provide anti-viral treatments and procedures in order to help the society and stop the virus spread. Here, we outline how advanced microscopy technologies such as high-throughput microscopy and electron microscopy played a major role in rapid response against SARS-CoV-2. General applicability of developed microscopy technologies makes them uniquely positioned to act as the first line of defence against any emerging infection in the future.


Subject(s)
COVID-19 , Microscopy/methods , SARS-CoV-2 , Antibodies, Viral/blood , Antiviral Agents/pharmacology , COVID-19/diagnosis , COVID-19/drug therapy , COVID-19/pathology , COVID-19/virology , COVID-19 Serological Testing , COVID-19 Vaccines , Cryoelectron Microscopy , Drug Development , High-Throughput Screening Assays , Humans , Microscopy, Electron , Pandemics , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , SARS-CoV-2/physiology , SARS-CoV-2/ultrastructure , Virus Replication
16.
Br J Dermatol ; 183(4): 729-737, 2020 10.
Article in English | MEDLINE | ID: covidwho-1081133

ABSTRACT

BACKGROUND: Chilblains ('COVID toes') are being seen with increasing frequency in children and young adults during the COVID-19 pandemic. Detailed histopathological descriptions of COVID-19 chilblains have not been reported, and causality of SARS-CoV-2 has not yet been established. OBJECTIVES: To describe the histopathological features of COVID-19 chilblains and to explore the presence of SARS-CoV-2 in the tissue. METHODS: We examined skin biopsies from seven paediatric patients presenting with chilblains during the COVID-19 pandemic. Immunohistochemistry for SARS-CoV-2 was performed in all cases and electron microscopy in one. RESULTS: Histopathology showed variable degrees of lymphocytic vasculitis ranging from endothelial swelling and endotheliitis to fibrinoid necrosis and thrombosis. Purpura, superficial and deep perivascular lymphocytic inflammation with perieccrine accentuation, oedema, and mild vacuolar interface damage were also seen. SARS-CoV-2 immunohistochemistry was positive in endothelial cells and epithelial cells of eccrine glands. Coronavirus particles were found in the cytoplasm of endothelial cells on electron microscopy. CONCLUSIONS: Although the clinical and histopathological features were similar to other forms of chilblains, the presence of viral particles in the endothelium and the histological evidence of vascular damage support a causal relation of the lesions with SARS-CoV-2. Endothelial damage induced by the virus could be the key mechanism in the pathogenesis of COVID-19 chilblains and perhaps also in a group of patients severely affected by COVID-19 presenting with features of microangiopathic damage. What is already known about this topic? Despite the high number of cases of chilblains seen during the COVID-19 pandemic, a definite causative role for SARS-CoV-2 has not yet been proven. Different pathogenetic hypotheses have been proposed, including coagulation anomalies, interferon release and external factors. What does this study add? The demonstration of SARS-CoV-2 in endothelial cells of skin biopsies by immunohistochemistry and electron microscopy confirms that these lesions are part of the spectrum of COVID-19. Virus-induced vascular damage and secondary ischaemia could explain the pathophysiology of COVID-19 chilblains. Our findings support the hypothesis that widespread endothelial infection by SARS-CoV-2 could have a pathogenetic role in the severe forms of COVID-19. Linked Comment: Wetter. Br J Dermatol 2020; 183:611.


Subject(s)
Chilblains/virology , Coronavirus Infections/complications , Endothelium, Vascular/pathology , Pneumonia, Viral/complications , Skin Diseases/virology , Vasculitis/virology , Betacoronavirus/isolation & purification , Betacoronavirus/pathogenicity , Biopsy , COVID-19 , Chilblains/pathology , Child , Coronavirus Infections/pathology , Coronavirus Infections/virology , Endothelial Cells/pathology , Endothelial Cells/ultrastructure , Endothelial Cells/virology , Endothelium, Vascular/virology , Humans , Immunohistochemistry , Microscopy, Electron , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , SARS-CoV-2 , Skin/blood supply , Skin/pathology , Skin/virology , Skin Diseases/pathology , Vasculitis/pathology
17.
18.
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
19.
Am J Surg Pathol ; 45(3): 293-303, 2021 03 01.
Article in English | MEDLINE | ID: covidwho-1010690

ABSTRACT

The novel coronavirus disease (COVID-19) is a rapidly spreading pandemic, secondary to severe acute respiratory syndrome coronavirus 2. The severity and the little knowledge that we have of the disease have made us focus mostly on the respiratory symptoms. As we bend the curve, other findings reported in association with COVID-19 become of importance for specialists to recognize. We describe the spectrum of clinicopathologic lesions in the skin that can be the only symptom or the first manifestation of COVID-19 and demonstrate the origin of the virus. We collected 25 patients with skin lesions in this context. We recognized 5 types of cutaneous manifestations including acute acroischemic or chilblain-like lesions (11), purpura palpable (2), exanthemas (9), urticarial eruptions (1), and other lesions (2) that might appear with more unspecific pictures. Chilblain-like lesions were the most common form of presentation, which tend to appear as self-healing, erythematous-necrotic plaques mostly on the feet, in young patients with no systemic symptoms associated. Importantly, we visualized viral particles with electron microscopy in 5 of 13 cases analyzed. In this study, we seek to draw a picture of the spectrum of clinicopathologic lesions that may appear in the skin in the context of COVID-19. Although apparently skin lesions are not correlated with disease severity, it may help in some cases to recognize and control the spread of the infection sooner.


Subject(s)
COVID-19/diagnosis , Endothelial Cells/virology , Skin Diseases/virology , Adolescent , Adult , Aged , Aged, 80 and over , COVID-19/complications , COVID-19/pathology , COVID-19 Testing , Endothelial Cells/pathology , Female , Humans , Male , Microscopy, Electron , Middle Aged , Retrospective Studies , Severity of Illness Index , Skin Diseases/diagnosis , Skin Diseases/pathology , Young Adult
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