Electron microscopy of SARS-CoV-2: a challenging task – Authors' reply
Authors: Zsuzsanna Varga, Andreas J Flammer, Peter Steiger, Martina Haberecker, Rea Andermatt, Annelies Zinkernagel, Mandeep R Mehra, Felix Scholkmann, Reto Schüpbach, Frank Ruschitzka, Holger Moch
Journal: The Lancet (2020)

We thank Cynthia Goldsmith and colleagues for their interest in our recent Correspondence.1Varga Z Flammer AJ Steiger P Endothelial cell infection and endotheliitis in COVID-19.Lancet. 2020; 395: 1417-1418Summary Full Text Full Text PDF PubMed Scopus (4567) Google Scholar We described autopsy findings from patients who had died from COVID-19 and showed a systemic endotheliitis with evidence of loss of integrity of the endothelial monolayer.1Varga Z Flammer AJ Steiger P Endothelial cell infection and endotheliitis in COVID-19.Lancet. 2020; 395: 1417-1418Summary Full Text Full Text PDF PubMed Scopus (4567) Google Scholar The framework of endotheliitis provides an explanation for the unique predilection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in those individuals with hypertension, diabetes, or established cardiovascular disease, a group known to have pre-existing endothelial dysfunction. COVID-19-endotheliitis could also explain impaired microcirculatory function across different organs and the frequently observed prothrombotic state with in-situ clot formation. Endothelial infection and injury by SARS-CoV-1 has been shown.2Ye J Zhang B Xu J et al.Molecular pathology in the lungs of severe acute respiratory syndrome patients.Am J Pathol. 2007; 170: 538-545Summary Full Text Full Text PDF PubMed Scopus (72) Google Scholar Our demonstration of viral particles using electron microscopy (EM) is supported by several reports independently describing ultrastructural round virus-like particles in the setting of a SARS-CoV-2 infection.3Su H Yang M Wan C et al.Renal histopathological analysis of 26 postmortem findings of patients with COVID-19 in China.Kidney Int. 2020; (published online April 9.)DOI:10.1016/j.kint.2020.04.003Summary Full Text Full Text PDF PubMed Scopus (1291) Google Scholar, 4Kissling S Rotman S Gerber C et al.Collapsing glomerulopathy in a COVID-19 patient.Kidney Int. 2020; (published online April 15.)DOI:10.1016/j.kint.2020.04.006Summary Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 5Zhu N Zhang D Wang W et al.A novel coronavirus from patients with pneumonia in China, 2019.N Engl J Med. 2020; 382: 727-733Crossref PubMed Scopus (18864) Google Scholar, 6Menter T Haslbauer JD Nienhold R et al.Post-mortem examination of COVID19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings of lungs and other organs suggesting vascular dysfunction.Histopathology. 2020; (published online May 4.)DOI:10.1111/his.14134Crossref PubMed Scopus (909) Google Scholar We demonstrated tubulo-reticular structures in the immediate vicinity of the spherical particles that are strikingly identical to SARS-CoV-1-associated membrane changes described by Goldsmith and colleagues in 2004.7Goldsmith CS Tatti KM Ksiazek TG et al.Ultrastructural characterization of SARS coronavirus.Emerg Infect Diseases. 2004; 10: 320-326Crossref PubMed Scopus (318) Google Scholar In our EM thin-section images, the virus-like particles were relatively large (mean diameter 180 nm [SD 10]). However, subsequent analysis of more EM images has revealed a mean particle size of 67 nm (SD 15 nm, median 65 nm, 95% CI 41–102; n=33). Zhu and colleagues5Zhu N Zhang D Wang W et al.A novel coronavirus from patients with pneumonia in China, 2019.N Engl J Med. 2020; 382: 727-733Crossref PubMed Scopus (18864) Google Scholar noted that SARS-CoV-2 virions ranged from "about 60 to 140 nm". In another recent study,6Menter T Haslbauer JD Nienhold R et al.Post-mortem examination of COVID19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings of lungs and other organs suggesting vascular dysfunction.Histopathology. 2020; (published online May 4.)DOI:10.1111/his.14134Crossref PubMed Scopus (909) Google Scholar virus-like particles in patients with confirmed SARS-CoV-2 infection were 70–110 nm in diameter. By comparison, SARS-CoV-1 viral particles analysed with the same technique (ultrathin EM imaging) were 50–80 nm in diameter.7Goldsmith CS Tatti KM Ksiazek TG et al.Ultrastructural characterization of SARS coronavirus.Emerg Infect Diseases. 2004; 10: 320-326Crossref PubMed Scopus (318) Google Scholar, 8Goldsmith CS Miller SE Modern uses of electron microscopy for detection of viruses.Clin Microbiol Rev. 2009; 22: 552-563Crossref PubMed Scopus (237) Google Scholar, 9Hieh WJ Hsiao CH Paddock CD et al.Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan.Hum Pathol. 2005; 36: 303-309Crossref PubMed Scopus (122) Google Scholar, 10Qinfen Z Jinming C Xiaojun H et al.The life cycle of SARS coronavirus in Vero E6 cells.J Med Virol. 2004; 73: 332-337Crossref PubMed Scopus (99) Google Scholar Goldsmith and colleagues have studied coronavirus isolates grown in cell culture, whereas our EM data of virus-like particles were obtained from a post-mortem kidney allograft obtained during autopsy. Since most other recent reports of patients with COVID-19 also describe postmortem findings, it remains unclear to what extent tissue type (cell culture, fresh biopsy material, or autopsy material), time to fixation, and postmortal autolysis alter subcellular structures in preparation for EM. This notwithstanding, these observed particles in patients with COVID-19 should be best designated as virus-like particles because definitive assignment of these structures as SARS-CoV-2 virions requires immuno-EM. Investigations with vascular organoids that preceded our observations1Varga Z Flammer AJ Steiger P Endothelial cell infection and endotheliitis in COVID-19.Lancet. 2020; 395: 1417-1418Summary Full Text Full Text PDF PubMed Scopus (4567) Google Scholar showed that SARS-CoV-2 can infect human blood vessels via the ACE2 pathways, providing the first and direct evidence that the virus can indeed invade human vasculature.11Monteil V Kwon H Prado P et al.Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2.Cell. 2020; (published online April 17.)DOI:10.1016/j.cell.2020.04.004Summary Full Text Full Text PDF PubMed Scopus (1616) Google Scholar Our findings have also been confirmed in descriptions of renal tropism of SARS-CoV-2, with detection of SARS-CoV-2 protein in human glomerular endothelial and epithelial cells.12Puelles VG Lutgehetmann M Lindenmeyer MT et al.Multiorgan and renal tropism of SARS-CoV-2.N Engl J Med. 2020; (published online May 13.)DOI:10.1056/NEJMc2011400Crossref PubMed Scopus (1308) Google Scholar Importantly, our demonstration of virus cell infection in the kidney and endotheliitis1Varga Z Flammer AJ Steiger P Endothelial cell infection and endotheliitis in COVID-19.Lancet. 2020; 395: 1417-1418Summary Full Text Full Text PDF PubMed Scopus (4567) Google Scholar points to a general host inflammatory response causing hyperinflammation as a principal participant in the vascular pathology of COVID-19. Endothelial cell dysfunction, which might subsequently induce a prothrombotic state, could thus explain the vascular microcirculatory complications seen in different organs in patients with COVID-19. We declare no competing interests. Electron microscopy of SARS-CoV-2: a challenging taskWe read with interest the Correspondence by Zsuzsanna Varga and colleagues1 on the possible infection of endothelial cells by SARS-CoV-2 using electron microscopic (EM) images as evidence. However, we believe the EM images in the Correspondence do not show coronavirus particles but instead show cross-sections of the rough endoplasmic reticulum (RER). These spherical structures are surrounded by dark dots, which might have been interpreted as spikes on coronavirus particles but are instead ribosomes. Full-Text PDF Endothelial cell infection and endotheliitis in COVID-19Cardiovascular complications are rapidly emerging as a key threat in coronavirus disease 2019 (COVID-19) in addition to respiratory disease. The mechanisms underlying the disproportionate effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on patients with cardiovascular comorbidities, however, remain incompletely understood.1,2 Full-Text PDF