Detection and identification of coronaviruses in human tissues using electron microscopy.

The identification of viral particles within a tissue specimen requires specific knowledge of viral ultrastructure and replication, as well as a thorough familiarity with normal subcellular organelles. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored how challenging the task of identifying coronavirus by electron microscopy (EM) can be. Numerous articles have been published mischaracterizing common subcellular structures, including clathrin- or coatomer- coated vesicles, multivesicular bodies, and rough endoplasmic reticulum, as coronavirus particles in SARS-CoV-2 positive patient tissue specimens. To counter these misinterpretations, we describe the morphological features of coronaviruses that should be used to differentiate coronavirus particles from subcellular structures. Further, as many of the misidentifications of coronavirus particles have stemmed from attempts to attribute tissue damage to direct infection by SARS-CoV-2, we review articles describing ultrastructural changes observed in specimens from SARS-CoV-2-infected individuals that do not necessarily provide EM evidence of direct viral infection. Ultrastructural changes have been observed in respiratory, cardiac, kidney, and intestinal tissues, highlighting the widespread effects that SARS-CoV-2 infection may have on the body, whether through direct viral infection or mediated by SARS-CoV-2 infection-induced inflammatory and immune processes. HIGHLIGHTS: The identification of coronavirus particles in SARS-CoV-2 positive tissues continues to be a challenging task. This review provides examples of coronavirus ultrastructure to aid in the differentiation of the virus from common cellular structures.

Lipid and Nucleocapsid N-Protein Accumulation in COVID-19 Patient Lung and Infected Cells.

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the coronavirus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS-CoV-2-infected Vero cells, processed identically. Correlative light and electron microscopy on semithick cryo-sections demonstrated induction of electron-lucent, lipid-filled compartments after SARS-CoV-2 infection in both lung and cell cultures. In lung tissue, the nonstructural protein 4 and the stable nucleocapsid N-protein were detected on these novel lipid-filled compartments. The induction of such lipid-filled compartments and the localization of the viral proteins in lung of patients with fatal COVID-19 may explain the extensive inflammatory response and provide a new hallmark for SARS-CoV-2 infection at the final, fatal stage of infection. IMPORTANCE Visualization of the subcellular localization of SARS-CoV-2 proteins in lung patient material of COVID-19 patients is important for the understanding of this new virus. We detected viral proteins in the context of the ultrastructure of infected cells and tissues and discovered that some viral proteins accumulate in novel, lipid-filled compartments. These structures are induced in Vero cells but, more importantly, also in lung of patients with COVID-19. We have characterized these lipid-filled compartments and determined that this is a novel, virus-induced structure. Immunogold labeling demonstrated that cellular markers, such as CD63 and lipid droplet marker PLIN-2, are absent. Colocalization of lipid-filled compartments with the stable N-protein and nonstructural protein 4 in lung of the last stages of COVID-19 indicates that these compartments play a key role in the devastating immune response that SARS-CoV-2 infections provoke.

SARS-CoV-2 Immunohistochemistry In Placenta.

Compared to the parental SARS-CoV-2 virus, infections by the now dominant Delta variant of SARS-CoV-2 appear to be more common and more severe in pregnant women. The need for a robust, cheap, and quick method for diagnosing placental infection by SARS-CoV-2 has thus become more acute. Here, we describe a highly sensitive and specific immunohistochemical assay for SARS-CoV-2 nucleocapsid protein for routine use in placental pathology practice.

Renal Considerations in COVID-19: Biology, Pathology, and Pathophysiology.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has emerged into a worldwide pandemic of epic proportion. Beyond pulmonary involvement in coronavirus disease 2019 (COVID-19), a significant subset of patients experiences acute kidney injury. Patients who die from severe disease most notably show diffuse acute tubular injury on postmortem examination with a possible contribution of focal macro- and microvascular thrombi. Renal biopsies in patients with proteinuria and hematuria have demonstrated a glomerular dominant pattern of injury, most notably a collapsing glomerulopathy reminiscent of findings seen in human immunodeficiency virus (HIV) in individuals with apolipoprotein L-1 (APOL1) risk allele variants. Although various mechanisms have been proposed for the pathogenesis of acute kidney injury in SARS-CoV-2 infection, direct renal cell infection has not been definitively demonstrated and our understanding of the spectrum of renal involvement remains incomplete. Herein we discuss the biology, pathology, and pathogenesis of SARS-CoV-2 infection and associated renal involvement. We discuss the molecular biology, risk factors, and pathophysiology of renal injury associated with SARS-CoV-2 infection. We highlight the characteristics of specific renal pathologies based on native kidney biopsy and autopsy. Additionally, a brief discussion on ancillary studies and challenges in the diagnosis of SARS-CoV-2 is presented.

Histopathological findings and clinicopathologic correlation in COVID-19: a systematic review.

The severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) pandemic has had devastating effects on global health and worldwide economy. Despite an initial reluctance to perform autopsies due to concerns for aerosolization of viral particles, a large number of autopsy studies published since May 2020 have shed light on the pathophysiology of Coronavirus disease 2019 (COVID-19). This review summarizes the histopathologic findings and clinicopathologic correlations from autopsies and biopsies performed in patients with COVID-19. PubMed and Medline (EBSCO and Ovid) were queried from June 4, 2020 to September 30, 2020 and histopathologic data from autopsy and biopsy studies were collected based on 2009 Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A total of 58 studies reporting 662 patients were included. Demographic data, comorbidities at presentation, histopathologic findings, and virus detection strategies by organ system were collected. Diffuse alveolar damage, thromboembolism, and nonspecific shock injury in multiple organs were the main findings in this review. The pathologic findings emerging from autopsy and biopsy studies reviewed herein suggest that in addition to a direct viral effect in some organs, a unifying pathogenic mechanism for COVID-19 is ARDS with its known and characteristic inflammatory response, cytokine release, fever, inflammation, and generalized endothelial disturbance. This study supports the notion that autopsy studies are of utmost importance to our understanding of disease features and treatment effect to increase our knowledge of COVID-19 pathophysiology and contribute to more effective treatment strategies.

Difficulties in Differentiating Coronaviruses from Subcellular Structures in Human Tissues by Electron Microscopy.

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.

Best practices for correctly identifying coronavirus by transmission electron microscopy.

This guidance provides clear, concise strategies for identifying coronaviruses by transmission electron microscopy of ultrathin sections of tissues or infected tissue cultures. These include a description of virus morphology as well as cell organelles that can resemble viruses. Biochemical testing and caveats are discussed. Numerous references provide information for documentation and further study.

Hunting coronavirus by transmission electron microscopy - a guide to SARS-CoV-2-associated ultrastructural pathology in COVID-19 tissues.

Transmission electron microscopy has become a valuable tool to investigate tissues of COVID-19 patients because it allows visualisation of SARS-CoV-2, but the 'virus-like particles' described in several organs have been highly contested. Because most electron microscopists in pathology are not accustomed to analysing viral particles and subcellular structures, our review aims to discuss the ultrastructural changes associated with SARS-CoV-2 infection and COVID-19 with respect to pathology, virology and electron microscopy. Using micrographs from infected cell cultures and autopsy tissues, we show how coronavirus replication affects ultrastructure and put the morphological findings in the context of viral replication, which induces extensive remodelling of the intracellular membrane systems. Virions assemble by budding into the endoplasmic reticulum-Golgi intermediate complex and are characterised by electron-dense dots of cross-sections of the nucleocapsid inside the viral particles. Physiological mimickers such as multivesicular bodies or coated vesicles serve as perfect decoys. Compared to other in-situ techniques, transmission electron microscopy is the only method to visualise assembled virions in tissues, and will be required to prove SARS-CoV-2 replication outside the respiratory tract. In practice, documenting in tissues the characteristic features seen in infected cell cultures seems to be much more difficult than anticipated. In our view, the hunt for coronavirus by transmission electron microscopy is still on.

SARS-CoV-2 causes a specific dysfunction of the kidney proximal tubule.

Coronavirus disease 2019 (COVID-19) is commonly associated with kidney damage, and the angiotensin converting enzyme 2 (ACE2) receptor for SARS-CoV-2 is highly expressed in the proximal tubule cells. Whether patients with COVID-19 present specific manifestations of proximal tubule dysfunction remains unknown. To test this, we examined a cohort of 49 patients requiring hospitalization in a large academic hospital in Brussels, Belgium. There was evidence of proximal tubule dysfunction in a subset of patients with COVID-19, as attested by low-molecular-weight proteinuria (70-80%), neutral aminoaciduria (46%), and defective handling of uric acid (46%) or phosphate (19%). None of the patients had normoglycemic glucosuria. Proximal tubule dysfunction was independent of pre-existing comorbidities, glomerular proteinuria, nephrotoxic medications or viral load. At the structural level, kidneys from patients with COVID-19 showed prominent tubular injury, including in the initial part of the proximal tubule, with brush border loss, acute tubular necrosis, intraluminal debris, and a marked decrease in the expression of megalin in the brush border. Transmission electron microscopy identified particles resembling coronaviruses in vacuoles or cisternae of the endoplasmic reticulum in proximal tubule cells. Among features of proximal tubule dysfunction, hypouricemia with inappropriate uricosuria was independently associated with disease severity and with a significant increase in the risk of respiratory failure requiring invasive mechanical ventilation using Cox (adjusted hazard ratio 6.2, 95% CI 1.9-20.1) or competing risks (adjusted sub-distribution hazard ratio 12.1, 95% CI 2.7-55.4) survival models. Thus, our data establish that SARS-CoV-2 causes specific manifestations of proximal tubule dysfunction and provide novel insights into COVID-19 severity and outcome.