SARS-CoV-2 infection of kidney tissues from severe COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests diverse clinical pathologies involving multiple organs. While the respiratory tract is the primary SARS-CoV-2 target, acute kidney injury is common in COVID-19 patients, displaying as acute tubular necrosis (ATN) resulting from focal epithelial necrosis and eosinophilia, glomerulosclerosis, and autolysis of renal tubular cells. However, whether any renal cells are infected by SARS-CoV-2 and the mechanism involved in the COVID-19 kidney pathology remain unclear. METHODS: Kidney tissues obtained at autopsy from four severe COVID-19 patients and one healthy subject were examined by hematoxylin and eosin staining. Indirect immunofluorescent antibody assay was performed to detect SARS-CoV-2 spike protein S1 and nonstructural protein 8 (NSP8) together with markers of different kidney cell types and immune cells to identify the infected cells. RESULTS: Renal parenchyma showed tissue injury comprised of ATN and glomerulosclerosis. Positive staining of S1 protein was observed in renal parenchymal and tubular epithelial cells. Evidence of viral infection was also observed in innate monocytes/macrophages and NK cells. Positive staining of NSP8, which is essential for viral RNA synthesis and replication, was confirmed in renal parenchymal cells, indicating the presence of active viral replication in the kidney. CONCLUSIONS: In fatal COVID-19 kidneys, there are SARS-CoV-2 infection, minimally infiltrated innate immune cells, and evidence of viral replication, which could contribute to tissue damage in the form of ATN and glomerulosclerosis.

Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp).

SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.

Molecular Profiling of Coronavirus Disease 2019 (COVID-19) Autopsies Uncovers Novel Disease Mechanisms.

Current understanding of coronavirus disease 2019 (COVID-19) pathophysiology is limited by disease heterogeneity, complexity, and a paucity of studies assessing patient tissues with advanced molecular tools. Rapid autopsy tissues were evaluated using multiscale, next-generation RNA-sequencing methods (bulk, single-nuclei, and spatial transcriptomics) to provide unprecedented molecular resolution of COVID-19-induced damage. Comparison of infected/uninfected tissues revealed four major regulatory pathways. Effectors within these pathways could constitute novel therapeutic targets, including the complement receptor C3AR1, calcitonin receptor-like receptor, or decorin. Single-nuclei RNA sequencing of olfactory bulb and prefrontal cortex highlighted remarkable diversity of coronavirus receptors. Angiotensin-converting enzyme 2 was rarely expressed, whereas basigin showed diffuse expression, and alanyl aminopeptidase, membrane, was associated with vascular/mesenchymal cell types. Comparison of lung and lymph node tissues from patients with different symptoms (one had died after a month-long hospitalization with multiorgan involvement, and the other had died after a few days of respiratory symptoms) with digital spatial profiling resulted in distinct molecular phenotypes. Evaluation of COVID-19 rapid autopsy tissues with advanced molecular techniques can identify pathways and effectors, map diverse receptors at the single-cell level, and help dissect differences driving diverging clinical courses among individual patients. Extension of this approach to larger data sets will substantially advance the understanding of the mechanisms behind COVID-19 pathophysiology.

Broad Severe Acute Respiratory Syndrome Coronavirus 2 Cell Tropism and Immunopathology in Lung Tissues From Fatal Coronavirus Disease 2019.

BACKGROUND: Coronavirus disease 2019 (COVID-19) patients manifest with pulmonary symptoms reflected by diffuse alveolar damage (DAD), excessive inflammation, and thromboembolism. The mechanisms mediating these processes remain unclear. METHODS: We performed multicolor staining for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and lineage markers to define viral tropism and lung pathobiology in 5 autopsy cases. RESULTS: Lung parenchyma showed severe DAD with thromboemboli. Viral infection was found in an extensive range of cells including pneumocyte type II, ciliated, goblet, club-like, and endothelial cells. More than 90% of infiltrating immune cells were positive for viral proteins including macrophages, monocytes, neutrophils, natural killer (NK) cells, B cells, and T cells. Most but not all infected cells were angiotensin-converting enzyme 2 (ACE2) positive. The numbers of infected and ACE2-positive cells are associated with extensive tissue damage. Infected tissues exhibited high levels of inflammatory cells including macrophages, monocytes, neutrophils, and NK cells, and low levels of B cells but abundant T cells consisting of mainly T helper cells, few cytotoxic T cells, and no regulatory T cells. Robust interleukin-6 expression was present in most cells, with or without infection. CONCLUSIONS: In fatal COVID-19 lungs, there are broad SARS-CoV-2 cell tropisms, extensive infiltrated innate immune cells, and activation and depletion of adaptive immune cells, contributing to severe tissue damage, thromboemboli, excess inflammation, and compromised immune responses.

Tissue-based SARS-CoV-2 detection in fatal COVID-19 infections: Sustained direct viral-induced damage is not necessary to drive disease progression.

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although viral infection is known to trigger inflammatory processes contributing to tissue injury and organ failure, it is unclear whether direct viral damage is needed to sustain cellular injury. An understanding of pathogenic mechanisms has been handicapped by the absence of optimized methods to visualize the presence and distribution of SARS-CoV-2 in damaged tissues. We first developed a positive control cell line (Vero E6) to validate SARS-CoV-2 detection assays. We then evaluated multiple organs (lungs, kidneys, heart, liver, brain, intestines, lymph nodes, and spleen) from fourteen COVID-19 autopsy cases using immunohistochemistry (IHC) for the spike and the nucleoprotein proteins, and RNA in situ hybridization (RNA ISH) for the spike protein mRNA. Tissue detection assays were compared with quantitative polymerase chain reaction (qPCR)-based detection. SARS-CoV-2 was histologically detected in the Vero E6 positive cell line control, 1 of 14 (7%) lungs, and none (0%) of the other 59 organs. There was perfect concordance between the IHC and RNA ISH results. qPCR confirmed high viral load in the SARS-CoV-2 ISH-positive lung tissue, and absent or low viral load in all ISH-negative tissues. In patients who die of COVID-19-related organ failure, SARS-CoV-2 is largely not detectable using tissue-based assays. Even in lungs showing widespread injury, SARS-CoV-2 viral RNA or proteins were detected in only a small minority of cases. This observation supports the concept that viral infection is primarily a trigger for multiple-organ pathogenic proinflammatory responses. Direct viral tissue damage is a transient phenomenon that is generally not sustained throughout disease progression.

Pathophysiology of SARS-CoV-2: the Mount Sinai COVID-19 autopsy experience.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated clinical syndrome COVID-19 are causing overwhelming morbidity and mortality around the globe and disproportionately affected New York City between March and May 2020. Here, we report on the first 100 COVID-19-positive autopsies performed at the Mount Sinai Hospital in New York City. Autopsies revealed large pulmonary emboli in six cases. Diffuse alveolar damage was present in over 90% of cases. We also report microthrombi in multiple organ systems including the brain, as well as hemophagocytosis. We additionally provide electron microscopic evidence of the presence of the virus in our samples. Laboratory results of our COVID-19 cohort disclose elevated inflammatory markers, abnormal coagulation values, and elevated cytokines IL-6, IL-8, and TNFα. Our autopsy series of COVID-19-positive patients reveals that this disease, often conceptualized as a primarily respiratory viral illness, has widespread effects in the body including hypercoagulability, a hyperinflammatory state, and endothelial dysfunction. Targeting of these multisystemic pathways could lead to new treatment avenues as well as combination therapies against SARS-CoV-2 infection.

A review of the main histopathological findings in coronavirus disease 2019.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has been declared by the World Health Organization as an emerging public health problem of global importance and classified as a pandemic. SARS-CoV-2 infection can result in diverse, multiorgan pathology, the most significant being in the lungs (diffuse alveolar damage in its different phases, microthrombi, bronchopneumonia, necrotizing bronchiolitis, viral pneumonia), heart (lymphocytic myocarditis), kidney (acute tubular injury), central nervous system (microthrombi, ischemic necrosis, acute hemorrhagic infarction, congestion, and vascular edema), lymph nodes (hemophagocytosis and histiocytosis), bone marrow (hemophagocytosis), and vasculature (deep vein thrombosis). An understanding of the spectrum and frequency of histologic findings in COVID-19 is essential for gaining a better understanding of disease pathophysiology and its ongoing impact on public health. To this end, we conducted a systematic meta-analysis of histopathologic observations to date and review the reported findings.

Central nervous system involvement by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

Neurologic sequelae can be devastating complications of respiratory viral infections. We report the presence of virus in neural and capillary endothelial cells in frontal lobe tissue obtained at postmortem examination from a patient infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Our observations of virus in neural tissue, in conjunction with clinical correlates of worsening neurologic symptoms, pave the way to a closer understanding of the pathogenic mechanisms underlying central nervous system involvement by SARS-CoV-2.