SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation.

Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.

SARS-CoV-2 spike protein dictates syncytium-mediated lymphocyte elimination.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is highly contagious and causes lymphocytopenia, but the underlying mechanisms are poorly understood. We demonstrate here that heterotypic cell-in-cell structures with lymphocytes inside multinucleate syncytia are prevalent in the lung tissues of coronavirus disease 2019 (COVID-19) patients. These unique cellular structures are a direct result of SARS-CoV-2 infection, as the expression of the SARS-CoV-2 spike glycoprotein is sufficient to induce a rapid (~45.1 nm/s) membrane fusion to produce syncytium, which could readily internalize multiple lines of lymphocytes to form typical cell-in-cell structures, remarkably leading to the death of internalized cells. This membrane fusion is dictated by a bi-arginine motif within the polybasic S1/S2 cleavage site, which is frequently present in the surface glycoprotein of most highly contagious viruses. Moreover, candidate anti-viral drugs could efficiently inhibit spike glycoprotein processing, membrane fusion, and cell-in-cell formation. Together, we delineate a molecular and cellular rationale for SARS-CoV-2 pathogenesis and identify novel targets for COVID-19 therapy.

COVID-19: the difference between the nose and the lung.

To the Editor An elegant study reported dysmorphic cells and syncytia in the deceased's lungs for COVID-19. The authors reasonably considered that most of these syncytia-forming cells were pneumocytes, as identified by specific biomarkers. However, cellular dysmorphism and syncytia are pathological features common in other respiratory infections caused by different viruses, including the human respiratory syncytial virus (HRSV) and Epstein-Barr virus (EBV), as correctly documented...

Postmortem lung biopsies from four patients with COVID-19 at a tertiary hospital in Cape Town, South Africa.

BACKGROUND: An outbreak of a novel coronavirus in China in late 2019 has resulted in a global pandemic. The virus (SARS-CoV-2) causes a severe acute respiratory syndrome and had been responsible for >14 000 deaths in South Africa (SA) at the time of writing, 30 August 2020. Autopsies in our setting have not been prioritised owing to the infective risks for staff, resulting in a lack of information on the histopathology of the disease in the SA setting. Postmortem biopsies are relatively quick and easy to perform and reduce the infective risk posed by full autopsies. OBJECTIVES: To determine whether postmortem biopsies of lung tissue could be used to determine cause of death in lieu of full autopsies in patients dying from COVID-19. METHODS: We performed postmortem biopsies of lung tissue on 4 patients with SARS-CoV-2 confirmed by reverse transcriptase polymerase chain reaction who died in the Tygerberg Hospital (Cape Town, SA) intensive care unit (ICU) in June - July 2020, in order to determine their cause of death. The biopsies were performed in the ICU with the necessary personal protective equipment within 2 hours after death. Clinical information was obtained from the hospital records and the histopathology was reviewed by two consultant histopathologists. Microbiology and electron microscopy were also performed on this tissue. RESULTS: All 4 patients were aged >50 years and had multiple comorbidities. Pulmonary pathology was present in only 3 cases, and the findings were surprisingly heterogeneous. One case demonstrated several findings including diffuse alveolar damage, extensive fibrin thrombi in pulmonary arteries with pulmonary infarction, organising pneumonia and bronchopneumonia. Other findings included type 2 pneumocyte hyperplasia, intra-alveolar macrophages and squamous metaplasia. An organising pneumonia was present in 2 other cases, although these findings were not deemed to be severe enough to be the cause of death. Fibrin thrombi were present in pulmonary arteries of 3 cases. One case showed no significant acute pulmonary pathology. The cause of death could only be determined in 1 case. CONCLUSIONS: The pulmonary findings we observed are in keeping with those described in the international literature. However, the pathology was surprisingly heterogeneous between cases, and was only deemed severe enough to be the cause of death in 1 of 4 cases. While lung-targeted, standardised postmortem biopsies may be safe, easy to perform and provide useful insights into the disease, they are not suitable to replace full autopsies in determining cause of death.

The anti-HIV drug nelfinavir mesylate (Viracept) is a potent inhibitor of cell fusion caused by the SARSCoV-2 spike (S) glycoprotein warranting further evaluation as an antiviral against COVID-19 infections.

Severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) is the causative agent of the coronavirus disease-2019 (COVID-19) pandemic. Coronaviruses enter cells via fusion of the viral envelope with the plasma membrane and/or via fusion of the viral envelope with endosomal membranes after virion endocytosis. The spike (S) glycoprotein is a major determinant of virus infectivity. Herein, we show that the transient expression of the SARS CoV-2 S glycoprotein in Vero cells caused extensive cell fusion (formation of syncytia) in comparison to limited cell fusion caused by the SARS S glycoprotein. Both S glycoproteins were detected intracellularly and on transfected Vero cell surfaces. These results are in agreement with published pathology observations of extensive syncytia formation in lung tissues of patients with COVID-19. These results suggest that SARS CoV-2 is able to spread from cell-to-cell much more efficiently than SARS effectively avoiding extracellular neutralizing antibodies. A systematic screening of several drugs including cardiac glycosides and kinase inhibitors and inhibitors of human immunodeficiency virus (HIV) entry revealed that only the FDA-approved HIV protease inhibitor, nelfinavir mesylate (Viracept) drastically inhibited S-n- and S-o-mediated cell fusion with complete inhibition at a 10-µM concentration. In-silico docking experiments suggested the possibility that nelfinavir may bind inside the S trimer structure, proximal to the S2 amino terminus directly inhibiting S-n- and S-o-mediated membrane fusion. Also, it is possible that nelfinavir may act to inhibit S proteolytic processing within cells. These results warrant further investigations of the potential of nelfinavir mesylate to inhibit virus spread at early times after SARS CoV-2 symptoms appear.

Dying with SARS-CoV-2 infection-an autopsy study of the first consecutive 80 cases in Hamburg, Germany.

Autopsies of deceased with a confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can provide important insights into the novel disease and its course. Furthermore, autopsies are essential for the correct statistical recording of the coronavirus disease 2019 (COVID-19) deaths. In the northern German Federal State of Hamburg, all deaths of Hamburg citizens with ante- or postmortem PCR-confirmed SARS-CoV-2 infection have been autopsied since the outbreak of the pandemic in Germany. Our evaluation provides a systematic overview of the first 80 consecutive full autopsies. A proposal for the categorisation of deaths with SARS-CoV-2 infection is presented (category 1: definite COVID-19 death; category 2: probable COVID-19 death; category 3: possible COVID-19 death with an equal alternative cause of death; category 4: SARS-CoV-2 detection with cause of death not associated to COVID-19). In six cases, SARS-CoV-2 infection was diagnosed postmortem by a positive PCR test in a nasopharyngeal or lung tissue swab. In the other 74 cases, SARS-CoV-2 infection had already been known antemortem. The deceased were aged between 52 and 96 years (average 79.2 years, median 82.4 years). In the study cohort, 34 deceased were female (38%) and 46 male (62%). Overall, 38% of the deceased were overweight or obese. All deceased, except for two women, in whom no significant pre-existing conditions were found autoptically, had relevant comorbidities (in descending order of frequency): (1) diseases of the cardiovascular system, (2) lung diseases, (3) central nervous system diseases, (4) kidney diseases, and (5) diabetes mellitus. A total of 76 cases (95%) were classified as COVID-19 deaths, corresponding to categories 1-3. Four deaths (5%) were defined as non-COVID-19 deaths with virus-independent causes of death. In eight cases, pneumonia was combined with a fulminant pulmonary artery embolism. Peripheral pulmonary artery embolisms were found in nine other cases. Overall, deep vein thrombosis has been found in 40% of the cases. This study provides the largest overview of autopsies of SARS-CoV-2-infected patients presented so far.