Ultrastructural study confirms the formation of single and heterotypic syncytial cells in bronchoalveolar fluids of COVID-19 patients.

BACKGROUND: SARS-CoV-2 was reported to induce cell fusions to form multinuclear syncytia that might facilitate viral replication, dissemination, immune evasion, and inflammatory responses. In this study, we have reported the types of cells involved in syncytia formation at different stages of COVID-19 disease through electron microscopy. METHODS: Bronchoalveolar fluids from the mild (n = 8, SpO2 > 95%, no hypoxia, within 2-8 days of infection), moderate (n = 8, SpO2 90% to ≤ 93% on room air, respiratory rate ≥ 24/min, breathlessness, within 9-16 days of infection), and severe (n = 8, SpO2 < 90%, respiratory rate > 30/min, external oxygen support, after 17th days of infection) COVID-19 patients were examined by PAP (cell type identification), immunofluorescence (for the level of viral infection), scanning (SEM), and transmission (TEM) electron microscopy to identify the syncytia. RESULTS: Immunofluorescence studies (S protein-specific antibodies) from each syncytium indicate a very high infection level. We could not find any syncytial cells in mildly infected patients. However, identical (neutrophils or type 2 pneumocytes) and heterotypic (neutrophils-monocytes) plasma membrane initial fusion (indicating initiation of fusion) was observed under TEM in moderately infected patients. Fully matured large-size (20-100 µm) syncytial cells were found in severe acute respiratory distress syndrome (ARDS-like) patients of neutrophils, monocytes, and macrophage origin under SEM. CONCLUSIONS: This ultrastructural study on the syncytial cells from COVID-19 patients sheds light on the disease's stages and types of cells involved in the syncytia formations. Syncytia formation was first induced in type II pneumocytes by homotypic fusion and later with haematopoetic cells (monocyte and neutrophils) by heterotypic fusion in the moderate stage (9-16 days) of the disease. Matured syncytia were reported in the late phase of the disease and formed large giant cells of 20 to 100 µm.

SARS-CoV-2 Delta (B.1.617.2) variant replicates and induces syncytia formation in human induced pluripotent stem cell-derived macrophages.

Alveolar macrophages are tissue-resident immune cells that protect epithelial cells in the alveoli from invasion by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the interaction between macrophages and SARS-CoV-2 is inevitable. However, little is known about the role of macrophages in SARS-CoV-2 infection. Here, we generated macrophages from human induced pluripotent stem cells (hiPSCs) to investigate the susceptibility of hiPSC-derived macrophages (iMΦ) to the authentic SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants as well as their gene expression profiles of proinflammatory cytokines during infection. With undetectable angiotensin-converting enzyme 2 (ACE2) mRNA and protein expression, iMΦ were susceptible to productive infection with the Delta variant, whereas infection of iMΦ with the Omicron variant was abortive. Interestingly, Delta induced cell-cell fusion or syncytia formation in iMΦ, which was not observed in Omicron-infected cells. However, iMΦ expressed moderate levels of proinflammatory cytokine genes in response to SARS-CoV-2 infection, in contrast to strong upregulation of these cytokine genes in response to polarization by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overall, our findings indicate that the SARS-CoV-2 Delta variant can replicate and cause syncytia formation in macrophages, suggesting that the Delta variant can enter cells with undetectable ACE2 levels and exhibit greater fusogenicity.

Vitamin D (1α,25(OH)2D3) supplementation minimized multinucleated giant cells formation and inflammatory response during Burkholderia pseudomallei infection in human lung epithelial cells.

Melioidosis is an infectious disease with high mortality rates in human, caused by the bacterium Burkholderia pseudomallei. As an intracellular pathogen, B. pseudomallei can escape from the phagosome and induce multinucleated giant cells (MNGCs) formation resulting in antibiotic resistance and immune evasion. A novel strategy to modulate host response against B. pseudomallei pathogenesis is required. In this study, an active metabolite of vitamin D3 (1α,25-dihydroxyvitamin D3 or 1α,25(OH)2D3) was selected to interrupt pathogenesis of B. pseudomallei in a human lung epithelium cell line, A549. The results demonstrated that pretreatment with 10-6 M 1α,25(OH)2D3 could reduce B. pseudomallei internalization to A549 cells at 4 h post infection (P < 0.05). Interestingly, the presence of 1α,25(OH)2D3 gradually reduced MNGC formation at 8, 10 and 12 h compared to that of the untreated cells (P < 0.05). Furthermore, pretreatment with 10-6 M 1α,25(OH)2D3 considerably increased hCAP-18/LL-37 mRNA expression (P < 0.001). Additionally, pro-inflammatory cytokines, including MIF, PAI-1, IL-18, CXCL1, CXCL12 and IL-8, were statistically decreased (P < 0.05) in 10-6 M 1α,25(OH)2D3-pretreated A549 cells by 12 h post-infection. Taken together, this study indicates that pretreatment with 10-6 M 1α,25(OH)2D3 has the potential to reduce the internalization of B. pseudomallei into host cells, decrease MNGC formation and modulate host response during B. pseudomallei infection by minimizing the excessive inflammatory response. Therefore, 1α,25(OH)2D3 supplement may provide an effective supportive treatment for melioidosis patients to combat B. pseudomallei infection and reduce inflammation in these patients.

Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibits the infection of SARS-CoV-2 variants and syncytium formation.

The continuous emergence of SARS-CoV-2 variants prolongs COVID-19 pandemic. Although SARS-CoV-2 vaccines and therapeutics are currently available, there is still a need for development of safe and effective drugs against SARS-CoV-2 and also for preparedness for the next pandemic. Here, we discover that astersaponin I (AI), a triterpenoid saponin in Aster koraiensis inhibits SARS-CoV-2 entry pathways at the plasma membrane and within the endosomal compartments mainly by increasing cholesterol content in the plasma membrane and interfering with the fusion of SARS-CoV-2 envelope with the host cell membrane. Moreover, we find that this functional property of AI as a fusion blocker enables it to inhibit the infection with SARS-CoV-2 variants including the Alpha, Beta, Delta, and Omicron with a similar efficacy, and the formation of syncytium, a multinucleated cells driven by SARS-CoV-2 spike protein-mediated cell-to-cell fusion. Finally, we claim that the triterpene backbone as well as the attached hydrophilic sugar moieties of AI are structurally important for its inhibitory activity against the membrane fusion event. Overall, this study demonstrates that AI is a natural viral fusion inhibitor and proposes that it can be a broad-spectrum antiviral agent against current COVID-19 pandemic and future outbreaks of novel viral pathogens.

The Integral Membrane Protein ZMPSTE24 Protects Cells from SARS-CoV-2 Spike-Mediated Pseudovirus Infection and Syncytia Formation.

COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a devastating impact on global public health, emphasizing the importance of understanding innate immune mechanisms and cellular restriction factors that cells can harness to fight viral infections. The multimembrane-spanning zinc metalloprotease ZMPSTE24 is one such restriction factor. ZMPSTE24 has a well-characterized proteolytic role in the maturation of prelamin A, precursor of the nuclear scaffold protein lamin A. An apparently unrelated role for ZMPSTE24 in viral defense involves its interaction with the interferon-inducible membrane proteins (IFITMs), which block virus-host cell fusion by rigidifying cellular membranes and thereby prevent viral infection. ZMPSTE24, like the IFITMs, defends cells against a broad spectrum of enveloped viruses. However, its ability to protect against coronaviruses has never been examined. Here, we show that overexpression of ZMPSTE24 reduces the efficiency of cellular infection by SARS-CoV-2 Spike-pseudotyped lentivirus and that genetic knockout or small interfering RNA-mediated knockdown of endogenous ZMPSTE24 enhances infectivity. We further demonstrate a protective role for ZMPSTE24 in a Spike-ACE2-dependent cell-cell fusion assay. In both assays, a catalytic dead version of ZMPSTE24 is equally as protective as the wild-type protein, indicating that ZMPSTE24's proteolytic activity is not required for defense against SARS-CoV-2. Finally, we demonstrate by plaque assays that Zmpste24-/- mouse cells show enhanced infection by a genuine coronavirus, mouse hepatitis virus (MHV). This study extends the range of viral protection afforded by ZMPSTE24 to include coronaviruses and suggests that targeting ZMPSTE24's mechanism of viral defense could have therapeutic benefit. IMPORTANCE The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has underscored the importance of understanding intrinsic cellular components that can be harnessed as the cell's first line of defense to fight against viral infection. Our paper focuses on one such protein, the integral membrane protease ZMPSTE24, which interacts with interferon-inducible transmembrane proteins (IFITMs). IFITMs interfere with virus entry by inhibiting fusion between viral and host cell membranes, and ZMPSTE24 appears to contribute to this inhibitory activity. ZMPSTE24 has been shown to defend cells against several, but not all, enveloped viruses. In this study, we extend ZMPSTE24's reach to include coronaviruses, by showing that ZMPSTE24 protects cells from SARS-CoV-2 pseudovirus infection, Spike protein-mediated cell-cell fusion, and infection by the mouse coronavirus MHV. This work lays the groundwork for further studies to decipher the mechanistic role of ZMPSTE24 in blocking the entry of SARS-CoV-2 and other viruses into cells.

An intra-cytoplasmic route for SARS-CoV-2 transmission unveiled by Helium-ion microscopy.

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.

Bronchial epithelia from adults and children: SARS-CoV-2 spread via syncytia formation and type III interferon infectivity restriction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.

Giant cell myocarditis after first dose of BNT162b2 - a case report.

Herein we report the case of a young man, admitted to the Department of Cardiology and Angiology at Hannover Medical School with shortness of breath and elevated troponin. Few weeks earlier the patient received the first dose of BioNTech's mRNA vaccine (Comirnaty, BNT162b2). After diagnostic work-up revealed giant cell myocarditis, the patient received immunosuppressive therapy. In the present context of myocarditis after mRNA vaccination we discuss this rare aetiology and the patient's treatment strategy in the light of current recommendations.

Supporting Cells of the Human Olfactory Epithelium Co-Express the Lipid Scramblase TMEM16F and ACE2 and May Cause Smell Loss by SARS-CoV-2 Spike-Induced Syncytia.

BACKGROUND/AIMS: Quantitative and qualitative alterations in the sense of smell are well established symptoms of COVID-19. Some reports have shown that non-neuronal supporting (also named sustentacular) cells of the human olfactory epithelium co-express ACE2 and TMPRSS2 necessary for SARS-CoV-2 infection. In COVID-19, syncytia were found in many tissues but were not investigated in the olfactory epithelium. Some studies have shown that syncytia in some tissues are formed when SARS-CoV-2 Spike expressed at the surface of an infected cell binds to ACE2 on another cell, followed by activation of the scramblase TMEM16F (also named ANO6) which exposes phosphatidylserine to the external side of the membrane. Furthermore, niclosamide, an approved antihelminthic drug, inhibits Spike-induced syncytia by blocking TMEM16F activity. The aim of this study was to investigate if proteins involved in Spike-induced syncytia formation, i.e., ACE2 and TMEM16F, are expressed in the human olfactory epithelium. METHODS: We analysed a publicly available single-cell RNA-seq dataset from human nasal epithelium and performed immunohistochemistry in human nasal tissues from biopsies. RESULTS: We found that ACE2 and TMEM16F are co-expressed both at RNA and protein levels in non-neuronal supporting cells of the human olfactory epithelium. CONCLUSION: Our results provide the first evidence that TMEM16F is expressed in human olfactory supporting cells and indicate that syncytia formation, that could be blocked by niclosamide, is one of the pathogenic mechanisms worth investigating in COVID-19 smell loss.

Fulminant Giant Cell Myocarditis following Heterologous Vaccination of ChAdOx1 nCoV-19 and Pfizer-BioNTech COVID-19.

A 48-year-old female patient underwent a heart transplantation for acute fulminant myocarditis, following heterologous vaccination with the ChAdOx1 nCoV-19 and Pfizer-BioNTech COVID-19. She had no history of severe acute respiratory syndrome coronavirus-2 infection. She did not exhibit clinical signs or have laboratory findings of concomitant infection before or after vaccination. Heart transplantation was performed because her heart failed to recover with venoarterial extracorporeal oxygenation support. Organ autopsy revealed giant cell myocarditis, possibly related to the vaccines. Clinicians may have to consider the possibility of the development of giant cell myocarditis, especially in patients with rapidly deteriorating cardiac function and myocarditis symptoms after COVID-19 vaccination.

Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is handled in biosafety level 3 (BSL-3) facilities, whereas the antiviral screening of pseudotype virus is conducted in BSL-2 facilities. In this study, we developed a SARS-CoV-2 spike-pseudotyped virus based on a semi-replication-competent retroviral (s-RCR) vector system. The s-RCR vector system was divided into two packageable vectors, each with gag-pol and env genes. For env vector construction, SARS-CoV-2 SΔ19 env was inserted into the pCLXSN-IRES-EGFP retroviral vector to generate pCLXSN-SΔ19 env-EGFP. When pCLXSN-gag-pol and pCLXSN-SΔ19env-EGFP were co-transfected into HEK293 T cells to generate an s-RCR virus, titers of the s-RCR virus were consistently low in this transient transfection system (1 × 104 TU/mL). However, a three-fold higher amounts of MLV-based SARS-CoV-2 pseudotyped viruses (3 × 104 TU/mL) were released from stable producer cells, and the spike proteins induced syncytia formation in HEK293-hACE2 cells. Furthermore, s-RCR stocks collected from stable producer cells induced more substantial syncytia formation in the Vero E6-TMPRSS2 cell line than in the Vero E6 cell line. Therefore, a combination of the s-RCR vector and the two cell lines (HEK293-hACE2 or Vero E6-TMPRSS2) that induce syncytia formation can be useful for the rapid screening of novel fusion inhibitor drugs.

Loss of Detection of sgN Precedes Viral Abridged Replication in COVID-19-Affected Patients-A Target for SARS-CoV-2 Propagation.

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.

A new screening system for entry inhibitors based on cell-to-cell transmitted syncytia formation mediated by self-propagating hybrid VEEV-SARS-CoV-2 replicon.

The extremely high transmission rate of SARS-CoV-2 and severe cases of COVID-19 pose the two critical challenges in the battle against COVID-19. Increasing evidence has shown that the viral spike (S) protein-driven syncytia may be responsible for these two events. Intensive attention has thus been devoted to seeking S-guided syncytium inhibitors. However, the current screening campaigns mainly rely on either live virus-based or plasmid-based method, which are always greatly limited by the shortage of high-level biosafety BSL-3 facilities or too much labour-intensive work. Here, we constructed a new hybrid VEEV-SARS-CoV-2-S-eGFP reporter vector through replacement of the structural genes of Venezuelan equine encephalitis virus (VEEV) with the S protein of SARS-CoV-2 as the single structural protein. VEEV-SARS-CoV-2-S-eGFP can propagate steadily through cell-to-cell transmission pathway in S- and ACE2-dependent manner, forming GFP positive syncytia. In addition, a significant dose-dependent decay in GFP signals was observed in VEEV-SARS-CoV-2-S-eGFP replicating cells upon treatment with SARS-CoV-2 antiserum or entry inhibitors, providing further evidence that VEEV-SARS-CoV-2-S-eGFP system is highly sensitive to characterize the anti-syncytium-formation activity of antiviral agents. More importantly, the assay is able to be performed in a BSL-2 laboratory without manipulation of live SARS-CoV-2. Taken together, our work establishes a more convenient and efficient VEEV-SARS-CoV-2-S-eGFP replicating cells-based method for rapid screening of inhibitors blocking syncytium formation.

Computationally prioritized drugs inhibit SARS-CoV-2 infection and syncytia formation.

The pharmacological arsenal against the COVID-19 pandemic is largely based on generic anti-inflammatory strategies or poorly scalable solutions. Moreover, as the ongoing vaccination campaign is rolling slower than wished, affordable and effective therapeutics are needed. To this end, there is increasing attention toward computational methods for drug repositioning and de novo drug design. Here, multiple data-driven computational approaches are systematically integrated to perform a virtual screening and prioritize candidate drugs for the treatment of COVID-19. From the list of prioritized drugs, a subset of representative candidates to test in human cells is selected. Two compounds, 7-hydroxystaurosporine and bafetinib, show synergistic antiviral effects in vitro and strongly inhibit viral-induced syncytia formation. Moreover, since existing drug repositioning methods provide limited usable information for de novo drug design, the relevant chemical substructures of the identified drugs are extracted to provide a chemical vocabulary that may help to design new effective drugs.

YAP1 nuclear efflux and transcriptional reprograming follow membrane diminution upon VSV-G-induced cell fusion.

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.

Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation.

During the current coronavirus disease 2019 (COVID-19) pandemic, a variety of mutations have accumulated in the viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and, at the time of writing, four variants of concern are considered to be potentially hazardous to human society1. The recently emerged B.1.617.2/Delta variant of concern is closely associated with the COVID-19 surge that occurred in India in the spring of 2021 (ref. 2). However, the virological properties of B.1.617.2/Delta remain unclear. Here we show that the B.1.617.2/Delta variant is highly fusogenic and notably more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates cleavage of the spike protein and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity compared with its parental virus. Our data suggest that the P681R mutation is a hallmark of the virological phenotype of the B.1.617.2/Delta variant and is associated with enhanced pathogenicity.