Pan-microscopic examination of monkeypox virus in trophoblasts cells reveals new insights into virions release through filopodia-like projections.

Vertical transmission has been described following monkeypox virus (MPXV) infection in pregnant women. The presence of MPXV has been reported in the placenta from infected women, but whether pathogens colonize placenta remains unexplored. We identify trophoblasts as a target cell for MPXV replication. In a pan-microscopy approach, we decipher the specific infectious cycle of MPXV and inner cellular structures in trophoblasts. We identified the formation of a specialized region for viral morphogenesis and replication in placental cells. We also reported infection-induced cellular remodeling. We found that MPXV stimulates cytoskeleton reorganization with intercellular extensions for MPXV cell spreading specifically to trophoblastic cells. Altogether, the specific infectious cycle of MPXV in trophoblast cells and these protrusions that were structurally and morphologically similar to filopodia reveal new insights into the infection of MPXV.

Preliminary landscape of Candidatus Saccharibacteria in the human microbiome.

Introduction: Candidate Phyla Radiation (CPR) and more specifically Candidatus Saccharibacteria (TM7) have now been established as ubiquitous members of the human oral microbiota. Additionally, CPR have been reported in the gastrointestinal and urogenital tracts. However, the exploration of new human niches has been limited to date. Methods: In this study, we performed a prospective and retrospective screening of TM7 in human samples using standard PCR, real-time PCR, scanning electron microscopy (SEM) and shotgun metagenomics. Results: Using Real-time PCR and standard PCR, oral samples presented the highest TM7 prevalence followed by fecal samples, breast milk samples, vaginal samples and urine samples. Surprisingly, TM7 were also detected in infectious samples, namely cardiac valves and blood cultures at a low prevalence (under 3%). Moreover, we observed CPR-like structures using SEM in all sample types except cardiac valves. The reconstruction of TM7 genomes in oral and fecal samples from shotgun metagenomics reads further confirmed their high prevalence in some samples. Conclusion: This study confirmed, through their detection in multiple human samples, that TM7 are human commensals that can also be found in clinical settings. Their detection in clinical samples warrants further studies to explore their role in a pathological setting.

The Role of Platelets in Infective Endocarditis.

Over the last decade, the incidence of infective endocarditis (IE) has increased, with a change in the frequency of causative bacteria. Early evidence has substantially demonstrated the crucial role of bacterial interaction with human platelets, with no clear mechanistic characterization in the pathogenesis of IE. The pathogenesis of endocarditis is so complex and atypical that it is still unclear how and why certain bacterial species will induce the formation of vegetation. In this review, we will analyze the key role of platelets in the physiopathology of endocarditis and in the formation of vegetation, depending on the bacterial species. We provide a comprehensive outline of the involvement of platelets in the host immune response, investigate the latest developments in platelet therapy, and discuss prospective research avenues for solving the mechanistic enigma of bacteria-platelet interaction for preventive and curative medicine.

Microscopic observations of SARS-CoV-2 like particles in different oral samples.

The emerging coronavirus pneumonia epidemic caused by the SARS-CoV-2 infection has spread rapidly around the world. The main routes of transmission of SARS-CoV-2 are currently recognised as aerosol/droplet inhalation. However, the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly known. The current data indicates the presence of viral RNA in oral samples, suggesting the implication of saliva in SARS-CoV-2 transmission, however, no direct observation of SARS-CoV-2 particles in different oral samples has been reported. In this study, we investigated whether particles of SARS-CoV-2 were present in oral samples collected from three symptomatic COVID-19 patients. Using scanning electron microscopy (SEM), the correlative strategy of light microscopy and electron microscopy and immunofluorescence staining, we showed the presence of SARS-like particles in RT-qPCR SARS-CoV-2-positive saliva, dental plaque and gingival crevicular fluid (GCF) samples. In the saliva samples, we demonstrated the presence of epithelial oral cells with morphogenetic features of SARS-CoV-2 infected cells. Inside those cells, vacuoles filled with nascent particles were observed, suggesting the potential infection and replication of SARS-CoV-2 in oral tissues. Our results corroborate previous studies and confirm that the oral cavity may be a potential niche for SARS-CoV-2 infection and a potential source of transmission.

Concentration of SARS-CoV-2-Infected Cell Culture Supernatants for Detection of Virus-like Particles by Scanning Electron Microscopy.

There is currently a need for new rapid viral diagnostic electron microscopy methods. Although the gold standard remains the transmission electron microscopy (TEM) negative staining method for electron microscopic examination of samples containing a virus, difficulties can arise when the virus particle content of the sample that has to be examined is poor. Such samples include supernatants of virus-infected cells that can be difficult to examine, as sometimes only a few virus particles are released in the culture medium upon infection. In addition to TEM, scanning electron microscopy (SEM) can also be used for visualizing virus particles. One advantage of SEM over TEM is its ability to rapidly screen several large specimens, such as microscopy slides. In this study, we investigated this possibility and tested different coating molecules as well as the effect of centrifugation for analyzing SARS-CoV-2-virus-infected cell culture supernatants deposited on microscopy glass slides by SEM. We found that centrifugation of 25XConcanavalinA-coated microscopy glass slides in shell vials provided an improved method for concentrating SARS-CoV-2-virus-infected cell supernatants for virus-like particle detection by SEM.

Microscopic Description of Platelet Aggregates Induced by Escherichia coli Strains.

In addition to their role in haemostasis, platelets are also involved in the inflammatory and antimicrobial process. Interactions between pathogens and platelets, mediated by receptors can lead to platelet activation, which may be responsible for a granular secretion process or even aggregation, depending on the bacterial species. Granular secretion releases peptides with bactericidal activity as well as aggregating factors. To our knowledge, these interactions have been poorly studied for Escherichia coli (E. coli). Few studies have characterised the cellular organization of platelet-E. coli aggregates. The objective of our study was to investigate the structure of platelet aggregates induced by different E. coli strains as well as the ultrastructure of platelet-E. coli mixtures using a scanning and transmission electron microscopy (SEM and TEM) approach. Our results show that the appearance of platelet aggregates is mainly dependent on the strain used. SEM images illustrate the platelet activation and aggregation and their colocalisation with bacteria. Some E. coli strains induce platelet activation and aggregation, and the bacteria are trapped in the platelet magma. However, some strains do not induce significant platelet activation and are found in close proximity to the platelets. The structure of the E. coli strains might explain the results obtained.

Limited permissibility of ENL-R and Mv-1-Lu mink cell lines to SARS-CoV-2.

The SARS-CoV-2 pandemic started in the end of 2019 in Wuhan, China, which highlighted the scenario of frequent cross-species transmission events. From the outbreak possibly initiated by viral spill-over into humans from an animal reservoir, now we face the human host moving globally while interacting with domesticated and peridomestic animals. The emergence of a new virus into the ecosystem leads to selecting forces and species-specific adaptations. The adaptation of SARS-CoV-2 to other animals represents a risk to controlling the dissemination of this coronavirus and the emergence of new variants. Since 2020, several mink farms in Europe and the United States have had SARS-CoV-2 outbreaks with human-mink and mink-human transmission, where the mink-selected variants possibly hold evolutionary concerning advantages. Here we investigated the permissibility of mink lung-derived cells using two cell lines, Mv-1-Lu and ENL-R, against several lineages of SARS-CoV-2, including some classified as variants of concern. The viral release rate and the infectious titers indicate that these cells support infections by different SARS-CoV-2 lineages. The viral production occurs in the first few days after infection with the low viral release by these mink cells, which is often absent for the omicron variant for lung cells. The electron microscopy reveals that during the viral replication cycle, the endomembrane system of the mink-host cell undergoes typical changes while the viral particles are produced, especially in the first days of infection. Therefore, even if limited, mink lung cells may represent a selecting source for SARS-CoV-2 variants, impacting their transmissibility and pathogenicity and making it difficult to control this new coronavirus.

A Massive Number of Extracellular Tropheryma whipplei in Infective Endocarditis: A Case Report and Literature Review.

Whipple's disease (WD) is a chronic multisystemic infection caused by Tropheryma whipplei. If this bacterium presents an intracellular localization, associated with rare diseases and without pathognomonic signs, it is often subject to a misunderstanding of its physiopathology, often a misdiagnosis or simply an oversight. Here, we report the case of a patient treated for presumed rheumatoid arthritis. Recently, this patient presented to the hospital with infectious endocarditis. After surgery and histological analysis, we discovered the presence of T. whipplei. Electron microscopy allowed us to discover an atypical bacterial organization with a very large number of bacteria present in the extracellular medium in vegetation and valvular tissue. This atypical presentation we report here might be explained by the anti-inflammatory treatment administrated for our patient's initial diagnosis of rheumatoid arthritis.

Control of CDH1/E-Cadherin Gene Expression and Release of a Soluble Form of E-Cadherin in SARS-CoV-2 Infected Caco-2 Intestinal Cells: Physiopathological Consequences for the Intestinal Forms of COVID-19.

COVID-19 is the biggest pandemic the world has seen this century. Alongside the respiratory damage observed in patients with severe forms of the disease, gastrointestinal symptoms have been frequently reported. These symptoms (e.g., diarrhoea), sometimes precede the development of respiratory tract illnesses, as if the digestive tract was a major target during early SARS-CoV-2 dissemination. We hypothesize that in patients carrying intestinal SARS-CoV-2, the virus may trigger epithelial barrier damage through the disruption of E-cadherin (E-cad) adherens junctions, thereby contributing to the overall gastrointestinal symptoms of COVID-19. Here, we use an intestinal Caco-2 cell line of human origin which expresses the viral receptor/co-receptor as well as the membrane anchored cell surface adhesion protein E-cad to investigate the expression of E-cad after exposure to SARS-CoV-2. We found that the expression of CDH1/E-cad mRNA was significantly lower in cells infected with SARS-CoV-2 at 24 hours post-infection, compared to virus-free Caco-2 cells. The viral receptor ACE2 mRNA expression was specifically down-regulated in SARS-CoV-2-infected Caco-2 cells, while it remained stable in HCoV-OC43-infected Caco-2 cells, a virus which uses HLA class I instead of ACE2 to enter cells. It is worth noting that SARS-CoV-2 induces lower transcription of TMPRSS2 (involved in viral entry) and higher expression of B0AT1 mRNA (that encodes a protein known to co-express with ACE2 on intestinal cells). At 48 hours post-exposure to the virus, we also detected a small but significant increase of soluble E-cad protein (sE-cad) in the culture supernatant of SARS-CoV-2-infected Caco-2 cells. The increase of sE-cad release was also found in the intestinal HT29 cell line when infected by SARS-CoV-2. Beside the dysregulation of E-cad, SARS-CoV-2 infection of Caco-2 cells also leads to the dysregulation of other cell adhesion proteins (occludin, JAMA-A, zonulin, connexin-43 and PECAM-1). Taken together, these results shed light on the fact that infection of Caco-2 cells with SARS-CoV-2 affects tight-, adherens-, and gap-junctions. Moreover, intestinal tissues damage was associated to the intranasal SARS-CoV-2 infection in human ACE2 transgenic mice.

GNS561 Exhibits Potent Antiviral Activity against SARS-CoV-2 through Autophagy Inhibition.

Since December 2019, SARS-CoV-2 has spread quickly worldwide, leading to more than 280 million confirmed cases, including over 5,000,000 deaths. Interestingly, coronaviruses were found to subvert and hijack autophagic process to allow their viral replication. Autophagy-modulating compounds thus rapidly emerged as an attractive strategy to fight SARS-CoV-2 infection, including the well-known chloroquine (CQ). Here, we investigated the antiviral activity and associated mechanism of GNS561/Ezurpimtrostat, a small lysosomotropic molecule inhibitor of late-stage autophagy. Interestingly, GNS561 exhibited antiviral activity of 6-40 nM depending on the viral strain considered, currently positioning it as the most powerful molecule investigated in SARS-CoV-2 infection. We then showed that GNS561 was located in lysosome-associated-membrane-protein-2-positive (LAMP2-positive) lysosomes, together with SARS-CoV-2. Moreover, GNS561 increased LC3-II spot size and caused the accumulation of autophagic vacuoles and the presence of multilamellar bodies, suggesting that GNS561 disrupted the autophagy mechanism. To confirm our findings, we used the K18-hACE2 mouse model and highlighted that GNS561 treatment led to a decline in SARS-CoV-2 virions in the lungs associated with a disruption of the autophagy pathway. Overall, our study highlights GNS561 as a powerful drug in the treatment of SARS-CoV-2 infection and supports the hypothesis that autophagy blockers could be an alternative strategy for COVID-19.

Incomplete tricarboxylic acid cycle and proton gradient in Pandoravirus massiliensis: is it still a virus?

The discovery of Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3 µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5 Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that eight putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all eight viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question "what is a virus?".

GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions.

Hepatocellular carcinoma is the most frequent primary liver cancer. Macroautophagy/autophagy inhibitors have been extensively studied in cancer but, to date, none has reached efficacy in clinical trials. In this study, we demonstrated that GNS561, a new autophagy inhibitor, whose anticancer activity was previously linked to lysosomal cell death, displayed high liver tropism and potent antitumor activity against a panel of human cancer cell lines and in two hepatocellular carcinoma in vivo models. We showed that due to its lysosomotropic properties, GNS561 could reach and specifically inhibited its enzyme target, PPT1 (palmitoyl-protein thioesterase 1), resulting in lysosomal unbound Zn2+ accumulation, impairment of cathepsin activity, blockage of autophagic flux, altered location of MTOR (mechanistic target of rapamycin kinase), lysosomal membrane permeabilization, caspase activation and cell death. Accordingly, GNS561, for which a global phase 1b clinical trial in liver cancers was just successfully achieved, represents a promising new drug candidate and a hopeful therapeutic strategy in cancer treatment.Abbreviations: ANXA5:annexin A5; ATCC: American type culture collection; BafA1: bafilomycin A1; BSA: bovine serum albumin; CASP3: caspase 3; CASP7: caspase 7; CASP8: caspase 8; CCND1: cyclin D1; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; CQ: chloroquine; iCCA: intrahepatic cholangiocarcinoma; DEN: diethylnitrosamine; DMEM: Dulbelcco's modified Eagle medium; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC: hepatocellular carcinoma; HCQ: hydroxychloroquine; HDSF: hexadecylsulfonylfluoride; IC50: mean half-maximal inhibitory concentration; LAMP: lysosomal associated membrane protein; LC3-II: phosphatidylethanolamine-conjugated form of MAP1LC3; LMP: lysosomal membrane permeabilization; MALDI: matrix assisted laser desorption ionization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKI67: marker of proliferation Ki-67; MTOR: mechanistic target of rapamycin kinase; MRI: magnetic resonance imaging; NH4Cl: ammonium chloride; NtBuHA: N-tert-butylhydroxylamine; PARP: poly(ADP-ribose) polymerase; PBS: phosphate-buffered saline; PPT1: palmitoyl-protein thioesterase 1; SD: standard deviation; SEM: standard error mean; vs, versus; Zn2+: zinc ion; Z-Phe: Z-Phe-Tyt(tBu)-diazomethylketone; Z-VAD-FMK: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone.

Presence of SARS-CoV-2 in a Cornea Transplant.

BACKGROUND: The SARS-CoV-2 pandemic has impacted tissue transplantation procedures since conjunctivas were found to be associated with coronavirus infection. Here, we investigated infection of a cornea graft from a COVID-19-positive donor. METHODS: In order to evaluate the presence of SARS-CoV-2 in the cornea graft we first carried out a qRT-PCR and then we investigated the presence of SARS-CoV-2 by fluorescence and electron microscopy. CONCLUSIONS: Although the cornea graft was found to be negative by qRT-PCR, we were able to show the presence of SARS-CoV-2 in corneal cells expressing the SARS-CoV-2 receptor, ACE2. Taken together, our findings may have important implications for the use of corneal tissue in graft indications and open the debate on SARS-CoV-2 transmissibility.

Generation of Infectious Mimivirus Virions Through Inoculation of Viral DNA Within Acanthamoeba castellanii Shows Involvement of Five Proteins, Essentially Uncharacterized.

One of the most curious findings associated with the discovery of Acanthamoeba polyphaga mimivirus (APMV) was the presence of many proteins and RNAs within the virion. Although some hypotheses on their role in Acanthamoeba infection have been put forward, none have been validated. In this study, we directly transfected mimivirus DNA with or without additional proteinase K treatment to extracted DNA into Acanthamoeba castellanii. In this way, it was possible to generate infectious APMV virions, but only without extra proteinase K treatment of extracted DNA. The virus genomes before and after transfection were identical. We searched for the remaining DNA-associated proteins that were digested by proteinase K and could visualize at least five putative proteins. Matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography-mass spectrometry comparison with protein databases allowed the identification of four hypothetical proteins-L442, L724, L829, and R387-and putative GMC-type oxidoreductase R135. We believe that L442 plays a major role in this protein-DNA interaction. In the future, expression in vectors and then diffraction of X-rays by protein crystals could help reveal the exact structure of this protein and its precise role.

Innovative Approach to Fast Electron Microscopy Using the Example of a Culture of Virus-Infected Cells: An Application to SARS-CoV-2.

Despite the development of new diagnostic methods, co-culture, based on sample inoculation of cell monolayers coupled with electron microscopy (EM) observation, remains the gold standard in virology. Indeed, co-culture allows for the study of cell morphology (infected and not infected), the ultrastructure of the inoculated virus, and the different steps of the virus infectious cycle. Most EM methods for studying virus cycles are applied after infected cells are produced in large quantities and detached to obtain a pellet. Here, cell culture was performed in sterilized, collagen-coated single-break strip wells. After one day in culture, cells were infected with SARS-CoV-2. Wells of interest were fixed at different time points, from 2 to 36 h post-infection. Microwave-assisted resin embedding was accomplished directly in the wells in 4 h. Finally, ultra-thin sections were cut directly through the infected-cell monolayers. Our methodology requires, in total, less than four days for preparing and observing cells. Furthermore, by observing undetached infected cell monolayers, we were able to observe new ultrastructural findings, such as cell-cell interactions and baso-apical cellular organization related to the virus infectious cycle. Our innovative methodology thus not only saves time for preparation but also adds precision and new knowledge about viral infection, as shown here for SARS-CoV-2.

Angiotensin II Receptor Blockers (ARBs Antihypertensive Agents) Increase Replication of SARS-CoV-2 in Vero E6 Cells.

Several comorbidities, including hypertension, have been associated with an increased risk of developing severe disease during SARS-CoV-2 infection. Angiotensin II receptor blockers (ARBs) are currently some of the most widely-used drugs to control blood pressure by acting on the angiotensin II type 1 receptor (AT1R). ARBs have been reported to trigger the modulation of the angiotensin I converting enzyme 2 (ACE2), the receptor used by the virus to penetrate susceptible cells, raising concern that such treatments may promote virus capture and increase their viral load in patients receiving ARBs therapy. In this in vitro study, we reviewed the effect of ARBs on ACE2 and AT1R expression and investigated whether treatment of permissive ACE2+/AT1R+ Vero E6 cells with ARBs alters SARS-CoV-2 replication in vitro in an angiotensin II-free system. After treating the cells with the ARBs, we observed an approximate 50% relative increase in SARS-CoV-2 production in infected Vero E6 cells that correlates with the ARBs-induced up-regulation of ACE2 expression. From this data, we believe that the use of ARBs in hypertensive patients infected by SARS-CoV-2 should be carefully evaluated.

Microscopic Observation of SARS-Like Particles in RT-qPCR SARS-CoV-2 Positive Sewage Samples.

The ongoing outbreak of novel coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 infection has spread rapidly worldwide. The major transmission routes of SARS-CoV-2 are recognised as inhalation of aerosol/droplets and person-to-person contact. However, some studies have demonstrated that live SARS-CoV-2 can be isolated from the faeces and urine of infected patients, which can then enter the wastewater system. The currently available evidence indicates that the viral RNA present in wastewater may become a potential source of epidemiological data. However, to investigate whether wastewater may present a risk to humans such as sewage workers, we investigated whether intact particles of SARS-CoV-2 were observable and whether it was possible to isolate the virus in wastewater. Using a correlative strategy of light microscopy and electron microscopy (CLEM), we demonstrated the presence of intact and degraded SARS-like particles in RT-qPCR SARS-CoV-2-positive sewage sample collected in the city of Marseille. However, the viral infectivity assessment of SARS-CoV-2 in the wastewater was inconclusive, due to the presence of other viruses known to be highly resistant in the environment such as enteroviruses, rhinoviruses, and adenoviruses. Although the survival and the infectious risk of SARS-CoV-2 in wastewater cannot be excluded from our study, additional work may be required to investigate the stability, viability, fate, and decay mechanisms of SARS-CoV-2 thoroughly in wastewater.

A neuronal blueprint for directional mechanosensation in larval zebrafish.

Animals have a remarkable ability to use local cues to orient in space in the absence of a panoramic fixed reference frame. Here we use the mechanosensory lateral line in larval zebrafish to understand rheotaxis, an innate oriented swimming evoked by water currents. We generated a comprehensive light-microscopy cell-resolution projectome of lateralis afferent neurons (LANs) and used clustering techniques for morphological classification. We find surprising structural constancy among LANs. Laser-mediated microlesions indicate that precise topographic mapping of lateral-line receptors is not essential for rheotaxis. Recording neuronal-activity during controlled mechanical stimulation of neuromasts reveals unequal representation of water-flow direction in the hindbrain. We explored potential circuit architectures constrained by anatomical and functional data to suggest a parsimonious model under which the integration of lateralized signals transmitted by direction-selective LANs underlies the encoding of water-flow direction in the brain. These data provide a new framework to understand how animals use local mechanical cues to orient in space.

Ultrastructure of a late-stage bacterial endocarditis valve vegetation.

Infective endocarditis (IE) remains a severe illness with high mortality rate, despite advances in antibiotic therapy and cardiac surgery. If infectious bacteria and platelets are two key players of human IE vegetation developmental process, their interactions and respective roles in fully developed late-stage IE vegetations remain obscure. The objective of this study was to better understand the organization of the different components of the IE vegetation and to provide a detailed description of this vegetation ultrastructure. A late stage Staphylococcal endocarditic vegetation was provided from a 13 years teenager patient. After reception of the surgical piece, we carried out a histological study using routine methods, notably the hematoxylin-eosin-saffron staining. Labeling with the anti-CD 61 antibody was also carried out. In a second step, we used transmission electron microscopy to describe the different regions making up the vegetation. Our ultrastructural study revealed vegetation was clearly composed by three different regions and identified the specific location of the bacteria and platelets in the vegetation tissues. Histological analysis showed that platelets and Staphylococcus aureus were not co-localized. Electron microscopy study confirmed that S. aureus were found at distance from platelets, as well from immune cells, embedded in a biofilm and/or a necrotic area. These results reveal a development of a deep bacteria-only niche in vegetation, raising questions about medication access to these microorganisms. Vegetation composed of three regions: a region rich in bacteria incorporated into the necrotic tissue, the second region composed of fibrin filaments and the third region rich in platelets and free of bacteria.

GNS561 exhibits potent in vitro antiviral activity against SARS-CoV-2 through autophagy inhibition.

Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-nCoV) has spread quickly worldwide, with more than 29 million cases and 920,000 deaths. Interestingly, coronaviruses were found to subvert and hijack the autophagic process to allow their viral replication. One of the spotlights had been focused on the autophagy inhibitors as a target mechanism effective in the inhibition of SARS-CoV-2 infection. Consequently, chloroquine (CQ) and hydroxychloroquine (HCQ), a derivative of CQ, was suggested as the first potentially be therapeutic strategies as they are known to be autophagy inhibitors. Then, they were used as therapeutics in SARS-CoV-2 infection along with remdesivir, for which the FDA approved emergency use authorization. Here, we investigated the antiviral activity and associated mechanism of GNS561, a small basic lipophilic molecule inhibitor of late-stage autophagy, against SARS-CoV-2. Our data indicated that GNS561 showed the highest antiviral effect for two SARS-CoV-2 strains compared to CQ and remdesivir. Focusing on the autophagy mechanism, we showed that GNS561, located in LAMP2-positive lysosomes, together with SARS-CoV-2, blocked autophagy by increasing the size of LC3-II spots and the accumulation of autophagic vacuoles in the cytoplasm with the presence of multilamellar bodies characteristic of a complexed autophagy. Finally, our study revealed that the combination of GNS561 and remdesivir was associated with a strong synergistic antiviral effect against SARS-CoV-2. Overall, our study highlights GNS561 as a powerful drug in SARS-CoV-2 infection and supports that the hypothesis that autophagy inhibitors could be an alternative strategy for SARS-CoV-2 infection.