WO2022023380A2 - Assay - Google Patents

Abstract

The present disclosure relates in one aspect to a methods for detecting an agent that modulates coronavirus-mediated cell fusion or syncytia formation, or for detecting an immune response against a coronavirus in a subject, comprising contacting cultured cells expressing a coronavirus Spike protein with the agent a sample from the subject; and detecting cell fusion or syncytia formation by automated microscopy. In further aspects, the disclosure relates to methods and pharmaceutical compositions for use in preventing or treating a coronavirus infection or coronavirus-associated disease, comprising an anoctamin (ANO)/transmembrane protein 16 (TMEM16) family member inhibitor; a cardiac glycoside; or an agent identified by the methods described above, preferably clofazimine or salinomycin.

Description

ASSAY

FIELD OF THE INVENTION

The present disclosure relates to the field of methods for detecting agents that modulate effects of coronaviruses on infected cells. The disclosure further relates to compositions and methods for treating and/or preventing coronavirus infection or coronavirus-mediated disease. The disclosure also relates to compositions and methods for treating and/or preventing cell fusion or syncytia formation associated with a coronavirus infection or coronavirus-mediated disease. The present invention is particularly concerned with assays for detecting the effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and agents to prevent these effects, including neutralizing antibodies and drugs that are useful for treating Coronavirus 19 disease (COVID-19).

BACKGROUND OF THE INVENTION

Coronavirus 19 disease (COVID-19) is now a major threat to public health throughout the world. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2), a new coronavirus first detected in China in November 2019. SARS-CoV-2 has spread to countries throughout Asia, Europe, North America, South America, Africa, and Oceania, leading to the World Health Organization declaration of a global pandemic. Human to human transmission of SARS-CoV-2 is likely to be caused by generation of respiratory droplets from an infected person, or indirect contact, such as touching a contaminated surface.

SARS-CoV-2 is related to severe acute respiratory syndrome coronavirus (SARS-CoV), the causative agent of the SARS outbreak in 2002-2003. SARS-CoV-2 belongs to the Coronaviridae family, genus Betacoronavirus (betaCoV) and may have emerged from a natural reservoir in bats. The virus has a ssRNA genome, 29,903 bp (Wuhan-Hu-1: Genbank Reference sequence: NC_045512.2) encoding for 25 non-structural protein and 4 structural proteins: Spike (S), envelope (E), membrane (M), nucleocapside (N). The virus has a variable size of between 60 to 140 nm in diameter. SARS-CoV-2 is enveloped and sensitive to UV, heat, and lipid solvents. SARS-CoV-2 has 89% nucleotide identity with bat SARS-like- CoVZXC21 and 81% nucleotide identity with human SARS-CoV.

Both SARS-CoV and SARS-CoV-2 infect cells by binding of the coronavirus Spike (S) protein to angiotensin-converting enzyme 2 (ACE2) on human cells, which involves processing of the S proteins by the cellular serine protease TMPRSS2 (see e.g. Li et ah, Nature (2003, 426: 450- 454); Hoffmann et al. Cell (2020) 181: 1-10; Glowacka et al., J. Virol. (2011) 85: 4122-4134; Matsuyama et al., J. Virol. (2010) 84: 12658-12664; Shulla et al. J. Virol. (2011, 85: 873-882; Shang et al. (2020), Nature volume 581, pages 221–224). In the case of SARS-CoV-2, however, other cellular proteases are involved in Spike priming at the plasma membrane. In particular, furin can cleave the S protein of these two viruses (Burkard, C, et al. 2014. PLoS Pathog 10, e1004502; Coutard, B, et al.2020. Antiviral Res 176, 104742; Hoffmann, M, et al. 2020. Cell 181, 271) at an amino acid sequence at the S1/S2 interface that is not present in SARS-CoV. As a consequence, cells expressing SARS-CoV-2 Spike can fuse with other cells expressing the ACE2 receptor and form large syncytia in vitro, while this property is markedly less pronounced for SARS-CoV S protein. Spike-mediated cell-cell fusion and formation of syncytia in SARS-CoV-2 -infected lungs and other organs are predicted to be responsible of some of the disease characteristics that are properties of COVID-19. In particular, these include high prevalence of diarrhoea and, most relevant, of lung and systemic thrombosis in the infected individuals (Chen, N, et al. 2020. Lancet 395, 507). Most current vaccine candidates against SARS-CoV-2 use the Spike protein as an immunogen, hence are predicted to block entry of the virus into cells expressing the receptor and proteases and to block cell-cell fusion when Spike is expressed on the plasma membrane of infected cells. Accordingly, there is a need for methods to identify agents, including donor antisera, neutralising antibodies and other types of drugs, that are effective against coronavirus-mediated disease (e.g. COVID-19) by blocking the pathological effects exerted by the Spike protein. At the same time, there is a need to detect or predict immunity against coronavirus infection in subjects, e.g. in order to determine the prevalence of exposure and/or immunity to the disease in a population, to predict susceptibility of individual subjects to the disease and to certify immunity, and to monitor the progression of the disease and/or response to vaccines or other treatments in subjects. SUMMARY OF THE INVENTION In one aspect, the present invention provides a method for detecting an agent that modulates coronavirus-mediated cell fusion or syncytia formation, preferably in an automated manner amenable to high throughput screening, comprising (a) contacting cultured cells expressing a coronavirus Spike protein with the agent; and (b) detecting cell fusion or syncytia formation. The method may be used for identifying an agent, for instance out of several thousands in a systematic manner, that modulates coronavirus-mediated cell fusion or syncytia formation, e.g. for screening multiple test agents for this activity, thereby identifying specific agents that modulate coronavirus-mediated cell fusion or syncytia formation. The method may thus be used to identify agents useful for treating or preventing coronavirus infection and/or coronavirus-mediated disease (e.g. SARS-CoV-2 infection or COVID-19). In another aspect, the present disclosure provides a method for detecting an immune response against a coronavirus in a subject, comprising contacting cultured cells expressing a coronavirus Spike protein with a sample from the subject; and detecting cell fusion or syncytia formation of the cultured cells. In such embodiments, inhibition of coronavirus-mediated cell fusion or syncytia formation in the presence of sample is indicative of an immune response against a coronavirus in the subject. The methods may be performed using microscopy, preferably automated microscopy, e.g. automated high content microscopy and may be coupled with robotic processing of samples for high throughput screening and analysis. In one embodiment, cell fusion or syncytia formation is detected by quantifying cytoplasmic and/or nuclear area of cells. In another embodiment, cell fusion or syncytia formation is detected by quantifying a number of nuclei per cytoplasm. In one embodiment, the method comprises preparing donor cultured cells expressing the coronavirus Spike protein and acceptor cultured cells that are capable of fusing with the donor cultured cells; contacting the donor and acceptor cultured cells with the agent; and detecting fusion of the donor and acceptor cultured cells. In such embodiments, fusion of the donor and acceptor cells may be determined by e.g. detecting cells labelled with markers specific for both the donor and acceptor cells. For instance, the donor cultured cells may be loaded with nanoparticles that emit light at a first wavelength and the acceptor cultured cells loaded with nanoparticles that emit light at a second wavelength. Fusion of the donor and acceptor cultured cells is detected by quantifying cells that emit light at both the first wavelength and second wavelength. In one embodiment, the nanoparticles are quantum dots. In some embodiments, the agent is an antibody or fragment thereof, e.g. a neutralising antibody against SARS-CoV-2. In another embodiment, the agent is a small organic molecule. Preferably the agent inhibits coronavirus-mediated disease in a subject, e.g. a mammal, preferably a human. In some embodiments, the sample from a subject comprises serum, or a component thereof. The serum sample preferably comprises neutralising antibodies against a coronavirus, e.g. SARS-CoV-2. In a further aspect, the present disclosure provides a method of treating or preventing a coronavirus infection or coronavirus-associated disease, comprising administering to a subject in need thereof a therapeutically effective amount of (i) an inhibitor of a member of the transmembrane protein 16 (TMEM16)/Anoctamin family, preferably an inhibitor of Anoctamin6 (ANO6)/transmembrane protein 16F (TMEM16F); (ii) a cardiac glycoside; or (iii) an agent identified by the methods described above, preferably clofazimine or salinomycin. In a further aspect, the present disclosure provides a pharmaceutical composition for use in treating or preventing a coronavirus infection or coronavirus-mediated disease, comprising (i) a member of the transmembrane protein 16 (TMEM16)/Anoctamin family, preferably an inhibitor of an inhibitor of Anoctamin6 (ANO6)/transmembrane protein 16F (TMEM16F); (ii) a cardiac glycoside; or (iii) an agent identified by the method described above, e.g. clofazimine and salinomycin. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients, diluents and/or carriers. In another aspect, the present disclosure provides a method of treating or preventing cell fusion or syncytia formation associated with a coronavirus infection or coronavirus-associated disease, said method comprising administering to a subject in need thereof a therapeutically effective amount of: (i) an anoctamin/transmembrane protein 16 (TMEM16) inhibitor, suitably wherein the TMEM16 inhibitor is niclosamide; (ii) clofazimine; or (iii) salinomycin. In a further aspect the present disclosure provides a pharmaceutical composition for use in treating or preventing cell fusion or syncytia formation associated with a coronavirus infection or coronavirus-mediated disease, comprising: (i) an anoctamin (ANO)/transmembrane protein 16 (TMEM16) inhibitor; (ii) clofazimine; or (iii) salinomycin; and optionally one or more pharmaceutically acceptable excipients, diluents and/or carriers. Preferably the coronavirus is SARS-CoV-2 or the disease is COVID-19. The method or composition may be used to treat or prevent cell-cell fusion or syncytia formation associated with a coronavirus infection. In some embodiments, the TMEM16 inhibitor (e.g. TMEM16A/F inhibitor) is selected from niclosamide, nitazoxanide, hexachlorophene, dichlorophen and gefitinib. In other embodiments, the cardiac glycoside is selected from digitoxin, ouabain, lanatoside C and digitoxigenin. In other embodiments, the agent is a syncytia inhibitor, e.g. clofazimine or salinomycin. In a further aspect, the present disclosure provides a method for detecting an agent that inhibits viral-mediated cell-cell fusion or syncytia formation, comprising: (a) preparing donor cells expressing a viral protein that mediates cell fusion and acceptor cells capable of fusing with the donor cells; (b) labelling the donor cells with a first marker and the acceptor cells with a second marker; (c) incubating the donor cells and acceptor cells with the agent; and (d) detecting cell fusion or syncytia formation by detecting cells labelled with the first marker and the second marker, preferably using high content, automated microscopy. In one embodiment, first and second markers emit light at different wavelengths, e.g. the first marker emits light at the first wavelength and the second marker emits light at a second wavelength. Preferably cell fusion or syncytia formation is detected by detecting cells that emit light at the first and second wavelengths. In one embodiment, the first and second markers are quantum dots. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Fusogenic properties of the SARS-CoV-2 Spike protein in COVID-19 patients and cell culture. a-c, Post-mortem analysis of COVID-19 patients shows the presence of multinucleated syncytia in the lungs, (a) histology, x40 (b) immunohistochemistry using the indicated antibodies, x40 (c) in situ hybridization using two LNA-modified RNA probes for the SARS- CoV-2 genome (upper four panels, including controls with the same probe on a non-infected individual and one control using a probe against the ubiquitous U6 human RNA) and immunohistochemistry with an anti-Spike antibody. Scale bar: 50 µM. Further information on the technique is in: https://medrxiv.org/cgi/content/short/2020.06.22.20136358v1. d, immunofluorescence (IF) detection of S-positive syncytia (panel b) after 24 hr expression of S in Vero cells (nuclei in white, cell contours in red using wheat germ agglutinin (WGA). Scale bar: 200 µM. e, IF visualisation of heterologous syncytia between U2OS cells expressing S and Vero cells expressing EGFP at 12 hr after transfection and co-culture; panel a is a control with un-transfected U2OS cells. Scale bar: 500 µM f, Cytological alterations induced by S. protein Vero cells were transfected with S protein and then cultured for 12 hr; S was then visualised by IF and cell body stained using HCS CellMask DeepRed. S-positive cells show marked morphological alterations, including presence of numerous filopodia (inset in panel a) and membrane protrusions (panels b-f) contacting the plasma membrane of neighbouring cells. Nuclei are stained with DAPI. Bar: 50 µM. g, Representative frames (numbered progressively from a total of 400) from a 12-hr time lapse movie of Vero cells co-transfected with S protein and GCaMP6s. Arrows point at different cells that are progressively recruited into the syncytium. Figure 2. Approved drug screenings using the CFIA and SIA assays. a, Cell Fusion Inhibition Assay (CFIA). b, Fluorescent images of cocultured U2OS (Green Q- dots) and Veri (Red Q-dots) cells. Cells are stained with HCS CellMask Blue; fused cells contain both green and red Q-dots in their cytoplasm. c, Quantification of the number of syncytia after 24 hr of coculture between Vero cells and U2OS cells expressing or not S- protein. d, Results of CFIA screening of FDA/EMA approved drug libraries. The percentage of syncytia normalized on total cells is plotted as z score. Compounds with a z-score ≤-2.58 (red dotted line, 0.005% tail of the distribution) are shown in red, those between ≤-1.96 (0.025% tail, blue dotted line) and -2.58 are in blue. e, Syncytia Inhibition Assay (SIA). f, Image analysis workflow; from the microscopy image, syncytia were defined as cells showing a cluster of nuclei with an area ≥5 times larger than the average of that of the nuclei of non- fused cells – cf. Methods for further information. g, Results of SIA screening of FDA/EMA approved drug libraries; display is as in panel d. Four drugs that were further studied with viral infection are indicated (NIC: Niclosamide; SAL: Salinomycin; CLO: clofazimine; SER: sertraline). h, Representative immunofluorescence images of the effect of Niclosamide compared to DMSO-treated, Control cells after 24 hr S expression in Vero cells. S-positive syncytia are in green, nuclei in blue, cell body in red using HCS CellMask DeepRed. The number of syncytia is shown at the bottom of each image pair as percentages of total nuclei. Scale bar: 500 µM Figure 3. Analysis of screening results. a, Representative images at two magnifications of the SIA screening showing the effect of the indicated drugs on the inhibition of syncytia formation. S protein is in green, nuclei in blue with DAPI, cell shape in red with Cell Mask. Scale bar: 500 µM. The number of syncytia is shown at the bottom of each image pair as percentages of total nuclei. Numbers on some of the images are those of the wells and are generated automatically by the high content microscope. b, plot showing the effect of individual drugs in the SIA screen grouped according to therapeutic classes. Data are z-scores from screening of both the Prestwick and the MS Discovery libraries (drugs present in both libraries are duplicated). The dotted lines are at z- scores ±2.58 (red; 0.005% tail of the distribution) and ±1.96 (blue; 0.025% tail). c, Venn diagram showing the drugs selected as significant at z-scores <-2.58 from the results of the CFIA (light blue) and SIA (light green) screenings using the Prestwick (left) and MS Discovery (right) drug libraries, as indicated. Drugs in bold are those that were considered further in the study with infectious SARS-CoV-2. Figure 4. Cell protection from SARS-CoV-2. a, Vero E6 cells were preincubated with drugs for 2 hr then infected with 100 TCID50 of IC19. After 3 days, inoculum was removed, and cells were fixed for imaging with 4% PFA. Cells survival was quantified by HC microscopy as the total cell area/well using DPC (Digital Phase Contrast) imaging (n=3). b, Cells were preincubated with either 2.5 µM Niclosamide, 10 µM Clofazimine or 5µ M Salinomycin for 2 hr. IC19 virus (100 TCID50) was inoculated and removed after 1 hr and media was replaced with drugs. Viral titre was calculated using TCID50 on supernatants collected every 24 hours. (n=3). Figure 5. Calcium oscillations, chloride channel activity and requirement of TMEM16F protein for Spike-mediated cell-cell fusion a, Representative frames (numbered progressively from of a total of 600) from a 12-hr time lapse movie of a co-culture of Vero cells expressing the GCaMP6s Ca2+ sensor and U2OS transfected to express the S protein and the mCherry fluorescent protein reporter. The arrows indicate Ca2+ spikes in GCaMP6s-ACE2 expressing cells when fusing to S-expressing cells. b, Frequent Ca2+ oscillations during syncytia formation. Representative frames (numbered progressively from of a total of 400) from a 12-hr time lapse movie of Vero cells transfected to express the S protein. Arrows point at cells progressively fusing to the syncytia and showing intense Ca2+ spikes. c, Increase of GCaMP intensity over time. Vero cells co-transfected with GCaMP6s and either an empty vector (left panels) or with S (right panels) and treated with the specified drugs. Data are expressed as ΔF/F over time (min); every line is one of at least twelve 180 μm2 ROIs per condition, representing a group of 4 GCaMP positive cells on average. d and e, Niclosamide-sensitive current is boosted by the expression of S/ACE2. In d, currents were measured using a 500 ms voltage ramp from -80 to +80 mV in HEK293 cells. Currents from control cells (C; black trace) were blocked by 2 µM Niclosamide (NIC; blue trace). Inset shows currents measured at +80 mV for control (C, n=30 cells) cells and cells treated with Niclosamide (NIC, n=7 cells), Salinomycin (SAL, n=8) and Clofazimine (CLO, n=9 cells). **P<0.01, Kruskal-Wallis test with Dunn’s post hoc multiple comparisons. In e, Currents were measured using a 500 ms voltage ramp from -80 to +80 mV in HEK293 cells expressing either EGFP (C; black trace, n= 0 cells) or co-expressing EGFP/Spike/Ace-2 (S/ACE2; red trace, n=29 cells). Inset shows currents measured at +80 mV. *P<0.05, Mann Whitney test. All values are displayed as mean±SEM. f and g, Detection of phosphatidylserine by fluorescent Annexin V in cells treated with the indicated drugs. A representative experiment is shown in f, quantification (mean±SEM) in g (n=5; **P<0.01, two-way ANOVA with Bonferroni post-hoc correction). The pictures in f show PS reactivity in basal conditions and upon 1 hr incubation with the drugs, with or without cell treatment with 5 µM ionomycin; the bottom panels show superimposition of Annexin V (green) with phase contrast at hither magnification. h, Annexin V reactivity (PS externalisation) of syncytia expressing S. Cells were transfected with the S- expressing plasmid and a plasmid expressing mCherry carrying a nuclear localisation signal. i and j, Inhibition of syncytia formation by selected siRNAs. Vero cells were first silenced for each of the indicated genes (ACE2, TMEM16A, TMEM16F, XKR8; efficiency of knock-down in Fig. 11; siNT1: non-targeting siRNA1 as negative control) and, after 24 hr, transfected to express the S-protein. After additional 24 hr, cells were immunostained for S-protein (green) and nuclei (blue). Representative images are in i, quantification in j. Data (mean±SEM; n=3) are plotted as percentage of total area of syncytia normalized on the total number of cells. **P<0.01, two-way ANOVA with Bonferroni post-hoc correction. k, Model showing the effect of selected drugs on syncytia formation and TMEM16F and TMEM16A activity. Infected cells expressing the S protein have increase Ca2+ (in orange) oscillations. This leads to increased activity of the plasma membrane TMEM16 proteins, which itself boosts Ca2+ levels. As a consequence, the chloride (blue) channel activity of these proteins is increased and phosphatidylserine (PS, pink) is externalised onto the outer leaflet of the plasma membrane. Drugs directly blocking TMEM16 proteins or blunting Ca2+ release are inhibitory of S- mediated syncytia formation. While PS externalisation is required for cell-cell fusion, activation of TMEM16A and TMEM16F might have relevance in COVID-19 pathogenesis. Figure 6 Results of CFIA a, CFIA image analysis pipeline. Images were analyzed using the Harmony software (PerkinElmer). The cytoplasmic area, stained by the HCS Cell Mask Blue, was defined using the “Find Cytoplasm” analysis module (Harmony) and the number of either green or red spots was counted using the “Find Spots” analysis module (Harmony). All the cells that scored a nuclear area greater than 5 times the average area of a single nucleus and were simultaneously positive for at least two red and two green dots were considered as fused (highlighted in green). Data were expressed as a percentage of fused cells/well. b, Analysis of CFIA results. A correlation chart between the percentage of syncytia/well and the total number of cells/well is shown for the two screened drug libraries. Drugs showing a total number of cells at ≥2 standard deviations lower than the average of all drugs were discarded as toxic. Distribution of control wells is shown by a green box. c, Distribution frequency of the % of syncytia is plotted as the number of drugs vs. the z score of the % of syncytia/well; toxic cells were excluded as in panel b. Blue dotted lines are at z scores ±1.96 and red dotted lines at z scores ±2.56. The numbers of drugs are shown. d, Correlation chart of the common drugs between the two libraries. Data are plotted as the z score of the % of syncytia/well. Blue and red lines are as above. Figure 7 Results of SIA a, Analysis of CFIA results. Correlation chart between the percentage of syncytia /well and the total number of cells/well. A correlation chart between the percentage of syncytia/well and the total number of cells/well is shown for the two screened drug libraries. Drugs showing a total number of cells at ≥2 standard deviations lower than the average of all drugs were discarded as toxic. Distribution of control wells is shown by a green box. d, Correlation chart between CFIA and SIA screenings for the two drug libraries. Data are plotted as the z-score of the % of syncytia/well. Blue dotted lines are at z scores ±1.96 and red dotted lines at z scores ±2.56. e, Representative immunofluorescence images for Spike protein (green), cell body (red, using HCS CellMask DeepRed) and nuclei (blue, Hoechst) of cells treated with the indicated drugs increasing syncytia formation compared to control, DMSO-treated cells. The number of syncytia is shown at the bottom of each image pair as percentages of total nuclei. Scale bar: 500 µM. Figure 8 Dose-response effect on syncytia formation of Niclosamide, Clofazimine and Salinomycin a, Effect of different doses of the indicated drugs on syncytia formed by Vero cells in response to S expression. Vero cells were transfected with pEC117-Spike-V5 and, 12 hr later, treated with the indicated drug concentrations for additional 12 hr. Syncytia were quantified by HC microscopy and are expressed as percentage of cell nuclei (n=6 wells/dose). Cell viability was assessed by HC counts of the number of nuclei in each well. b, same as in panel a in HEK293 cells. Figure 9 Cell protective ability of drugs against SARS-CoV-2. a, List of drugs (CAS # and chemical name) shortlisted from the syncytia assays and tested with infectious SARS-CoV-2. b, Effect of drugs on cell viability. Vero E6 cells were preincubated with a selection of drugs at 10 µM for 2 hr before 100 TCID50 of IC19 was added. After 5 days, cells were fixed in 4% PFA. Cells survival was quantified by densitometry as the total cell area/well (average of 2 replicates). Drugs labeled with Tox showed a cytotoxic effect at the used concentration independent of viral infection. Drugs in green were further tested for dose-dependent response. c, Dose-dependent cell protection of selected drugs against SARS- CoV-2 infection. Cells were preincubated with drugs or a negative DMSO control at different concentrations for 2 hr before 100 TCID50 of IC19 was added. After 3 days, cells were fixed in 4% PFA and cell survival was quantified by HC microscopy as the total cell area/well using DPC (Digital Phase Contrast) imaging (n=3). The image shows one of the replicates. d, Virus production in response to different drug concentrations. Cells were preincubated with drugs or a negative DMSO control at different concentrations for 2 hr before 200 TCID50 of IC19 was added. The inoculum was removed after 1 hr and replaced with media containing drugs. Viral titre was measured after 2 days by TCID50 (n=3). Figure 10 Effect of drugs on Ca2+ oscillations and chloride current a, Amplitude of calcium transient peaks per group of 4 GCaMP-positive cells in average in at least 12180 μm2 ROI per condition. Vero cells co-transfected with GCaMP6s and either an empty vector (grey bars) or with S (green bars) and treated with the specified drugs. Data are expressed as ΔF/F, **P<0.01. b, Distribution of transient frequencies of single cells in 400 min analysis. Data are expressed as percentage of cells. Results from at least 50 cells per condition are shown. *P<0.05, **P<0.01, two-way ANOVA with Bonferroni post-hoc correction. c, Results of whole-cell voltage-clamp recordings of HEK293 cells treated with the benchmark TMEM16 chloride channel antagonist benzbromarone (BENZ, n=3) or an siRNA against TMEM16F (n=4). C: untreated control cells (n=7). Currents were measured using a 500 ms voltage ramp from -80 to +80 mV as in Figs.5c and 5d. The inset shows currents measured at +80 mV. All values are displayed as mean±SEM. Figure 11 Expression profiles of TMEM16s family members and RNAi. a, Relative expression of TMEM16s family members in HEK293 and Vero cells. The amount of TMEM16s are normalized over the GAPDH housekeeping gene. Data are mean±SEM of 3 independent experiments. b, Silencing of the indicated genes by RNAi was confirmed by qPCR by using TaqMan probes. The expression levels of cellular ACE2, TMEM16A and TMEM16F genes are shown after RNAi in HEK293 cells upon normalization over the GAPDH housekeeping gene. Data are mean±SEM of 4 independent experiments. c, Silencing of cellular ACE2 and TMEM16F after RNAi in Vero cells. Experiments were performed as in panel b. d, Western blotting analysis showing TMEM16F and ACE2 levels. Amount of TMEM16F and ACE2 by immunoblotting after cells treatment with the respective siRNAs. Tubulin was used as a loading control. Figure 12 Inhibition of syncytia formation in HEK293 cells by downregulation of TMEM16F. a and b, Inhibition of syncytia formation by selected siRNAs. HEK293 cells were first silenced for each of the indicated genes (ACE2, TMEM16A, TMEM16F, XKR8) or treated with siNT1 (non-targeting siRNA1 as negative control) and then, after 24 hr, transfected to express the S- protein. After additional 24 hr, cells were immunostained for S-protein (green) and nuclei (blue). Representative images are in a, quantification in b. Data (mean±SEM; n=3) are plotted as percentage of total area of syncytia normalized on the total number of cells. *P<0.05, **P<0.01, two-way ANOVA with Bonferroni post-hoc correction. The siRNA against TMEM16A exerted a cytotoxic effect in these experiments and was not considered (n.c.) further. Figure 13 Effect of drugs on syncytial formation induced by different Spike protein variants. A. Representative images and B. Quantification. Experiments were performed by transfecting Vero cells with expression vectors for the indicated Spike protein variants in the presence of the indicated drugs. At 20 hr after transfection, cells were fixed and stained for Spike protein. Syncytia were defined as cells showing a cluster of nuclei with an area ≥5 times larger than the average of the area of the nuclei of non-fused cells. Treatment was with Niclosamide 100 nM, Clofazimine or Salinomycin 500 nM. Experiments were performed in 6 replicates per condition. The P2 Spike variant has a two consecutive amino acid mutation that enhances Spike trimer stability. This mutation, which is introduced in some of the currently available vaccines, does not induce fusion. Niclosamide, Clofazimine and Salinomycin were all effective against all the Spike variants tested. LIST OF SEQUENCES SEQ ID NO: 1 - Severe acute respiratory syndrome coronavirus 2 surface (Spike) glycoprotein (Wuhan-Hu-1) (GenBank: QHD43416) SEQ ID NO:2 - Angiotensin-converting enzyme 2 precursor [Homo sapiens] (NCBI Reference Sequence: NP_001358344.1) SEQ ID NO:3 - Transmembrane protease serine 2 isoform 1 [Homo sapiens] (NCBI Reference Sequence: NP_001128571.1) SEQ ID NO:4 - Anoctamin-6 isoform a [Homo sapiens] (NCBI Reference Sequence: NP_001020527.2) SEQ ID NO:5 – Forward PCR primer used in Example 1 below SEQ ID NO:6 – Reverse PCR primer used in Example 1 below SEQ ID NO: 7 - Severe acute respiratory syndrome coronavirus 2 protein-coding sequence (Wuhan-Hu-1) (GenBank NC_045512.2, position 21563-25384). SEQ ID NO: 8 - Severe acute respiratory syndrome coronavirus 2 protein-coding sequence (D614G variant) SEQ ID NO: 9 - Severe acute respiratory syndrome coronavirus 2 protein-coding sequence (Alpha variant/UK lineage B.1.1.7) SEQ ID NO: 10 - Severe acute respiratory syndrome coronavirus 2 protein-coding sequence (Beta variant/South African variant B.1.351) SEQ ID NO: 11 - Severe acute respiratory syndrome coronavirus 2 protein-coding sequence (Gamma variant/Brazil lineage B.1.1.248.1) SEQ ID NO: 12 - Severe acute respiratory syndrome coronavirus 2 protein-coding sequence (P2 spike variant, vaccine) SEQ ID NO: 13 – Severe acute respiratory syndrome coronavirus 2 surface (Spike) glycoprotein (delta variant) (GenPept: QXP08802.1) SEQ ID NO: 14 – Severe acute respiratory syndrome coronavirus 2 surface (Spike) glycoprotein (beta variant/South African lineage B.1.351) SEQ ID NO: 15 - Severe acute respiratory syndrome coronavirus 2 S protein (Alpha variant/UK lineage B.1.1.7) SEQ ID NO: 16 - Severe acute respiratory syndrome coronavirus 2 S protein (Gamma variant/Brazil lineage B.1.1.248.1) DETAILED DESCRIPTION OF THE INVENTION Embodiments of the present invention utilize automated microscopy to monitor virus (especially coronavirus)-mediated cell fusion or syncytia formation. The methods may be based on high content microscopy and are particularly suited to high throughput analysis. This enables the rapidly screening for agents that modulate (e.g. inhibit) coronavirus-mediated disease such as COVID-19. Traditional assays for detecting neutralising antibodies and/or antiviral drugs include methods such as plaque reduction neutralisation test (PRNT assay) or plaque assay. Such methods essentially rely on detecting inhibition of viral replication. The present methods are particularly advantageous as they are capable of identifying agents effective against SARS- CoV-2 and other coronaviruses without the need to monitor viral replication. The present methods are instead based on detecting a pathological response seen in human subject cells to the viral S protein (i.e. cell fusion/syncytia formation). The present assays can therefore be performed safety in a laboratory environment without e.g. level 3 biosafety containment measures, making them much more amenable to high throughput screening. Methods In a broadest aspect, the methods disclosed herein relate to detecting, measuring or quantifying virus-mediated cell fusion or syncytia formation. Typically the methods utilize microscopy, e.g. automated high content microscopy, to monitor cell fusion or syncytia formation. The methods may be performed using cultured cells that undergo cell fusion or syncytia formation due to expression of a viral protein, e.g. a coronavirus Spike protein. The methods may be used to detect agents that modulate cell fusion or syncytia formation. For instance, levels of cell fusion or syncytia formation may be quantified in the presence and absence of the agent. A difference between levels of cell fusion or syncytia formation in the presence and absence of the agent may be used to identify modulatory agents. By “modulates” it is meant that the agent increases or decreases cell fusion or syncytia formation. In preferred embodiments, the agent decreases cell fusion or syncytia formation. For instance, the agent may decrease cell fusion or syncytia formation by at least 5%, 10%, 20%, 30%, 50%, 70% or 90%, e.g. compared to a level of cell fusion or syncytia formation occurring in the absence of the agent. In one embodiment, the method is used to identify an agent that modulates (e.g. inhibits) virus- mediated cell fusion or syncytia formation. For instance, the method may be applied to high throughput screening of antisera, antibodies (including purified immunoglobulins and fragments thereof) or other drugs or therapeutic agents (including small organic molecules) to identify those which inhibit virus-mediated cell fusion or syncytia formation. In some embodiments, the identified antisera, neutralising antibodies or other drugs may be used for treatment of viral infection or virus-associated disease, e.g. COVID-19. In other embodiments, the method may be used to detect and/or predict an immune response against a virus (e.g. a coronavirus such as SARS-CoV-2) in a subject. By this it is meant that the method may be used to determine whether an immune response has occurred in the subject (in the past), is currently occurring or is likely to occur (in the future) in response to exposure to the virus. For instance, the method may be used to determine whether a subject has been infected with a coronavirus (e.g. SARS-CoV-2) and/or whether the subject is immune to coronavirus infection. Thus in some embodiments, the method may be used for mass screening of populations of subjects, e.g. to determine the prevalence of exposure and/or immunity to the disease in a population, or to monitor the efficacy or development of vaccines. Virus In one aspect, the virus may be any virus that induces host cell fusion and/or syncytia formation. Thus the cultured cells express a protein from the virus that mediates cell fusion. Preferably the virus is a coronavirus, e.g. e.g. SARS-CoV or e.g. SARS-CoV-2, more preferably e.g. SARS-CoV-2. Thus the virus may comprise a Spike protein as shown in SEQ ID NO:1 below, or having at least e.g. 85%, 90% or 95% sequence identity thereto. In other embodiments, the virus comprises a Spike protein as shown in any one of SEQ ID NO:s 13- 16, or a sequence having at least e.g. 85%, 90%, 95% or 99% sequence identity thereto. In further embodiments, the virus comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID Nos: 7 to 12 (or an RNA equivalent thereof, i.e. wherein T residues are replaced by U). In other embodiments, the virus comprises a Spike protein encoded by any one of SEQ ID NO:s 7-12, or a sequence having at least e.g.85%, 90%, 95% or 99% sequence identity thereto. Cultured cells Embodiments of the present invention utilise cultured cells, i.e. cells that are maintained in tissue culture ex vivo or in vitro. Typically the cells are animal cells and preferably mammalian (e.g. human or primate) cells. Various suitable mammalian cell lines are available for tissue culture and are well known to a skilled person. For instance, in particular embodiments the cells may be Vero cells or U-2 OS cells. At least some of the cells express a viral protein that mediates cell fusion and/or syncytia formation. Typically the cells express a coronavirus Spike protein, e.g. SARS-CoV-2 S protein. The sequences of coronavirus Spike proteins are known and are available from publicly accessible databases. One example of a SARS-CoV-2 S protein sequence is shown in SEQ ID NO:1: SEQ ID NO: 1 Severe acute respiratory syndrome coronavirus 2 surface glycoprotein (GenBank: QHD43416)

5

Further SARS-CoV-2 S protein sequences that may be used are shown in SEQ ID Nos 13 to 16 below. Thus the SARS-CoV-2 S protein sequence may be from a variant virus, e.g. an alpha, beta, gamma or delta variant. In other embodiments, the SARS-CoV-2 S protein has at least 85%, at least 90%, at least 95% or at least 99% sequence identity to (i) any one of SEQ ID Nos 1, 13, 14, 15 or 16, or (ii) a polypeptide sequence encoded by any one of SEQ ID Nos: 7-12. Expression of SARS-CoV-2 S protein or other viral proteins may be induced in the cultured cells by using standard techniques, e.g. transfection with a suitable expression vector. For instance, in one embodiment, the SARS-CoV-2 S protein may be introduced into the cultured cells using an adeno-associated virus (AAV) expression vector or plasmid, or a lentiviral vector. Suitable expression vectors and cell expression systems are disclosed in e.g. Ou et al. (2020), Nature Communications volume 11, Article number: 1620; Walls et al. (2020), Cell Volume 181, Issue 2, Pages 281-292.e6; and Xia et al. (2020), Cell Research 30:343–355. In some embodiments, the cultured cells may comprise a single population of cells. For instance, expression of the viral protein in the cells (e.g. Vero cells) may lead to syncytia formation, due to the fusogenic property of the protein (e.g. SARS-CoV-2 S protein). Thus in this embodiment, the cultured cells may express both the viral protein and a cell surface receptor for the viral protein (e.g. angiotensin-converting enzyme 2 (ACE2)), as well as any further molecules (e.g. proteases) that are required for cell fusion. In one embodiment, the cultured cells express ACE2 and/or TMPRSS2. The sequences of human ACE2 and TMPRSS2 are available from the UniProt database under accession numbers Q9BYF1 and O15393 respectively. The sequence of human ACE2 precursor is shown in SEQ ID NO:2:

5

The sequence of human TMPRSS2 (isoform 1) is shown in SEQ ID NO:3 below:

TMPRSS2 is a type II transmembrane serine protease (TTP) that belongs to the hepsin/TMPRSS subfamily and which plays a role in SARS-CoV-2 entry into host cells (Hoffmann et al., Cell (2020) 181: 1-10). It is thought that following attachment of the SARS- CoV-2 virus, TMPRSS2 cleaves the S protein (“priming”), exposing a fusion peptide that promotes fusion of the viral envelope and the cell membrane. The SARS-CoV-2 virus RNA is then released into the cell, which is then copied and SARS-CoV-2 viral particles produced. Thus where a single population of cells is used in the assay, the cells may be from a cell line that expresses both ACE2 and TMPRSS2, e.g. Vero cells. In alternative embodiments, the cultured cells may comprise two or more subpopulations of cells. For instance, in one embodiment the cultured cells may comprise donor cells and acceptor cells. “Donor cells” refers here to cells that express a viral protein (e.g. SARS-CoV- 2 S protein) that mediates cell fusion. “Acceptor cells” refers to cells that are capable of fusing with the donor cells, e.g. cells that express a cell surface receptor (e.g. angiotensin-converting enzyme 2 (ACE2)) that binds to the viral protein and/or any necessary proteases (e.g. TMPRSS2). Thus the acceptor cells are typically capable of supporting viral entry and replication on exposure to the viral protein. In one embodiment, the donor cells may be from a cell line that does not express a cell surface receptor for the viral protein and/or cellular proteases involved in cleavage of the viral protein. For instance, the donor cell may lack expression of angiotensin-converting enzyme 2 (ACE2) and/or TMPRSS2. Thus the donor cells may be from a cell line such as U2OS osteoblastoma cells (which have undetectable levels of ACE2 and TMPRSS2 and do not undergo viral fusion), typically that have been transfected with SARS-CoV-2 Spike protein. In one embodiment, the acceptor cells express both ACE2 and/or TMPRSS2, e.g. Vero cells. Agents In embodiments of the present invention, the cultured cells are contacted with one or more test agents. The test agent(s) may be e.g. any formulation, compound or combination of compounds that it is desired to test for antiviral activity. For instance, in some embodiments, a library of antibodies, nucleic acids (e.g. aptamers or inhibitory RNAs) or small organic molecules may be screened using the method. In one embodiment, the agent is an antibody or fragment thereof (e.g. Fab, Fab’ or scFv), including immunoglobulins from any class thereof (e.g. IgG, IgM, IgA or IgE). The antibody may be e.g. a monoclonal (e.g. mouse) antibody, a chimeric antibody, a humanized antibody or a fully human antibody. The present methods are particularly suitable for screening for neutralising immunoglobulins (e.g. an IgG) present in antibody libraries or polyclonal antisera having antiviral activity. Neutralising antibodies and fragments thereof identified using the assays can then be selected, purified and used for e.g. COVID-19 treatment. In some embodiments, the agent is a polyclonal antiserum, e.g. a serum sample or fraction thereof from a subject (e.g. a subject who has been exposed to a coronavirus, or who is immune to coronavirus infection). Polyclonal antisera that contain neutralising antibodies to e.g. SARS- CoV-2 may potentially be used directly in treatment of subjects with COVID-19. In embodiments of the present invention, the agent is contacted with the cultured cells. For instance, the agent may be added to a culture medium or solution in which the cells are present. The agent may be contacted with the cells at any suitable concentration or a range of concentrations, e.g. at concentrations that may be therapeutically relevant. Typically the concentration of the agent in the culture medium is at least 1 pM, at least 1 nM, or at least 1 µM. A suitable range of concentrations of the agent may be e.g.0.1 nM to 1 mM, e.g.1 nM to 100 µM or 1 nM to 10 µM. The cells may be contacted with the agent under suitable laboratory conditions (e.g. at 37°C) and for a predetermined time period (e.g. at least 1 hour, 2 hours, 12 hours or 24 hours, preferably 1 to 48 hours or 2 to 24 hours). Detecting cell fusion/syncytia formation The present methods involve detecting cell fusion or syncytia formation. By this it is meant that fusion of two or more of the cultured cells is detected, and/or that cytoplasmic masses derived from two or more cells (i.e. syncytia) are detected in the cultured cells. Cell fusion or syncytia formation may be detected using microscopy, e.g. by obtaining one or more microscopic images of the cells. Various automated microscopy systems may be used for detecting cell fusion/syncytia formation in the present invention. In preferred embodiments, the present methods use automated high content screening microscopy. Suitable systems are known in the art and available from various sources, e.g. Perkin Elmer, Inc. (Waltham, MA). One example of a suitable system is an Operetta CLS high content screening microscope (available from Perkin Elmer, Inc.). Image analysis software is also available for automated analysis of such microscopic images, e.g. identification of subcellular structures (nuclei, cytoplasm etc.), detection of specific markers and labels, quantification of fluorescence and so on. Cell fusion/syncytia formation can be detected in various ways. In one embodiment, cell fusion or syncytia formation is detected by quantifying cytoplasmic size or nuclear size. For instance, cytoplasm and nuclei of cells in the image may be identified by their characteristic morphology and or specific markers, e.g. using known histochemical techniques. Cells may be classified according to an area represented by their cytoplasm or nuclei in an image of the cells. Individual cells having an area of cytoplasm or nuclei significantly different from the average area represented by a single (non-fused) cell can thus be identified as fused cells or syncytia. For instance, a cut-off value representing e.g. 2x, 3x or 5x an average area represented by a non-fused cell may be defined. Cells having an area of cytoplasm or nuclei above that value can be classified as fused cells or syncytia. In other embodiments, cell fusion or syncytia formation is detected by quantifying a number of nuclei per cytoplasm. For instance, cells having two or more (e.g. at least 2, 3, 4, 5 or more) nuclei can be classified as syncytia. In a further embodiment, fusion of donor and acceptor cells (as described above) is determined. In such embodiments, fusion of donor and acceptor cells may be detected using unique markers or labels present on each cell type. For instance, a first marker may be present on the donor cells and a second marker may be present on the acceptor cells. Cell fusion is determined by detecting cells expressing the first marker and the second marker. The first and second markers may be e.g. a protein or other molecule that is expressed on either the donor or acceptor cells, but not both. Such markers may be visualised by using suitable staining techniques well known to a skilled person, e.g. immunocytochemistry and the like. In some embodiments, the first and second markers may be a fluorescent label or tag. Typically a pair of fluorescent labels may be used, e.g. that emit light at different wavelengths. Thus fused cells may be quantified by identifying cells that emit light at wavelengths associated with both markers, i.e. a first fluorescent marker associated with the donor cells and a second fluorescent marker associated with the acceptor cells. In one embodiment, the donor and acceptor cells are independently labelled with nanoparticles that emit light at different wavelengths. Suitable nanoparticles include e.g. quantum dots, which are nanoparticles (typically e.g.1-10 nm in diameter) formed from semiconductors such as lead sulfide, cadmium selenide and indium phosphide. On illumination with UV light, quantum dots of different sizes may emit light of different wavelengths, including in the visible range. In one embodiment, donor cultured cells may be loaded with nanoparticles (e.g. quantum dots) that emit light at a first wavelength and acceptor cultured cells may be loaded with nanoparticles that emit light at a second wavelength. Fusion of the donor and acceptor cultured cells is then detected by quantifying cells that emit light at the first wavelength and second wavelength. In some embodiments, cell fusion or syncytia formation may be determined using any combination of the above methods, e.g. quantifying cytoplasmic size or nuclear size, number of nuclei per cytoplasm and/or co-detection of markers derived from donor and acceptor cells. Typically a level of cell fusion or syncytia formation is determined in the presence and absence of the agent, e.g. a level of cell fusion or syncytia formation after a defined period of incubation with the agent may be compared to a level of cell fusion or syncytia formation after the same period of incubation in the absence of the agent. The present methods may be performed using any suitable containers, plates, arrays and so on suitable for holding the cultured cells, particularly those adapted for high throughput screening. Therapeutic methods and compositions In further aspects, the present disclosure relates to methods of treating or preventing a coronavirus infection or coronavirus-associated disease, as well as pharmaceutical compositions for use in such methods. Such methods and compositions may use, for example, one or more agents identified by the present assay methods, i.e. agents that are capable of inhibiting coronavirus-mediated cell fusion or syncytia formation. In one embodiment, the agent is an inhibitor of one or more members of the transmembrane protein 16 (TMEM16) family of proteins (see e.g. Whitlock and Hartzell, Annu Rev Physiol 2017; 79:119-143; Oh and Jung, Pflugers Arch 2016;468(3):443-53). These proteins are calcium-dependent chloride channels and scramblases and may also be referred to as anoctamins. Preferably the agent is an inhibitor of anoctamin-6 (TMEM16F), see e.g. Wu et al. Cell Reports, Volume 30, Issue 4, 28 January 2020, Pages 1129-1140.e5; Alvadia et al., eLife 2019;8:e44365; Le et al., Nature Communications volume 10, Article number: 1846 (2019) and/or anoctamin-1 (TMEM16A). As used herein, the term ‘inhibitor’ refers to an agent that reduces the expression, activity and/or function of the target. Therefore, the agent may inhibit the TMEM16 family (including TMEM16A and TMEM16F) directly (i.e., by binding to the TMEM16 member and preventing normal function) or indirectly (i.e., by inhibiting the expression, activity and/or function of a target that in turn inhibits TMEM16 member). Therefore, the term ‘TMEM16F inhibitor’ includes agents that exclusively inhibit TMEM16F, or agents that inhibit TMEM16F in addition to other targets (i.e, TMEM16A). The TMEM16F inhibitor may be one of the agents disclosed herein. Inhibition may be reported, for example, as the half-maximal inhibitory concentration (IC50). IC50 refers to the quantity of the drug (or ‘inhibitor’) is needed to inhibit the biological process by half. Therefore, the IC50 of an inhibitor of a TMEM16 family member may be quantified, for example, in relation to the peak chloride current using standard techniques in the art. Similarly, IC50 may be used to report the quantity of a drug (or inhibitor) required to inhibit syncytia formation by half. IC₅₀ values may be calculated using standard statistical techniques, such as nonlinear regression analysis using a dose–response (variable slope) equation. TMEM16 inhibition may also be quantified by determining a binding affinity of the agent to TMEM16. As used herein, ‘binding’ refers to a non-covalent interaction between macromolecules (e.g., between a drug and a protein). While in a state of non-covalent interaction, the macromolecules are said to be ‘associated’ or ‘interacting’ or ‘binding’ (e.g., when a molecule X is said to interact with a molecule Y, it means that the molecule X binds to molecule Y in a non-covalent manner). Binding interactions are generally characterized by a dissociation constant (Kd) of less than 10-3M, less than 10-6M, less than 10-7M, less than 10- 8M, less than 10-9M, less than 10-10M, less than 10-11M, less than 10-12M or less than 10-15M. Kd is dependent on environmental conditions, e.g., pH and temperature, as is known by those in the art. Thus the TMEM16 (e.g. TMEM16F) inhibitor preferably has a Kd for binding to TMEM16 within one of the ranges set out above (e.g.10-3M to 10-15M, 10-6M to 10-12M or 10- 7M to 10-10M. The sequences of human anoctamin-6 (TMEM16F) and other members of the anoctamin/TMEM16 family are available in public databases, e.g. under Uniprot accession no.s Q4KMQ2 or W6JLH6. The sequence of human anoctamin-6 isoform a is shown in SEQ ID NO:4 below:

Anoctamin/TMEM16 inhibitors, including TMEM16F inhibitors, are known in the art or can be identified using known assay methods. Suitable inhibitors may include e.g antibodies, inhibitory RNAs (e.g. shRNA or siRNA) and expression vectors coding therefor, and small organic molecules. For instance, anoctamin inhibitors and assays are described in Oh et al., Molecular Pharmacology (2013) 84, 5, 726-735); Hwang et al., Br J Pharmacol. 2016 Apr; 173(8): 1339–1349; Song et al., Cell Death & Disease volume 9, Article number: 703 (2018); Jo et al., FASEB Journal 2020, Vol.34, S1, Supplement. Assays for determining TMEM16F inhibiotors are also described in Martins et al. (2011. PNAS, 108(44): 18168-18172), Kmit et al. (2013. Cell Death Dis., 4(4): e611) and Schreiber et al. (2018. J Physiol, 596(2): 217-229). WO2018/202471 discloses further TMEM16 inhibitors and assays therefore, e.g. niflumic acid, flufenamic acid (FFA, N-(3-[trifluoro- methyl]phenyl)anthranilic acid), meclofenamic acid (MFA, 2-[(2,6- dichloro-3-methylphenyl)amino]benzoic acid) mefloquine ((R*,S*)-(±)-a-2- piperidinyl-2,8-bis(trifluoro- methyl)-4-quinolinemethanol), dichlorophen (2,2'- methylenebis(4- chlorophenol), hexachlorophene (2,2'-methylenebis(3,4,6-trichlorphenol)), hexachlorophene (2,2'-Methylenebis(3,4,6-trichlorphenol)); miconazole ((RS)-l-[2,4-dichlor- - (2,4-dichlorbenzyloxy)-phenethyl]imidazol), plumbagin (5-hydroxy-2-methyl- naphthalene- 1 ,4-dione), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), benzbromarone ((2-ethyl-3- benzofuranyl)-(3,5-dibrom-4-hydroxyphenyl)ketone), idebenone (2-(10-hydroxydecyl)-5,6- dimethoxy-3-methyl-cyclohexa-2,5-dien-l,4-dion), tannic acid (2,3-dihydroxy-5-({[(2R,3R, 4S,5R,6R)-3,4,5,6-tetrakis({3,4-dihydroxy-5-[(3,4,5-trihydroxyphenyl)carbonyloxy]phenyl}- carbonyl- oxy)oxan-2-yl]methoxy}carbonyl)phenyl 3,4,5-trihydroxybenzoate), CaCCinh-AOl (6-(l, l-Dimethylethyl)-2-[(2-furanylcarbonyl)amino]-4,5,6,7- tetrahydrobenzo[b]thiophene-3- carboxylic acid), T16Ainh-A01 (2-[(5-Ethyl- l,6-dihydro-4-methyl-6-oxo-2-pyrimidinyl)thio]- N- [4-(4-methoxyphenyl)-2-thiazolyl]-acetamide). Preferred TMEM16 inhibitors include e.g. niclosamide (5-chloro-N-(2-chloro-4-nitrophenyl)- 2-hydroxybenzamide), nitazoxanide ([2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl] acetate), hexachlorophene, dichlorophen, benzobromarone and gefitinib (N-(3-chloro-4-fluoro-phenyl)- 7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine). In a further embodiment, the TMEM16 inhibitor may be a phenothiazine antipsychotic, e.g. trifluoperazine (10-[3-(4- methylpiperazin-1-yl)propyl]-2-(trifluoromethyl)-10H-phenothiazine) or ivermectin (22,23- dihydroavermectin B1a and/or 22,23-dihydroavermectin B1b). Most preferably the agent is niclosamide. In another embodiment, the agent is a TMEM16 inhibitor (e.g. TMEM16A or TMEM16F inhibitor) other than niclosamide. In preferred embodiments, the agent may be one of those defined above that inhibits both TMEM16F and TMEM16A. In embodiments, the agent is selected from one or more of: niclosamide or a salt thereof, ani6, ani9, idebenone, benzbromarone, clofazimine, salinomycin, tannic acid, niflumic acid, or CaCCinhAO1. In embodiments, the agent is an indirect inhibitor of TMEM16F, such as N-(p- amylcinnamoyl) anthranilic acid (ACA) or bromoenol lactone (BEL). In embodiments the agent may also be selected from: nitazoxanide, hexachlorophene, gefitinib, dichlorophen, flufenamic acid, NPPB plumbagin, dichlorophen, miconazole, mefloquine, T16Ainh-A01, meclofenamic acid, a phenothiazine antipsychotic, e.g. rifluoperazine or ivermectin. In another embodiment, the agent is an anti-anoctamin/TMEM16 antibody. The generation of antibodies to a certain protein is well known in the art, and standard techniques can be employed to obtain an antibody directed against TMEM16 family members, e.g. TMEM16F. Illustrative examples of an antibody directed against a TMEM16 family member have been disclosed in US 2013/323252. Some antibodies disclosed therein specifically bind to a peptide of sequence KLIRYLKLKQ or RYKDYREPPWS. In embodiments the antibody may be an antibody against TMEM16F. In embodiments the antibody may be selected from one or more of: Abcam ab234422, Invitrogen PA5-35240, NeuroMab 73-418, Sigma-Aldrich HPA038958, or Sigma-Aldrich MABN1866, In another embodiments, the agent may be an inhibitory RNA, e.g. the expression of anoctamin/TMEM16 may be inhibited using RNA interference. Thus the agent may be e.g. a small hairpin RNA (shRNA) or small interfering RNA (siRNA) against anoctamin/TMEM16 (e.g. TMEM16F), e.g. encoded by a suitable vector (e.g. a lentiviral vector). Inhibitory RNAs against TMEM16F are disclosed in e.g. Kmit et al. Cell Death Dis. 2013 Apr 25;4(4):e611; Ousingsawat et al. Nature Communications volume 6, Article number: 6245 (2015); and Schenk et al. Cell Physiol Biochem 2016;38:2452-2463. In further embodiments, expression of anoctamin/TMEM16 (e.g. TMEM16A or TMEM16F may be inhibited using CRISPR/Cas9 gene editing (e.g. as described in Bricogne et al., Nature Scientific Reports volume 9, Article number: 619 (2019); Zhang et al., Sci. Adv.2020; 6 : eaba0310; Gyobu et al. PNAS (2017), Characterization of the scrambling domain of the TMEM16 family, ). In embodiments, TMEM16F may be inhibited using genome editing. In embodiments, TMEM16F may be inhibited using a method disclosed in Bricogne et al., (2019); Zhang et al., (2020) or Gyobu et al. (2017), incorporated by reference herein. In another embodiment, the agent is a cardiac glycoside (see e.g. Botelho et al. Toxicon Volume 158, February 2019, Pages 63-68). The cardiac glycoside may be selected from, for example, digitoxin ((3β,5β)-3-[(O-2,6-dideoxy-β-D-ribo-hexapyranosyl-(1->4)-2,6-dideoxy-β-D-ribo- hexopyranosyl)oxy]-14-hydroxycard-20(22)-enolide), digoxin (3β-[(O-2,6-dideoxy-β-D-ribo- hexopyranosyl-(1→4)-O-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-ribo- hexopyranosyl)oxy]-12β,14-dihydroxy-5β-card-20(22)-enolide), ouabain (4-[(1R,3S,5S,8R, 9S,10R,11R,13R,14S,17R)-1,5,11,14-tetrahydroxy-10-(hydroxymethyl)-13-methyl-3-((2R, 3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yloxy)hexadecahydro-1H- cyclopenta[a]phenanthren-17-yl]furan-2(5H)-one), lanatoside C ([6-[6-[6-[[12,14-dihydroxy- 10,13-dimethyl- 17-(5-oxo-2H-furan-3-yl)- 1,2,3,4,5,6,7,8,9,11,12,15,16,17- tetradecahydro- cyclopenta[a]phenanthren- 3-yl]oxy]- 4-hydroxy- 2-methyloxan- 3-yl]oxy- 4-hydroxy- 2- methyloxan-3-yl]oxy- 2-methyl- 3-[3,4,5-trihydroxy- 6-(hydroxymethyl)oxan-2-yl]oxyoxan- 4-yl] acetate) and digitoxigenin (3-[(3S,5R,8R,9S,10S,13R,14S,17R)-3,14-dihydroxy-10,13- dimethyl-1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetradecahydrocyclopenta[a]phenanthren-17-yl]- 2H-furan-5-one). In another embodiment, the agent is clofazimine (N,5-bis(4-chlorophenyl)-3-(1- methylethylimino)-5H-phenazin-2-amine) or salinomycin ((2R)-2-[(2R,5S,6R)-6- [(2S,3S,4S,6R)-6-[(3S,5S,7R,9S,10S,12R,15R)-3-[(2R,5R,6S)-5-ethyl-5-hydroxy-6- methyloxan-2-yl]-15-hydroxy-3,10,12-trimethyl-4,6,8-trioxadispiro[4.1.57.35]pentadec-13- en-9-yl]-3-hydroxy-4-methyl-5-oxooctan-2-yl]-5-methyloxan-2-yl]butanoic acid), both of which inhibit syncytia formation stimulated by coronavirus S protein. Although less preferred, in other embodiments, the agent may be deptropine (3a-[(10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5-yl)oxy]1aH,5aH-tropane) or sertraline ((1S,4S)-4-(3,4- Dichlorophenyl)-N-methyl-1,2,3,4-tetrahydronaphthalen-1-amine). In embodiments of the present invention, the agent is used to treat the disease by inhibiting an anoctamin/TMEM16 family member (e.g. TMEM16A/F), e.g. thereby inhibiting cell-cell fusion and/or syncytia formation in infected tissues of the subject. Without being by theory, as demonstrated herein in the examples suitable agents that inhibit an anoctamin/TMEM16 family member may block phosphatidylserine (PS) externalization in cells expressing the viral S protein. Thus agents as described herein may be used to block PS externalization in the treatment of infected subjects. The use of the present agents to inhibit cell-cell fusion and/or syncytia formation associated with coronavirus infection relates to a different therapeutic application to treatments intended to inhibit viral replication. In particular, the present agents are used to treat the pathology caused by the virus, rather than to target the virus itself. Therefore the present agents may be used in a different clinical frame, e.g. to treat different subjects or at a different stage of the disease, compared to applications where it is desired to inhibit viral replication of coronaviruses (e.g. SARS-CoV-2). For instance, the agents may be used to treat subjects suffering from severe or chronic disease and/or ongoing lung pathology associated with coronavirus (e.g. SARS-CoV-2) infection, rather than simply to prevent viral replication in the early stages after infection). In embodiments, the subject has, or is suspected to have coronavirus infection or coronavirus- mediated disease. In embodiments, the subject in need thereof has tested positive for a coronavirus infection. In embodiments, the subject in need thereof has symptoms of a coronavirus infection or coronavirus-mediated disease (e.g. COVID-19). The agents described herein have been advantageously demonstrated to show effects against a number of variants of SARS-CoV-2. Thus the subject to be treated may be infected with any known variant of coronavirus, e.g. of SARS-CoV-2. In particular embodiments, the subject is infected with a SARS-CoV-2 variant selected from Wuhan-Hu-1, D614G, alpha (B.1.1.7), beta (B.1.351), gamma (B.1.1.248.1) and delta (B.1.617.2). Thus the subject may be infected with a coronavirus comprising a spike (S) protein sequence as defined in any one of SEQ ID NOs: 1, 13, 14, 15 or 16, or a sequence having at least 85%, 90%, 95% or 99% sequence identity thereto. The present disclosure describes the presence of syncytia in post-mortem COVID-19 positive samples (i.e., severe COVID-19 cases). Therefore, the present invention also provides a method of treating a subgroup of subjects suffering from severe COVID-19 disease. The subjects to be treated may be suffering from severe-critical COVID-19 symptoms, and/or subjects at risk of developing severe-critical covid symptoms. Severe-critical COVID-19 status may be determined by a positive test for COVID-19 infection (such as a lateral flow test or PCR test) and/or the presence of pneumonia, acute respiratory distress syndrome (ARDS), systemic inflammation, multiple organ failure, lung thrombosis, and hospitalisation. Subjects at risk of developing severe-critical COVID-19 may be determined by a positive test for COVID-19 infection (such as a lateral flow test or PCR test) and the presence of one or more comorbidities selected from: older age (≥65), sex (male), race (Black, Hispanic or South Asian), cardiovascular diseases or conditions (including heart failure, coronary artery disease, cardiomyopathies or hypertension), diabetes (Type 1 or Type 2), chronic lung diseases (including moderate to severe asthma, chronic obstructive pulmonary disease, emphysema, scarred or damaged lung tissue, chronic bronchitis, interstitial lung disease, cystic fibrosis, and pulmonary hypertension), overweight (BMI >25 kg/m2), pregnancy, blood disorders (sickle cell disease or thalassemia), stroke, cerebrovascular disease, substance use disorder, smoking, Down’s syndrome, immunocompromised, chronic kidney disease, cancer and chronic liver disease. Because the present agents are typically administered to e.g. treat the severe lung pathology that occurs following coronavirus (e.g. SARS-CoV-2) infection, the timing of their administration may be different to the timing of administration of agents intended to prevent viral replication. For instance, the agents may be administered to the subject after viral infection, and typically after viral replication has occurred. Thus the agents may be administered to the subject to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days after viral infection. In some embodiments, the agents may be administered less than 12 months, 6 months, 3 months, 2 months or 1 month after viral infection. For instance, the agents may be administered 1-90 days, 5-60 days or 10-30 days after coronavirus (preferably SARS-CoV-2) infection. In further embodiments, the subject has one or more symptoms of COVID-19 infection including loss of smell and/or taste. It has been found that cells of the respiratory epithelium that are responsible for olfaction become infected with SARS-CoV-2. These cells express TMEM16 proteins, e.g. TMEM16A and TMEM16F, and therefore the agents described herein may be used to treat loss of smell and/or taste associated with coronavirus (e.g. COVID-19) infection. In other embodiments, the present invention provides a method of treating a post-COVID-19 syndrome, otherwise known as chronic COVID syndrome or ‘long COVID’. Such subjects may include patients that have symptoms for more than four weeks. Long COVID may be diagnosed by ongoing COVID-19 symptoms that last for four to 12 weeks. Long COVID may also refer to COVID-19 symptoms that last more than 12 weeks. Long COVID may refer to symptoms that occur without current COVID-19 infection, i.e., a subject initially tests positive for COVID-19 infection and the symptoms remain even when the subject subsequently tests negative for a COVID-19 infection. COVID-19 infection may be diagnosed by standard techniques, such as a lateral flow test or PCR. Pharmaceutical compositions of the present disclosure, can be prepared in accordance with methods well known and routinely practiced in the art (see e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co.20th ed.2000; and Ingredients of Vaccines – Fact Sheet from the Centers for Disease Control and Prevention, e.g., adjuvants, enhancers, preservatives, and stabilizers). Pharmaceutical compositions are preferably manufactured under GMP conditions. The present invention also encompasses the use of pro-drugs or derivatives of any of the compounds mentioned above, including e.g. pharmaceutically acceptable salts, esters and other modified forms thereof. The compositions described herein may be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Such formulations typically further comprise one or more physiologically or pharmaceutically acceptable carriers, excipients and/or diluents. Such excipients, carriers, diluents, and buffers include any pharmaceutical agent that can be administered without undue toxicity. Definitions As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise. The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The term also encompasses “consisting of” and “consisting essentially of”. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of and from the specified value, in particular variations of +/-10% or less, preferably +/-5% or less, more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed. Whereas the term “one or more”, such as one or more members of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any ^3, ^4, ^5, ^6 or ^7 etc. of said members, and up to all said members. The term “nucleic acid” or "polynucleotide" refers to a (e.g. polymeric) form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single- stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double- stranded form. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulphide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labelling component. Polypeptides such as anti- angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a gene product to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein. A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol.266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith- Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443- 453 (1970) Of interest is the BestFit program using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981) to determine sequence identity. The gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many embodiments will be 3. The gap extension penalty will generally range from about 0.01 to 0.20 and in many instances will be 0.10. The program has default parameters determined by the sequences inputted to be compared. Preferably, the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA. Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp.127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: Mismatch Penalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; and Joining Penalty: 30.0. "Recombinant," as used herein means that the vector, polynucleotide, polypeptide or cell is the product of various combinations of cloning, restriction or ligation steps (e.g. relating to a polynucleotide or polypeptide comprised therein), and/or other procedures that result in a construct that is distinct from a product found in nature. A recombinant virus or vector is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct. By "pharmaceutically acceptable" it is meant a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing any undesirable biological effects. Thus, such a pharmaceutical composition may be used, for example, in administering an active agent directly to a subject. The term “one or more physiologically or pharmaceutically acceptable carriers, excipients and/or diluents” as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and/or carrier is a diluent, excipient, and/or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the US and/or European Pharmacopeia or other generally recognised pharmacopeia for use in animals, including humans as well as non-human mammals. The term diluent, excipient, and/or “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluent, excipient, and/or carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions may be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and/or excipients include starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, may also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsion, sustained release formulations and the like. Examples of suitable pharmaceutical diluent, excipient, and/or carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. The formulation should suit the mode of administration. The appropriate diluent, excipient, and/or carrier will be evident to those skilled in the art and will depend in large part upon the route of administration. Pharmaceutically acceptable salts may be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7(th) ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3 rd ed. Amer. Pharmaceutical Assoc. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations may be sterilised, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilising agents in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the encapsulated or unencapsulated composition is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulphate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents. Medicaments described herein may be provided as a kit which comprises at least one container and a package insert. The container contains at least one dose of a medicament comprising a composition as described herein. The package insert, or label, comprises instructions for treating a patient using the medicaments as described herein. The kit may further comprise other materials that may be useful in administering the medicaments, such as diluents, filters, IV bags and lines, needles and syringes. As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing or reversing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions may be included to better appreciate the teaching of the present invention. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. The following examples are provided to illustrate certain embodiments of the invention. It is not intended to limit the invention in any way. EXAMPLES There is a strong interest in detecting the presence of antibodies protecting from infection in sera of patients with COVID19 or who have recovered from the disease or in whom the disease was asymptomatic. While antibodies and their titres are indicators of previous exposure to the virus, their mere presence cannot predict whether they are protective against re-exposure to the virus. The detection of neutralising antibodies thus appears essential 1) to understand the kinetics of immune response in patients and their correlation with disease progression; 2) to identify donor serum for immunotherapy; 3) to certify immunity; 4) to monitor vaccine development; 5) to monitor vaccine efficacy. At the same time, there is a strong and urgent interest in screenings for drugs that block fusion of the virus envelope with the target cells and of the infected cells with other cells (cell-fusion). Neutralising antibodies target the Spike (S) protein of the virus, while cell fusion events are based on the fusogenic activity of same protein. In the course of our current research on SARS-CoV-2, we developed two cell-based assays that are prone to automation (high throughput analysis), are based on high content microscopy and can be used systematically to detect the presence of neutralising antibodies in human and animal serum and to assess the efficacy of drugs in inhibiting formation of cell fusion (syncytia). The first assay (Cell Fusion Inhibition Assay, CFIA) is based on SARS-CoV-2 Spike-protein- mediated cell fusion. Briefly, “Donor” human U2OS cells (which do not express the viral ACE2 receptor and are not susceptible to SARS-CoV-2 infection) are transfected to transiently express the Spike protein and then co-cultured with “Acceptor” Vero cells, which supports SARS-CoV-2 replication. The donor is loaded with quantum dots (Q-dots) emitting at 525 nm (green) while the acceptor with Q-dots emitting at 800 nm (red). After 24 hr, the fused cells have 2 or more nuclei and a cytoplasm with both red and green Q-dots. Fusion events are measured using a fusogenic index based on the automated detection of cells having more than one nucleus, cytoplasm over 2 SD from the culture mean and both red and green Q-dots in the cytoplasm. In the second assay (Syncytia Inhibition Assay, SIA), the Spike protein cDNA is delivered into Vero cells, which determines the formation of massive, progressively growing syncytia based on the fusogenic property of this protein. In this case, the fusogenic index is calculated from the size of cytoplasm (as above) and number of nuclei. In both cases, the formation of Spike-mediated cell fusion is visualized by automated High Content Microscopy and is thus amenable to automation. Both assays may be used in a robotic workstation to screen for the effect of several thousands of drugs and small nucleic acids. The same assays are also used for the systematic detection of neutralising antibodies from large number of sera in a robust and automated manner. The developed assays permit automation, are quantitative, are applicable to large number of samples and are robust and highly reproducible. The assays may be applied for the detection of neutralising antibodies against SARS-CoV-2, e.g. (i) for the detection of neutralising sera for the treatment of COVID-19 disease; (ii) for the future follow up of patients vaccinated against SARS-CoV-2 (most of the currently developed vaccines are based on the Spike protein); for certifying immunity to SARS-CoV-2; and for producing antisera and purified immunoglobulins for therapy of COVID-19. EXAMPLE 1. Summary COVID-19 is a disease with unique characteristics. In addition to the clinical features common to interstitial pneumonias and other causes of acute respiratory distress syndrome (ARDS) 1, this condition is characterized by lung thrombosis 2, frequent diarrhoea 3, and a set of unexplained symptoms, such as unperceived oxygen saturation (happy hypoxia), loss of smell and taste and rapid deterioration of lung function consistent with alveolar oedema 4. These features suggest that the disease has a more complex explanation than simply death of pneumocytes caused by SARS-CoV-2 replication. While some of these findings can be attributed to abnormal activation of the inflammatory or immune response 5, the pathological substrate for this response still remains largely elusive. Here we show that the lungs of patients with COVID-19 show the long-term presence of infected pneumocytes with abnormal morphological characteristics and frequent multinucleation. Generation of these syncytia likely results from the expression and activation of the SARS-CoV-2 Spike protein on the infected cell plasma membrane. Based on these observations, we performed two high-content microscopy-based, high-throughput screenings with over ~3000 approved drugs to search for inhibitors of Spike-driven syncytia. We converged on the identification of 83 drugs that inhibited Spike-mediate cell-cell fusion, several of which belonged to defined pharmacological classes. Of these, we focussed our attention on effective drugs that also protected the cells against virus-induced death. In particular, we found that Niclosamide, the top drug in our screenings, markedly blunted calcium oscillations and chloride channel currents in Spike- expressing cells by blocking the activation of members of the TMEM16/Anoctamin family of proteins. Consistent with this observation, downregulation of the ion channel and scramblase TMEM16F blocked Spike-induced syncytia formation. Together, these findings support the possibility that COVID-19 disease is due to the prolonged persistence of virus-infected cells, suggest potential mechanisms for COVID-19 disease pathogenesis and sustain the repurposing of Niclosamide for therapy. Introduction One of the defining features of coronavirus biology is the coordinated process by which the virus binds and enters the host cell, which involved both docking to receptors at the cell surface (DPP4 for MERS-CoV 6 and ACE2 for SARS-CoV 7 and SARS-CoV28), and proteolytic activation of the Spike protein S by host encoded proteases at two distinct sites 9. The first activation step is carried out by a cellular protease that cleaves S at the S1/S2 boundary. This can occur either before or after receptor binding. The second proteolytic activation happens after a conformation change triggered by receptor engagement that exposes the S2 portion, and primes S2 for fusion of the virus envelope with the cellular membrane. The protease priming event at this S2’ site and subsequent fusion can occur after endocytosis, in which case the cleavage is carried out by endosomal low pH-activated proteases such as cathepsin B and cathepsin L 10 or at the plasma membrane level, where cleavage is by TMPRSS211-13. The S proteins of MERS-CoV and SARS-CoV-2 possess an amino acid sequence at the S1/S2 interface that is not present in SARS-CoV 14 that also allows cleavage by the ubiquitously expressed serine protease furin 15-17. As a consequence, cells expressing MERS-CoV and SARS-CoV-2 S protein at the plasma membrane can fuse with other cells expressing the respective DDP4 and ACE2 receptors and form syncytia, while this property is markedly less pronounced for S from SARS-CoV. The possible pathogenic significance of these differences has remained so far unexplored. In the case of MERS-CoV, furin cleavage at the S1/S2 region is known to promote subsequent TMPRSS2 activation-dependent viral infection 18,19. Of note, while infection with coronaviruses in cell culture can occur through endocytosis and cathepsin B/L activation, S protein priming at the plasma membrane by TMPRSS2 plays a crucial role for viral spread and pathogenesis in vivo 20-22. These observations highlight the relevance of events occurring at the plasma membrane, including formation of syncytia, for coronavirus- induced disease. Methods Patients Patients’ lung samples are from post-mortem analysis of 6 patients who died of COVID-19 at the University Hospital in Trieste, Italy after intensive care support. These are from a more extensive cohort of 41 consecutive patients, all deceases of the disease. Of these, 25 males and 16 females; the average age was 77 for males and 84 for females. During hospitalization, all patients scored positive for SARS-CoV-2 on nasopharyngeal swab and presented symptoms and imaging data indicative of interstitial pneumonia related to COVID-19 disease. Extensive characterization of these patients is reported in ref. https://medrxiv.org/cgi/content/short/2020.06.22.20136358v1. In these patients, dysmorphic and syncytial pneumocytes were present and abundant in the lungs of 20 patients (50%), including all 6 patients requiring intensive care, and occasional in additional 16 patients (39%). Use of these samples for investigation was approved by the competent Joint Ethical Committee of the Regione Friuli Venezia Giulia, Italy. Cells Vero(WHO) Clone 118 cells (ECACC 88020401) were cultured in Dulbecco’s modified Eagle medium (DMEM, Life Technologies) with 1 g/L glucose (Life Technologies) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Life Technologies) plus a final concentration of 100 IU/ml penicillin and 100 (μg/ml) streptomycin or without antibiotics where required for transfection. Cells were incubated at 37°C, 5% CO2. Vero E6 cells were grown in DMEM (Gibco) containing 10% FBS, 1% Non-essential Amino Acids (Gibco) and 1% penicillin-streptomycin (Thermo Fisher Scientific). Cells were incubated at 37°C, 5% CO2. U-2 OS (U2OS) cells (ATCC HTB-96) and HEK293T cells (ATCC CRL-3216) were cultured in Dulbecco’s modified Eagle medium (DMEM) with 1 g/L glucose (Life Technologies) supplemented with 10% foetal bovine serum (FBS) (Life Technologies) plus a final concentration of 100 IU/ml penicillin and 100 (μg/ml) streptomycin or without antibiotics where required for transfections. The stable U2OS cell clone expressing mCherry was obtained by transduction with lentiviral particles for constitutive expression of mCherry (rLV.EF1.mCherry-9, Takara 0037VCT) at 10 M.O.I with 4 µg/ml polybrene (Sigma-Aldrich TR-1003-G). The medium was replaced after 4 hr. Selection of the transduced cells was performed adding 1 µg/ml puromycin (InvivoGen ant- pr-1) starting 48 hr after transduction. Expression of the transgene was verified by fluorescence microscopy. Antibodies Antibodies against the following proteins were used: TMEM16A (Abcam, ab64085), TMEM16B (Abcam ab113443), TMEM16F (Abcam ab234422 and Sigma-Aldrich HPA038958-100UL), ACE2 (Abcam ab87436 and ab15348), TMPRSS2 (Abcam ab92323 and ab109131), α-tubulin (Sigma-Aldrich T5168), V5 (Thermo Fisher Scientific R96025), V5-488 (Thermo Fisher Scientific 377500A488), mouse-HRP (Abcam ab6789), rabbit-HRP (Abcam ab205718), β-actin-HRP (Sigma-Aldrich A5316), Napsin (Roche 760-4867), Surfactant B (Thermo Fisher Scientific MS-704-P0), mouse-Biotin (Vector Laboratories BA-9200), SARS- CoV-2 Spike protein (GeneTex GTX632604) Cell Fusion Inhibition Assay (CFIA) “Donor” U2OS cells were seeded in 10-cm Petri dishes (2 million cells per dish) to reach 70- 80% confluency on the subsequent day. Transfection was performed using 10 µg of either pEC117-Spike-V5 (expressing the V5-tagged Spike protein, codon optimised) or a control vector using 35 µl of FuGENE HD Transfection Reagent (Promega E2311) in 500 µl of Opti- MEM medium (Life Technologies). After overnight transfection, cells were loaded with 10 nM Qtracker 525 Cell Labeling Kit (Invitrogen Q25041MP) in 1ml of medium for 1 hr at 37°C in 5% CO₂. After extensive washes with 1xPBS, cells were detached with 1x Versene (Thermo Fisher Scientific 15040066). “Acceptor” Vero cells were prepared as follows. Cells were seeded in 10 cm Petri dishes (1.2 million cells per dish) the day before the assay. Cells were then loaded with 10 nM Qtracker 800 Cell Labelling Kit (Invitrogen Q25071MP) in 1 ml medium for 1 hr at 37°C in 5% CO₂. After extensive washes with PBS, cells were detached with 0.05% Trypsin-EDTA (Sigma-Aldrich T4049). Vero (1000 cells/well) and U2 OS cells (800 cells/well) were mixed at the final ratio of 5:4 and diluted at the final concentration of 20x103 cells/ml in Dulbecco’s modified Eagle medium (DMEM) with 1 g/L glucose (Life Technologies) supplemented with 10% heat-inactivated foetal bovine serum (FBS; Life Technologies). Diluted cells (50 µl/well) were seeded in twelve 384-well microplates (CellCarrierUltra 384, Perkin Elmer) using a Multidrop dispenser. Three hours after seeding, 3700 FDA approved drugs (Prestwick FDA Library (1200) and MS discovery Spectrum collection (2500) were dispensed on top of cells at the final concentration of 10 µM (1% DMSO final). Twenty-four hours later plates were washed in 50 µl/well PBS and fixed in 40 µl 4% PFA for 10 min at room temperature (RT). After fixation, cells were washed two times in 50 µl/well PBS and permeabilized in 0.1% Triton X100 (Sigma-Aldrich 1086431000) for 10 min at RT. Cells were washed two times in 50 µl/well of PBS and stained with HCS CellMask Blue (Thermo Fisher Scientific H32720) and Hoechst (H3570), according to the manufacturer’s instructions. Image acquisition was performed using an Operetta CLS high content screening microscope (Perkin Elmer) with a Zeiss 20x (NA=0.80) objective. A total of 25 fields per well were imaged at 3 different wavelengths: 1) Excitation (Ex) 365-385nm, Emission (Em) 430-500nm (nucleus and cytoplasm “Blue”); 2) Ex 460-490nm, Em 500-550 nm (Donor quantum dots “Green”); 3) Ex 615-645nm Em 655-750nm (Acceptor quantum dots “Red”). Images were subsequently analysed, using the Harmony software package (PerkinElmer). Images were first flatfield- corrected and nuclei were segmented using the “Find Nuclei” analysis module (Harmony). The thresholds for image segmentation were adjusted according to the signal-to-background ratio. Splitting coefficient was set in order to avoid splitting of overlapping nuclei (fused cells). The cytoplasm area, stained by the HCS Cell Mask Blue, was defined using the “Find Cytoplasm” analysis module (Harmony) and the number of either green or red spots was counted using the “Find Spots” analysis module (Harmony). All the cells that had a nuclear area greater than 4 times the average area of a single nucleus and were simultaneously positive for at least two red and two green dots were considered as fused. Data were expressed as a percentage of fused cells by calculating the average number of fused cells normalized on the total number of cells per well. Syncytium Inhibition Assay (SIA) Vero cells were seeded in 10-cm Petri dishes (1.2 million cells per dish) to reach 70-80% confluency on the subsequent day. Transfection was performed using 10 µg pEC117-Spike-V5 using 30 µl and FuGENE HD Transfection Reagent (Promega E2311) in 500 µl of Opti-MEM medium (Life Technologies). Twelve hours after transfection, cells were detached, washed in 1xPBS and diluted to the final concentration of 12x103 cells/ml in Dulbecco’s modified Eagle medium (DMEM) with 1 g/L glucose (Life Technologies) supplemented with 10% heat- inactivated FBS (Life Technologies). Fifty µl diluted cell suspension (600 cells/well) were seeded in twelve 384-well microplates (CellCarrierUltra 384, Perkin Elmer) using a Multidrop dispenser. Three hours after seeding, 3700 FDA/EMA approved drugs (Prestwick FDA Library and MS discovery Spectrum collection were dispensed on top of the cells at the final concentration of 10µM (1% DMSO final). At 24 hr after drug treatment, plates were washed in 50 µL/well of 1xPBS and fixed in 40 µl 4% PFA for 10 min at RT. After fixation cells were processed for immunofluorescence anti V5, Hoechst (H3570) and HCS CellMask Red (Thermo Fisher Scientific H32712), according to the manufacturer’s instructions. Image acquisition was performed using the Operetta CLS high content screening microscope (Perkin Elmer) with a Zeiss 20x (NA=0.80) objective. A total of 25 fields per well were imaged at 3 different wavelengths: 1) Excitation (Ex) 365-385nm, Emission (Em) 430-500nm (nucleus “Blue”); 2) Ex 460-490nm, Em 500-550nm (Spike protein “Green”); 3) Ex 615-645nm Em 655-750nm (HCS Cell Mask “Red”). Images were subsequently analysed, using the Harmony software (PerkinElmer). Images were first flatfield-corrected and nuclei were segmented using the “Find Nuclei” analysis module (Harmony). The thresholds for image segmentation were adjusted according to the signal-to-background ratio. Splitting coefficient was set in order to avoid splitting of overlapping nuclei (fused cells). The cytoplasm area, stained by the HCS Cell Mask Red, was defined using the “Find Cytoplasm” analysis module (Harmony) and the intensity of the green fluorescence was calculated using the “Calculate Intensity Properties” module (Harmony). All the cells that had a nuclear area greater than 5 times the average area of a single nucleus and were simultaneously positive for green signal in the cytoplasm area were considered as fused. Data were expressed as a percentage of fused cells by calculating the average number of fused cells normalized on the total number of cells per well. Immunofluorescence To stain for the V5 epitope in the Spike protein, after fixation cells were washed two times in 50 µl/well (384 well-plate) or 100 µl/well (96-well plate) of 1xPBS and then permeabilized in same volumes of 0.1% Triton X100 (Sigma-Aldrich 1086431000) for 10 min at RT. Cells were then washed two times 1xPBS and blocked in 1% BSA for 1 hr at RT. After blocking, the supernatant was removed and 20 µl/well (384 well-plate) or 40 µl/well (96-well plate) diluted (1:1000 in 1%BSA) V5 Tag Alexa Fluor 488 Monoclonal Antibody (Thermo Fisher Scientific 37-7500-A488) was added to each well and incubated at RT for 2 hr. Cells were then washed two times in 1xPBS before microscopy. To visualize the actin cytoskeleton with phalloidin, after fixation cells were washed two times in 50 µl/well (384 well-plate) or 100 µL/well (96-well plate) of 1xPBS and then permeabilized in same volumes of 0.1% Triton X100 (Sigma-Aldrich 1086431000) for 10 min at RT. Cells were then washed two times 1xPBS and blocked in 1% BSA for 1 hr at RT. After blocking, the supernatant was removed and 20 µl/well (384 well-plate) or 40 µl/well (96-well plate) diluted (1:1000 in 1%BSA) Alexa488 Phalloidin (Thermo Fisher Scientific A12379) was added to each well and incubated at RT for 1 hr. Cells were washed two times in 1xPBS before microscopy. Histology and immunohistochemistry Samples from COVID-19 autopsies were fixed in 10% formalin for at least 50 hours and then embedded in paraffin. Four-micron sections were deparaffinized in xylene, rehydrated and processed for hematoxylin-eosin or immunohystochemical stainings. Antigen retrieval was performed in boiling sodium citrate solution (0.01 M, pH 6.0) for 20 min. Sections were allowed to cool down and permeabilized for 10 min in 1% Triton X-100 in 1xPBS, followed by blocking in 2% BSA (Roche) and overnight staining at 4°C with the primary antibodies diluted in blocking solution. After endogenous peroxidase inhibition with 3% H2O2, sections were incubated with appropriate biotin-conjugate secondary antibody for 1 hr at room temperature. Following signal amplification with avidin–biotin-complex–HRP (Vectastain), DAB solution (Vector) was applied for 2-3 min. Haematoxylin (Bioptica) was further used to stain nuclei and Bluing reagent was used on Ventana automated stainings. Images were acquired using a Leica ICC50W light microscope. In situ hybridization In situ hybridization (ISH) was performed using locked nucleic acid (LNA) probes for U6 snRNA 15 and SARS-CoV-2 RNA, designed to target the sense strand of ORF1ab and Spike regions of the viral genome. Scrambled sequences were used as control. Experiments were performed using a dedicated ISH kit for Formalin-fixed paraffin-embedded (FFPE) tissues (Qiagen) according to the manufacturer’s protocol. Briefly, FFPE tissue slides were deparaffinized in xylene, treated with proteinase-K (15 µg/ml) for 5 min at 37°C and incubated with either SARS-CoV-2 (40 nM) or U6 probes (2 nM) for one hour at 54°C in a hybridizer. After washing with SSC buffer, the presence of SARS-CoV-2 RNA was detected using an anti- DIG alkaline phosphatase (AP) antibody (1:500) (Roche Diagnostics) supplemented with sheep serum (Jackson Immunoresearch) and bovine serum albumin (BSA). Hybridization was detected by adding NBT-BCIP substrate (Roche Diagnostics). Nuclei were counterstained with nuclear fast red. Plasmids The expression plasmid pEC117-Spike-V5 was generated as follows: the SARS-CoV-2 wild type protein (NCBI accession number NC_045512.2, position 21563-25384) was codon- optimised and synthesized in two fragments of approximately 2 kb each as gBlock DNA fragments (IDT Integrated DNA Technologies) with the in-frame addition of the V5 tag at the C-terminus, and then cloned into the pZac 2.1 backbone under the control of the cytomegalovirus (CM) IE promoter. The lentiviral plasmid pEC119-S-protein-BSD was generated by cloning the S protein in the pWPI-BLR, a derivative of the bicistronic lentiviral vector pWPI (Addgene 12254) harboring the blasticidin resistance gene. The construct DNA sequences were verified by Sanger sequencing. Expression vectors for hACE2 (Addgene 1786) and TMPRSS2 (Addgene 53887) were obtained from Addgene, pGCaMP6S (Addgene 40753) was provided by J. Burrone (King’s College London), pCMV-EGFP by L. Zentilin (ICGEB, Trieste, Italy) and the ClopHensor (Arosio D et al., Nat Methods.2010 Jul; 7(7):516-8) by D. Arosio (IBF-CNR Trento, Italy). Pseudotyped viral particle production The expression plasmid pEC120-S-D19-V5 lacking the SARS-CoV-2 ER retention signal and harboring the V5 tag at the C-terminus was generated by PCR with the following primers:

and cloned into the pEC117-S-protein-V5 vector. The construct was verified by Sanger sequencing. Pseudotyped viral particles were produced as follows: HEK293T (2.5 x 106 cells) were seeded in a 100 mm-dish and co-transfected with 10 µg pLVTHM/GFP (Addgene 12247), 7.5 µg psPAX2 (Addgene 12260), and 6 µg pMD2.G (Addgene 12259) or pEC120-S-D19-V5 for a total of 13.5 µg for each dish. Transfection was performed with FuGENE HD (Promega) as indicated by the manufacturer. Forty-eight hr post-transfection, the medium was collected and centrifuged at 3000 rpm for 5 min. The supernatant was then filtered with a 0.45 nm pore- size filter, aliquoted and stored at -80°C. Phosphatidylserine (PS) externalisation assay Vero(WHO) Clone 118 cells were seeded in 96-well microplates with a flat and clear bottom (CellCarrierUltra 96, Perkin Elmer) at a density of 6500 cells/well. After 2 hr, cells were treated with different drug concentrations. One hr after treatment, cells were washed once with 1xPBS and once with Annexin V binding Buffer (140 mM NaCl, 10 mM Hepes, and 2.5mM CaCl₂, pH 7.4) at RT and then incubated with 100 µl 1:100 Annexin V-Alexa Fluor 488 (Abcam ab219904) and with 1:5000 propidium iodide (PI; Sigma-Aldrich P4864) for 15 min at RT, with or without 5 µM ionomycin. At the end of the incubation period, 50 µl were removed from each well and 250 µl Annex V binding buffer were added. Cells were then immediately imaged by fluorescence microscopy using an Operetta CLS microscope mounting a 20x NA1.1 lens. Nine images/well were automatically acquired at 3 different wavelengths: 1) Ex 460- 490nm, Em 500-550nm (AnnexinV “Green”); 2) Ex 530-560, Em 570-650nm (PI “Red”); 3) Brightfield. The sum of the area covered by the green fluorescence was measured. Analysis of cell viability Cell viability was assessed using the ATP light assay. Cells were seeded in 96-well microplates with a white opaque bottom (CulturePlate96, Perkin Elmer) at a density of 6500 cells/well. After 12 hr, cells were treated with different drug concentrations. At 24, 48 and 72 hr after treatment, plates were processed using the 1Step ATP-light kit (Perkin-Elmer 6016736) according to the manufacturer instruction. Luminescence levels were measured using an Envision 2015 Multimode Plate Reader (Perkin-Elmer). Calcium imaging Cells were seeded in 10-cm Petri dishes (1.2 million cells per dish) to reach 70-80% confluency on the subsequent day. Co-transfection was performed using 5 µg pGCaMP6S and 5 µg either pEC117-Spike-V5 or an empty pZac 2.1 vector using 30 µl FuGENE HD Transfection Reagent (Promega #E2311) in 500 µl of Opti-MEM medium (Life Technologies). Sixteen hr after transfection, cells were detached, washed in 1xPBS and diluted to the final concentration of 6x104 cells/ml in Dulbecco’s modified Eagle medium (DMEM) with 1 g/L glucose (Life Technologies) supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Life Technologies). A total of 6000 cells/well (100 µl) were seeded in 96-well microplates with a flat and clear bottom (CellCarrierUltra 96, Perkin Elmer). Three hours after seeding, cells where treated with either 1 µM Niclosamide (EP #N0560000), 5 µM Clofazimine (EP #Y0000313), 5 µM Salinomycin (SIGMA #S4526-5) or 1% DMSO. Image acquisition was performed using the Operetta CLS high content screening microscope (Perkin Elmer) with a Zeiss 20x (NA=0.80) objective at 37°C and 5% CO2. A total of 3 fields per well were imaged every 2 minutes at Ex 460-490 nm, Em 500-550 nm (GCaMP6S sensor “Green”). Images were subsequently analysed with the ImageJ software. For each frame, fluorescence intensity (F) was subtracted from the same frame in the preceding acquisition to remove the background signal and to calculate ΔF/F0 index, where F0 is the median baseline fluorescence and ΔF = F−F0. For intensity analysis, more than twelve 180-µm2 Regions Of Interest (ROI) per condition were considered, compiling a total of at least 50 GCaMP6S- positive cells per condition. For each ROI, positive ΔF values (i.e. increase) were taken into account with the BAR script “Find peaks”. Among these, values outside 1.5 times the interquartile range above the upper quartile were considered as “spikes”. Single cell spike frequency was then counted for the whole observation period. siRNA transfection The siRNAs (Dharmacon siGENOME SMARTpools, 4 siRNAs per gene target) targeting ACE2 (M-005755-00-0005), ANO1/TMEM16A (M-027200-00- 0005), TMEM16B (M- 016745-01- 0005) and TMEM16F/ANO6 (M-003867-01- 0005) were dispensed onto the bottom of 96-well microplates (CellCarrierUltra 96, Perkin Elmer); siRNA buffer and a non- targeting siRNA were used as controls. Briefly, the transfection reagent (Lipofectamine RNAiMAX, Life Technologies) was diluted in Opti-MEM (Life Technologies) and added to each siRNA in the microplate array. Thirty min later, 6.5×103 Vero cells or 8×103 HEK293T cells were seeded in each well. All siRNAs were tested at different concentrations ranging from 12.5 nM to 50 nM. Twenty-four hr after siRNA transfection, 100 ng of either pEC117-Spike- V5 or pCMV-EGFP expression plasmid was transfected using a standard forward transfection protocol. Briefly, pDNA was diluted in Opti-MEM (Life Technologies), mixed with the transfection reagent (FuGENE HD, Promega) using the following ratios: 1 µg pDNA:3µl FugeneHD. The mix was incubated 20 min at RT and added to the siRNA-transfected plates. After 24 hr, cells were fixed in 4% PFA and processed for immunofluorescence. For gene silencing experiments, the indicated siRNAs were transfected into Vero/HEK cells using a standard reverse transfection protocol, at a final concentration of 25 nM. Briefly, the transfection reagent (Lipofectamine RNAiMAX, Life Technologies) was diluted in Opti-MEM (Life Technologies) and added to the siRNAs arrayed in 12-well plates; 30 min later, ~200- 100×103 cells were seeded per well. After 48-72 hr, cells lysates were analysed by qPCR and/or western blotting, as detailed later. RNA extraction and real-time PCR Total mRNA was isolated from HEK293T or Vero cells 48-72 hrs after siRNA transfection using a standard Trizol RNA extraction protocol. The RNA obtained (1 μg) was reverse- transcribed using MLV-RT (Invitrogen) with random hexameric primers (10 μM) in a 20-μl reaction following the manufacturer’s instructions. Quantification of TMEM16A (Hs00216121_m1), TMEM16B (Hs00220570_m1), TMEM16E (Hs01381106_m1) and TMEM16F (Hs03805835_m1) gene expressions was performed by quantitative Real-Rime PCR using Taqman probes. Expression of the housekeeping gene GAPDH (Hs02786624_g1) was used for normalization. Amplifications were performed using a QuantStudio3 machine using TaqMan™ Gene Expression Master Mix (Applied Biosystems 4369016). The Real-Time qPCR profile was programmed with a standard protocol, according to the manufacturer’s instructions. Western blotting and immunoprecipitation After 48-72 hr siRNAs transfection, HEK293 cells were harvested and homogenized in RIPA lysis buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate supplemented with protease inhibitors (Roche)) for 10 min at 4 °C and sonicated by using Bioruptor (Diagenode) for 30 min. Equal amounts of total cellular proteins (15-20 μg), as measured with the Bradford reagent (Biorad), were resolved by electrophoresis in 4-20% gradient polyacrylamide gels (Mini-PROTEAN, Biorad) and transferred to nitrocellulose/PVDF membranes (GE Healthcare). Membranes were blocked at RT for 60 min with PBST (PBS + 0.1% Tween-20) with 5% skim milk powder (Cell signalling, 9999). Blots were then incubated (4 °C, overnight) with primary antibodies against ACE2 (diluted 1:1000), TMPRSS2 (diluted 1:500), TMEM16F (1:1000), α-tubulin (diluted 1:10000), or TMEM16F (diluted 1:500), TMEM16A (diluted 1:500), TMEM16B (diluted 1:1000) and TMEM16E (diluted 1:500). Blots were washed three times (5 min each) with PBST. For standard Western blotting detection, blots were incubated with either anti-rabbit HRP- conjugated antibody (1:5000) or anti-mouse HRP-conjugated antibody (1:10,000) for 1 hr at RT. After washing three times at RT with PBST (10 min each), blots were developed with ECL (Amersham). For immunoprecipitation, anti-V5 agarose beads (S190-119, Thermo Fisher Scientific) or Protein A-magnetic beads (10002D, Thermo Fisher Scientific) were conjugated with the indicated antibodies according to the manufacturer's instructions. Cell pellets were lysed in lysis buffer (Cell Signaling 9803S) containing protease inhibitors (Roche). Protein A magnetic beads-conjugated antibodies were incubated 4 hr at 4°C with cell extracts (1 mg). After incubation, beads were washed 5 times with lysis buffer, then processed for SDS-PAGE and western blotting using the indicated antibodies. The immunocomplexes were analysed by western blotting using the indicated antibodies. Electrophysiology Whole cell patch clamp recordings were carried out on HEK293 cells grown on glass cover slips coated with poly-L lysine. Following plating, cells were transfected the following day with either EGFP or a combination of EGFP, Spike and Ace-2. Recordings were typically carried out 12-36 hours after transfection. Whole-cell recordings were performed in an HBS extracellular solution (pH 7.3, 315 mOsm) that contained (in mM): 139 NaCl, 2.5 KCl, 10 HEPES, 10 Glucose, 1.3 MgCl2 and 2 CaCl2. Patching electrodes were made from thick- walled borosilicate glass (O.D: 1.5mm, I.D: 0.86 um, Sutter Instruments) obtaining a resistance between 3-4 M^ and filled with an intracellular solution containing (in mM): 130 CsCl, 10 HEPES, 10 EGTA, 1 MgATP, 1 MgCl2 and free Calcium adjusted to 28 micromolar using CaCl2 according to calculations using MAXchelator software. Solution was adjusted to a pH of 7.4 using CsCl and osmolarity to 290 mOsm. Recordings were obtained with a Multiclamp 700B amplifier (Molecular Devices) and digitised with the Digidata 1440A digitizer (Molecular Devices). Data was acquired with the software Clampex (Molecular Devices) and Axon Multiclamp Commander Software (Molecular Devices). Signals were sampled at 5 kHz and filtered at 2.5 kHz. Pipette offsets were nulled before seal formation and pipette capacitance was compensated in the cell-attached configuration once a giga-seal was obtained. We used responses to hyperpolarising and depolarising steps to estimate the series resistance (Rs) of the recording, the membrane resistance (Rm; from the steady holding current at the new voltage) and membrane capacitance (Cm; from the area under the exponentially decaying current from peak to holding). Recordings were excluded if access resistance exceeded 15 MΩ and if input resistance was lower than 150 MΩ. All recordings were carried out at room temperature. Cells were held at 0 mV and ramps consisted of either a short 20 ms step to -80 mV followed by 500 ms ramp from -80 to +80 mV or a longer 300 ms step to -80 mV followed by 500 ms ramp from -80 to +80 mV. Data was pooled from both as there was no significant difference in the current responses. To remove the leak current from the ramp protocols, a line was fit to the current measured from -80 to -20 mV and subtracted from the entire curve. The resulting ramps showed clearly identifiable outwardly rectifying currents that reversed at 0 mV. Analysis was carried out using custom written routines in MATLAB and statistics was performed on Prism (GraphPad, USA). SARS-CoV-2 infection SARS-CoV-2/England/IC19/2020 (IC19) was isolated on Caco2 cells from a clinical sample collected from a patient admitted to St. Mary’s Hospital in London, United Kingdom. For the drug inhibition assays, drugs were diluted in DMSO and added to DMEM with 1% penicillin- streptomycin and 0.3% BSA fraction V (Thermo Fisher Scientific). Vero E6 cells were preincubated with drugs for 2 hr. Virus (100 or 200 TCID50) was added and left for 1 hr and either left on the cells for the duration of the experiment or removed and replaced with media containing drugs. Viral titre was measured using TCID50. Cell survival was measured by fixing the cells in 4% PFA, followed by quantification, using HC microscopy, of the total cell area/well using DPC (Digital Phase Contrast) imaging. Results Post-mortem analysis of lungs from COVID-19 patients reveals the presence of numerous pneumocyte syncytia. We examined the organs of 41 consecutive patients who died from COVID-19 in the period March-May 2020 at the University Hospital in Trieste, Italy. Detailed post-mortem analysis of these patients is reported elsewhere (https://medrxiv.org/cgi/content/short/2020.06.22.20136358v1). Histological observation of the lungs revealed diffuse alveolar damage (DAD), with significant alveolar oedema and intra- alveolar fibrin deposition. The vast majority of patients showed massive thrombosis of the small and large pulmonary vessels, with signs of different thrombus organization, indicative of an endogenous thrombotic process in the lung (https://medrxiv.org/cgi/content/short/2020.06.22.20136358v1). In addition to these disease features, we noticed that a peculiar finding in COVID-19 lungs was the presence, in the damaged alveolar spaces of almost 90% of patients, of atypical cells with the characteristic of syncytia, showing a large cytoplasm containing a variable number of nuclei, ranging from 2 to over 20 (shown in Fig.1a for three different patients). Most of these syncytial cells were bona fide pneumocytes, as they expressed two pneumocyte-specific markers (napsin and surfactant B; Fig.1b). In situ hybridisation with two LNA-modified RNA probes for the SARS-CoV-2 genome revealed cytoplasmic positivity, indicating that these syncytia were infected by the virus (Fig. 1c, panels c-f at different magnifications). Lung samples from individuals who died in 2018 for other causes served as a negative control for this analysis, while a positive control was hybridization with a probe for the ubiquitous U6 RNA, as per our previous experience 23, which showed nuclear localization in all cells (shown for one of each controls in Fig.1c, panels a and b respectively). Fusogenic properties of the SARS-CoV-2 Spike protein The presence of fused cells in the lungs of COVID-19 patients likely results from the fusogenic activity of the SARS-CoV-2 S protein. In vitro, we observed that the expression of a codon- optimised S protein cDNA in African green monkey (AGM) Vero cells, which express ACE2 receptors and are permissive to SARS-CoV-2 infection, leads to the conspicuous presence of syncytia at 24 hr after transfection (Fig. 1d). We found that the presence of S on the plasma membrane could also trigger fusion of heterologous cells when these express the ACE2 receptor. The effect of co-culturing, for 12 hr, S-transfected human U2OS osteoblastoma cells (which have undetectable levels of ACE2 and do not undergo fusion) with EGFP-transfected Vero cells, which express ACE2, was studied. Progressive cell fusion continued in the 12-hr observation period. At the end of the experiment, large, heterologous syncytia were clearly visible (Fig.1e panel b). Fusion strictly depended on SARS-CoV-2 S expression in U2OS cells (Fig. 1e panel a). No formation of syncytia was observed when SARS-CoV S protein was expressed, consistent with the observation that the S protein of this virus is not pre-activated for fusion at the plasma membrane level (not shown). At higher magnification, we observed that S-expressing Vero cells and S-positive syncytia projected a remarkable number of plasma membrane protrusions, showing filopodia extensions, as recently shown in wild-type virus infection 24, and contacting the plasma membrane of neighbouring, non-S-expressing cells (Fig. 1f). These S-expressing cells and syncytia displayed sudden calcium (Ca2+) transients inside the cells (cf. later). A 12-hr, time- lapse movie of Vero cells expressing the S protein and transfected with the GCaMP6s fluorescent Ca2+ sensor was produced. Individual frames from this movie, along with the indication of the cells undergoing progressive fusion into the syncytium main body, are shown in sequence in Fig.1g. Screening for FDA/EMA-approved drugs that inhibit Spike-mediated heterologous cell fusion We wanted to screen for clinically approved small molecules that inhibit SARS-CoV-2 Spike protein-mediated cell-cell fusion. We developed two assays that are amenable to high throughput screening (HTS) using High Content (HC) imaging. The first assay is based on the co-culture, for 12 hr, of Vero cells with U2OS cells transiently transfected to express Spike. To permit quantitative assessment of cell fusion by HC microscopy, the Spike “donor” U2OS cells were loaded with quantum dots (Q-dots) emitting at 525 nm (green) while the “acceptor” Vero cells with Q-dots emitting at 800 nm (red; scheme in Fig. 2a). Heterokaryons are identified as cells having more than two nuclei and carrying both red and green Q-dots in their cytoplasm - the basal level of cell fusion in the co-cultures is almost undetectable (Fig. 2b). This heterologous cell fusion assay permits quantification by HC image analysis, through the automatic detection first of cell boundaries and then of the presence of the Q-dots of different colours (Fig. 6a). The assay has >5-fold dynamic range; Fig. 2c) and thus is amenable to perform automated screenings for inhibitors. Using such Cell Fusion Inhibition Assay (CFIA), we screened two FDA/EMA-approved drug libraries, The Spectrum Collection, MS Discovery System, Inc. and the Prestwick Chemical Library, Prestwick Chemical; 2545 and 1280 individual small molecules respectively. As 776 drugs are common in the two libraries, this amounts to 3049 different molecules. Screenings were performed in 384-well plates by co-plating Q-dot loaded “donor” and “acceptor” cells, followed by drug treatment at 10 µM standard concentration. After 24 hours, cells were fixed and processed by HC microscopy. Multiple control with no drugs were included in each plate. The cumulative results of the screening are shown in Fig. 6b for the two libraries, showing total number of cells and the percentage of fusions per well; drugs reducing the total number of cells by more than 2 standard deviations from the mean of all the tested drugs were not included in the subsequent analysis. The distribution of the results for each of the two individual libraries is shown in Fig. 6c. To permit comparison, z scores were then evaluated. In the Prestwick collection, 34 drugs inhibited S-mediated fusion with a z score <-1.96 (0.025% tail of the distribution), of which 8 with a z score of <-2.58 (0.005% tail; shown in red in Fig. 2d). In the MS Discovery Spectrum collection, 54 drugs performed at a z score <-1.96 of which 14 with a z score of <-2.58. All untreated controls for both libraries had a z score in the ± 0.40 range. For each of the 803 common drugs, the correlation between the z scores in the two screenings is shown in Fig. 6d (P<0.0001; R2=0.22). The results of this screening are collectively discussed later. Screening for FDA/EMA-approved drugs inhibiting syncytium formation The CFIA assay scores for the effect of drugs on the fusion of heterologous cells and detects inhibitors that block fusion of as few as 2 cells. We also wanted to test the same drug libraries in a screening aimed at assessing the inhibition of larger syncytia. For this purpose, we transiently transfected the S protein directly in Vero cells and screened for the inhibition of syncytia formation (Syncytium Inhibition Assay, SIA; Fig.2e). At 15 hr after S transfection, cells were incubated with the drugs for additional 24 hr and then imaged. For automated HC quantification, we took advantage of the property of S-transfected cell nuclei to cluster together at the centre of the syncytium (cf. Fig. 1d panel b and Fig. 1f panel b); we thus defined as a syncytium a group of nuclei with an area ≥5 times larger than the average of that of the nuclei of non-fused cells (Fig.2f). As for the previous screening, drugs reducing the total number of cells by more than 2 standard deviations from the mean of all the tested drugs were not included in the subsequent analysis (shown in Fig.7a for the two libraries). Based on these criteria, the screening revealed that 57 drugs from the Prestwick library and 84 drugs from the MS Discovery library inhibited Spike-mediated fusion with a z score <-1.96 (0.025% tail of the distribution), of which 37 and 47 respectively with a z score of <-2.58 (0.005% tail; show in red in Fig.2g). All DMSO-treated (Controls) wells for both libraries had a z score in the ± 0.25 range. The distribution of the results for each of the two individual libraries is shown in Fig. 7b. As for CFIA, there was a statistically significant correlation of effect for the 803 common drugs in the two libraries (Fig. 7c; P<0.0001; R2=0.18). Representative images from the screening are shown in Fig.2h for Niclosamide, the top hit in the Prestwick library and second one in the MS Discovery library, and in Fig. 3a for other effective drugs, including Clofazimine, Deptropine, Salinomycin and Sertraline, along with two other drugs that have been previously considered for COVID-19 treatment based on in vitro testing (Chloroquine and Ivermectin), which were less effective on syncytia formation. The heterologous cell fusion assay and the homologous syncytium assay are intrinsically different, as the former tests for plasma membrane fusion events between single cells while the latter scores for larger syncytia formed by at least 5 different nuclei. In addition, in CFIA, the S protein is expressed by non-permissive human U2OS cells while in SIA S is produced by permissive Vero cells, which also express ACE2 and proteases. Thus, the two assays provide complementary but not necessarily overlapping information. Thus, the correlation between the efficacy of the individual drugs in the two assays was statistically significant (P<0.0001; R2=0.10) but only partial (Fig.7d). We analysed the results of the SIA screening according to therapeutic drug classes. Antipsychotics, antidepressants and H1 histamine receptor antagonists all had a trend towards exerting a negative effect on S-mediated cell fusion (Fig.3b), possibly indicative of a common mechanism of action on syncytia formation. Other interesting categories were the antifungal drugs of the imidazole class, which tended to increase the frequency of syncytia, and cardiac glycosides, all of which instead exerted an inhibitory effect. Of these, Digitoxin, Ouabain, Lanatoside C and Digitoxigenin were all in the 0.025% tail of the distribution when used at 10 µM in the screening. A few drugs were found to increase syncytia formation. These included, among others, Econazole and Mebendazole from the SIA screening. The function of these drugs on syncytia formation deserves future investigation. Dose-response curves for syncytia inhibition for three selected drugs that were further investigated in this study (Niclosamide, Clofazimine and Salinomycin) are shown in Fig. 8a and 8b for Vero and HEK293 cells respectively. Effect of shortlisted drugs on SARS-CoV-2 infection We focused our attention on the inhibitory drugs. The drugs effective in CFIA and SIA with a z score <-2.58 from the two libraries are shown in the Venn diagram in Fig.3c. The two assays cumulatively identified 83 drugs, of which 9 were common in both screenings. The choice for further studies took in consideration the following criteria: 1) common inhibitory effect in the two screening assays and from both libraries (when duplicated); 2) relative efficacy within specific drug classes, 3) pharmacological suitability to clinical application. Based on these criteria, we started to assess the effect of 43 drugs, among the 83 selected, on wild type SARS- CoV-2 infection (drug list in Fig. 9a). Vero E6 cells were infected with SARS-CoV-2 strain IC19/2020 (100 TCID50 per well) in the presence of 10 µM drugs in 96-well plates. The ability of drugs to protect cells from viral cytopathic effects was analysed after 5 days without drug re-administration (Fig. 9b). Of the drugs that were the most effective, we selected one of the anti-histaminic (Deptropine), one of the antidepressants (Sertraline) and Clofazimine for further studies. Niclosamide and Salinomycin resulted cytotoxic in the absence of the virus at 10 µM but were also shortlisted as these were among the most effective in our screenings. The five selected drugs were further assessed in a dose-response study. Cells were challenged with the same amount of virus (100 TCID50 per well) in the presence of a range of drug doses, followed by the analysis of cell survival after 5 days. Representative raw cell survival images are shown in Fig. 9c; quantification is in Fig. 4a. Niclosamide and Salinomycin protected against virus-induced cell lysis across concentrations from 5 µM to 32 nM for Niclosamide, from 10 µM to 125 nM for Salinomycin. Clofazimine showed a dose dependent inhibition with IC50 ~1.3 µM. Sertraline and Deptropine showed no protective effect below 2.5 µM. Based on these results, we focussed our attention on Niclosamide, Clofazimine and Salinomycin and tested whether the three drugs exerted a direct antiviral effect. Cells were infected with SARS-CoV-2 (200 TCID50) in 24-well plates in the presence of 2.5 µM Niclosamide, 5 µM Salinomycin or 10 µM Clofazimine in triplicate; at different times after infection (24-96 hr) the amount of virus released in the culture supernatants was measured by TCID50. We found that Clofazimine suppressed viral replication at all time points, while Niclosamide and Salinomycin significantly slowed virus replication showing significant decrease in virus yield until 72 hr post-infection (>13- and >63-fold reduction for Niclosamide and Salinomycin at 48 hr respectively; Fig.4b). Finally, based on the 48 hr-post-infection time point, we assessed virus production upon cell treatment with different drug doses. All three drugs conferred antiviral activity at the highest concentrations tested (10 µM, 5 µM and 2.5 µM for Clofazimine, Salinomycin and Niclosamide respectively; Fig.9d). Mechanisms of action of drugs blocking syncytia formation We wanted to understand the mechanism(s) by which the selected drugs inhibit syncytia formation. We were intrigued by the observation that our assays had selected for specific drug classes. In particular, there were 11 antipsychotics, 8 antidepressants and 5 first-generation histamine 1 (H1) receptor antagonists among the highly statistically effective drugs cumulatively. A characteristic common to these drugs is to regulate intracellular Ca2+ levels. The H1 receptor, M1, M2 and M5 muscarinic receptors, the 5-HT2 serotonin receptors, all of which are known to be expressed in respiratory epithelial cells, signal through a Gq ^ subunit to activate PLCß, which in turn hydrolyses PIP2 to generate Ins(1,4,5)P3 (IP3). IP3 acts as a second messenger to activate Ca2+ release into the cytoplasm from the endoplasmic reticulum (ER) stores upon binding to IP3R 25. Besides H1R antagonists and anticholinergics, phenothiazine antipsychotics and other tricyclics also act, to a different extent, on these receptors. In addition, phenothiazines and other related antipsychotics inhibit the effects of the Ca2+-binding protein calmodulin 26. Cyclosporin A forms a complex with cyclophilin, which, in platelets, inhibits Store-Operated-Ca2+ Entry (SOCE) into the cells through Ca2+release- activated Ca2+ (CRAC) channels 27. Finally, the L-type Ca2+ channel blockers and most of the tricyclic antidepressants 28 potently inhibit the voltage-dependent L-type calcium channels, which are also expressed in epithelial cells, including HEK293 cells 29. Based on these considerations, we wanted to explore the dynamics of Ca2+ levels in cells expressing S and during cell-cell fusion. We started by performing time-lapse imaging of Vero cells expressing the genetically encoded calcium indicator GCaMP6s30, co-cultured with U2OS cells expressing S and the mCherry fluorescent protein. We observed that the S-mediated fusion events correlated with numerous oscillations in intracellular Ca2+ levels. A movie of these events was produced; individual frames from this movie are in Fig. 5a. To obtain a quantitative measurement of these Ca2+ oscillations, we expressed GCaMP6s in Vero cells, with or without the S protein, and imaged these cells for 400 min. Over this period, the cells expressing S progressively fused and these events were again accompanied by frequent Ca2+ oscillations (individual frames of movie in Fig. 5b). The calcium traces for over 50 cells (or syncytia in the case of fused cells) are overlaid for each condition (Control or Spike; Fig. 5c, upper two panels) to show the remarkable heterogeneity of spontaneous Ca2+ responses in these cells. The presence of the S protein significantly increased the amplitude of Ca2+ transients in individual cells (mean peak level per cell ±SEM: 2.22±0.13 in untreated cells vs. 3.12±0.17 in S-expressing cells; P<0.01; Fig. 10a), without a significant difference in frequency, suggesting the expression of S acts to amplify spontaneous Ca2+ transients. To study the effect of Niclosamide, Clofazimine and Salinomycin on these Ca2+ events, we performed the same experiment in the presence of 1 µM Niclosamide and 5 µM of the other two drugs. We found that both Niclosamide and Clofazimine markedly blunted these Ca2+ oscillations in both control and S-treated cells (Fig. 5c). In particular, Niclosamide markedly reduced both the amplitude and frequency of Ca2+ transients in both control and S-expressing cells (P<0.01; Fig. 5c and Fig. 10b respectively), while Clofazimine had a significant effect only in S-expressing cells. Salinomycin, while still inhibiting syncytia formation, did not affect Ca2+ transients in these experiments. Together, these results suggest that whereas Niclosamide can strongly block spontaneous calcium transients, Clofazimine appears to exclusively block the amplification of Ca transients by S. Next we wanted to understand whether the suppression of Ca2+ oscillations might be causally involved in the inhibition of syncytia by which Niclosamide and Clofazimine had originally been selected. In this respect, we were intrigued by the observation that Niclosamide was recently identified as a potent inhibitor of the Ca2+-activated TMEM16/Anoctamin family of chloride channels and scramblases 31. This is family of 10 proteins sharing a common multi- pass transmembrane organisation, which were independently discovered in 2008 by three different groups as the long sought Ca2+-dependent chloride channel (reviewed in refs.32-34). We thus wondered whether the S protein and/or the selected drugs modify the activity or levels of TMEM16 proteins. The levels of TMEM16A and TMEM16B (the two main pure chloride channels in the family) as well as those of TMEM16F (which also acts as a scramblase responsible for phosphatidylserine - PS - externalisation on the outer leaflet of the plasma membrane35) remained unchanged in both Vero and HEK293 cells in response to S (not shown). Since TMEM16F is highly expressed in both HEK293 and Vero cells (Fig. 11a) we performed whole-cell voltage-clamp recordings of HEK293 cells to measure the endogenous currents carried by this channel. We measured small but clearly detectable outwardly rectifying currents in response to voltage ramps. This current was insensitive to CsCl (included in the intracellular solution), required high intracellular calcium (40 µM), was blunted by the targeted knockdown of TMEM16F by RNA interference (Fig. 10c) and blocked by the TMEM16F inhibitor benzobromarone (10 µM; Fig. 10c; P<0.01). Taken together, these results suggest these currents reflect TMEM16F activity. We found that, whereas acute administration of Niclosamide (2 µM) readily blocked this current (Fig.5d; P<0.01), both Clofazimine (5 µM) and Salinomycin (5 µM) had no significant effect (Fig. 5d), suggesting that modulation of TMEM16F activity by these drugs may be achieved by both direct and indirect routes. For example, although Niclosamide may be blocking TMEM16F activity directly, Clofazimine may be acting through the modulation of cytoplasmic Ca2+ rises to indirectly alter its activity (Fig. 5d). Importantly, we found that HEK293 cells expressing S and ACE2 showed an increased current in response to voltage ramps, which was significantly different when tested at +80 mV (Fig.5e; P<0.05). This finding is consistent with the activation of the ion channel activity of TMEM16 proteins by the S protein and is likely to act synergistically with the increases in calcium transients described above (Fig.5c). These results indicate that SARS-CoV-2 S leads to the activation of TMEM16 proteins and that Niclosamide inhibits this activity. We thus wanted to explore the relationship between this effect and the suppression of syncytia. Besides acting as an ion channel, TMEM16F is a main cellular Ca2+-activated scramblase. By studying reactivity of Vero cells to fluorescent Annexin V, we found that a 1 hr treatment with either Niclosamide and Clofazimine (but not Salinomycin) significantly reduced the levels of PS present on the outer leaflet of the plasma membrane in both basal conditions and upon treatment with 5 µM ionomycin, which increases intracellular Ca2+ levels (representative images in Fig.5f and quantification in Fig.5g). Over 95% of cell nuclei remained negative to propidium iodide in these experiments, ruling out the induction of cell apoptosis. Consistent with the possibility that the S protein activates TMEM16F, several S-induced syncytia exposed PS on their plasma membrane (shown in Vero cells co-transfected with S and a nuclear mCherry protein to visualize syncytia in Fig.5h). The observation that Niclosamide and Clofazimine blocks both cell-cell fusion and PS externalization and that Niclosamide acts as an inhibitor of TMEM16 proteins pointed to a specific involvement of TMEM16F in syncytia formation. To confirm the involvement of TMEM16F in cell-cell fusion driven by SARS-CoV-2 S protein, we knocked down, by RNA interference, ACE2, TMEM16A, TMEM16F and the XKR8, a major scramblase involved PS externalization during apoptosis 36 in both Vero and HEK293 cells. Effective knock-down of ACE2 and TMEM16F was verified by RT-PCR in both cell lines (Fig. 11b and 11c for HEK293 and Vero cells respectively) and also by western blotting in HEK293 cells (Fig.11d). We found that the downregulation of TMEM16F markedly blunted syncytia formation in cells expressing SARS-CoV-2 S, to an extent similar to that of the anti-ACE2 siRNA (representative images shown at two magnifications in Fig.5i and quantification in Fig.5j for Vero cells). The downregulation of XKR8 showed no effect, denoting a specific involvement of TMEM16F. An analogous inhibitory effect of TMEM16F knock down on syncytia formation was observed in HEK293 cells (Figs. 12a and 12b). Thus, TMEM16F is required for the formation of S- stimulated cell-cell fusion and formation of syncytia. Discussion A remarkable property of the SARS-CoV-2 S protein is the efficient cleavage of its S1 and S2 subunits by proprotein convertases such as furin, which enhances cell entry by facilitating the downstream events that lead to fusion between virus and cell membrane. While this might have conferred the virus a selective advantage and permitted its adaptation to humans 16, a consequence of this feature is the potentiation of the fusogenic capacity of SARS-CoV-2 S protein when present on the membrane of infected cells, which is far superior to that of SARS- CoV 37. Our findings indicate that this fusogenic capacity of SARS-CoV-2 i) correlates with the presence of fused cells and syncytia in the lungs of infected patients, ii) requires the TMEM16F cell scramblase for PS externalisation, iii) leads to the activation of Ca2+- dependent chloride channels, iv) can be inhibited by selected drugs. From a mechanistic point of view, these finding are consistent with a model (Fig. 5k) by which cells expressing the SARS-CoV-2 S protein have increased Ca2+ oscillations and augmented activity of the plasma membrane TMEM16 proteins, which leads to PS externalization and chloride secretion. While the former event is required for plasma membrane fusion, chloride secretion might have relevance in COVID-19 pathogenesis. Drugs blocking TMEM16F or TMEM16A, or blunting Ca2+ levels reduce cell-cell fusion and syncytia formation. We can envisage at least three mechanisms by which S could activate TMEM16 proteins. This can occur directly on the S-expressing (infected) cells in cis or upon binding to the ACE2 receptor and activating protease in trans, or indirectly through the activation of Ca2+ release. Further molecular experiments are clearly needed to discern among these possibilities. As far as the interplay between Ca2+ levels and TMEM16 proteins is concerned, TMEM16 activation by SARS-CoV-2 S appears to amplify the amplitude of spontaneous Ca2+ signals. This is in line with previous reports showing that, much like its yeast homologue Ist238, TMEM16A tethers the ER to the plasma membrane via its interaction with IP3R responsible for the release of Ca2+ from intracellular stores 39; 40. Both TMEM16A and TMEM16F augment intracellular Ca2+ signals by increasing the filling of ER stores and augmenting IP3R-induced Ca2+ release, thus amplifying Ca2+ signals activated by GPCRs 40. TMEM16F is an essential component of the outwardly rectifying chloride channel, which is ubiquitously present in several cell types 41. Its role in SARS-CoV-2 S-protein-induced syncytia formation is consistent with the previously proposed role of PS exposure, and of TMEM16 scramblases in particular, in most other physiological cell-cell fusion events. Macrophages fusing into inflammatory giant cells 42 or to form bone-reabsorbing osteoclasts 43, myoblasts committed to fuse into myotubes 44, cytotrophoblasts becoming syncytiotrophoblasts 45 and, finally, sperm cells during egg fertilisation 46 all expose PS at the cell surface. In the cases of myoblasts 47 and of placental development, specific involvement of TMEM16F 48 and TMEM16E 47 respectively was recently reported. Besides Niclosamide, other drugs known to inhibit the Ca2+-dependent chloride channels of the anoctamin/TMEM16 family also scored positive in inhibiting S-mediated syncytia formation, including Nitazoxanide 31, Hexachlorophene and Dichlorophen, which all were statistically highly significant in SIA. Gefitinib, which is known to block TMEM16A-activated EGFR on the plasma membrane 49, also blocked S-driven fusion. In addition phenothiazine antipsychotics inhibit calmodulin, which participates in TMEM16A channel activation 50,51 (reviewed in ref. 33). TMEM16A currents are strongly inhibited by trifluoperazine 51 and by serotonin reuptake inhibitors 52-54. Finally, the anti-helminthic drug Ivermectin, which scored positive in our SIA and was proposed as an anti-COVID-19 drug 55, was also recently reported to inhibit TMEM16A 56. Similar to Niclosamide and Nitazoxanide 31 it appears likely that the inhibitory effect of these drugs against TMEM16A might also extend to TMEM16F. Thus, our screening for syncytia inhibition appears to have disclosed a common, essential mechanism for S-dependent, cell-cell fusion. The activation of members of the TMEM16 family by the SARS-CoV-2 S protein might have specific relevance in the context of COVID-19 pathogenesis, especially in connection with the observation that S-expressing, virus-infected cells and syncytia persist for long periods in the lungs of infected patients (https://medrxiv.org/cgi/content/short/2020.06.22.20136358v1). In pancreatic cells, TMEM16A promotes NK- ^B activation and IL-6 secretion as a consequence of Ca2+ level elevation 57. TMEM16F is essential for lipid scrambling in platelets during blood coagulation 58,59. Externalised PS serves as anchoring sites for the assembly of the tenase and prothrombinase complex, which jointly enhance the rate of thrombin generation by 5 to 10 orders of magnitude 60. As platelets express the ACE2 receptor (our unpublished observations), it is tempting to speculate that S-driven, TMEM16F activation in these cells might trigger thrombotic events in COVID-19 lungs. Additionally, TMEM16A contributes to dysfunction of endothelial cells 61, which are a frequent target of SARS-CoV-2 infection in both lungs and other organs (https://medrxiv.org/cgi/content/short/2020.06.22.20136358v1)62). Finally, activation of chloride secretion might be responsible of alveolar oedema and diarrhoea in the course of pulmonary or gut infection respectively (our still unpublished data show the presence of virus RNA-positive in gut epithelial cells of COVID-19 patients with diarrhoea). There is an incomplete correlation in terms of potency between the effect of the drugs we selected as inhibitors of S-induced syncytia, their efficacy at protecting from virus induced cell death and their capacity to inhibit viral replication (cf. Figs 9c and 9d). This underscores the concept that syncytia formation is an S-driven pathological process that does not necessarily reflect viral replication itself. As there is increasing evidence that COVID-19 disease might be related to the persistence of virus infected cells, rather than to direct cell death due to viral replication, the identified drugs are worth considering for COVID-19 therapy. In particular, Niclosamide is a synthetic salicylanilide developed in the 1950s as a molluscicide against snails 63. The drug was later approved for humans and used for over 50 years against cestode (tapeworm) infection, in which it mainly works by inhibiting oxidative phosphorylation 64. In mammalian cells, Niclosamide increases autophagic flux 65 and blocks Wnt/ß-catenin signalling and cancer cell proliferation (recently reviewed in ref. 66). Niclosamide was also reported to be active against various enveloped and non-enveloped viruses, in particular against SARS-CoV 67 and MERS-CoV 65. Although the drug has relatively low solubility, there is evidence of considerable absorption, with serum levels that can reach 1-20 μM 68. In a colorectal cancer clinical trial 69, administration of 2 g daily Niclosamide resulted in plasma concentrations of ~0.5-1 µg/ml (https://ascopubs.org/doi/abs/10.1200/JCO.2018.36.15_suppl.e14536), which correspond to ~1.5-3 µM, which is well in the range at which Niclosamide blocks coronavirus replication and syncytia formation in our experiments. Together, these considerations appear to provide a mechanism and a rationale for repurposing of Niclosamide to treat patients with COVID-19. EXAMPLE 2 METHODS Production of SARS-CoV2 Spike Variant Vector SARS-CoV-2 wild-type spike protein-coding sequence (NCBI accession number NC_045512.2, position 21563-25384) was codon-optimized (SEQ ID NO: 7) and synthesized with the V5 tag at the C-terminus and cloned into the pZac 2.1 AAV backbone under the control of the cytomegalovirus (CMV) IE promoter as described in Example 1. Spike variants D614G (SEQ ID NO: 8), Alpha (SEQ ID NO: 9), Beta (SEQ ID NO: 10), Gamma (SEQ ID NO: 11) and P2-Spike (SEQ ID NO: 12) were generated using the aforementioned codon-optimized spike sequences and inserting mutations or deletions depending on the type of variant. In further examples, SARS-Cov2 spike variant vectors are produced using the described method for the delta variant spike protein (SEQ ID NO: 13). Syncytium inhibition assay Vero cells were cultured as described in Example 1. Vero cells (1x104/well) were then reverse transfected with wild type spike coding vector and the variants described above using Fugene HD transfection reagent (Promega) following the manufacturer protocol (3:1 lipids (0.3 μl) and plasmid (100 ng) ratio). Eight hours after transfection, cells were treated with drugs (2.5 µM Niclosamide, 5 µM Clofazimine or 5 µM Salinomycin) in DMEM with 1 g/L glucose (Life Technologies) supplemented with 10% heat-inactivated FBS (Life Technologies). 20 hours after drug treatment, cells were washed with 100 μl per well PBS and fixed in 50 μl 4% PFA for 10 minutes at room temperature. After fixation, cells were processed for immunofluorescence using anti V5, Hoechst (H3570), according to the manufacturer’s instructions. Image acquisition and analysis of syncytia Image acquisition was performed using the Operetta CLS high content screening microscope (Perkin Elmer) with a Zeiss 20× (NA = 0.80) objective, a total of 25 fields were acquired per wavelength, well and replicate. Images were subsequently analysed using the Harmony software (version 4.9, PerkinElmer). Images were first flatfield-corrected, and nuclei were segmented using the “Find Nuclei” analysis module (Harmony software, version 4.9, PerkinElmer). The thresholds for image segmentation were adjusted according to the signal to background ratio. The splitting coefficient was set to avoid the splitting of overlapping nuclei (fused cells). The intensity of the green fluorescence (spike) was calculated using the ‘Calculate Intensity Properties’ module (Harmony software, version 4.9, PerkinElmer). All cells that had a nuclear area greater than 4 times the average area of a single nucleus and simultaneously showed a green signal (spike) in the cytoplasm were considered as syncytia. RESULTS Vero cells transfected with SARS-CoV-2 Wuhan and the Alpha, Beta, Gamma and D614G variants formed syncytia (Figure 13A and 13B). The P2-spike variant did not result in a significant increase in the percentage of syncytia, which is expected as the P2 Spike variant has a two consecutive amino acid mutation that enhances Spike trimer stability and is known not to induce cell fusion. As demonstrated in Figures 13A and 13B, treatment of Vero cells infected with either the Wuhan, Alpha, Beta, Gamma, D614G or P2-spike variants with Niclosamide, Clofazimine and Salinomycin inhibited syncytia formation. As demonstrated in Figure 13B, the most effective treatment for SARS-CoV-2 Wuhan and all variants appeared to be Niclosamide. Clofazamine and Salinomycin seemed to have similar efficacies at reducing syncytia formation in all cases. In further examples, the assay is performed by infecting Vero cells with the delta variant, and Niclosamide, Clofazimine and Salinomycin are all shown to reduce syncytia formation for this variant. In further examples, the assay is performed using other TMEM16F inhibitors. FURTHER ASPECTS In further aspects, the present invention provides embodiments as described in the following numbered paragraphs: 1. A method for identifying an agent that modulates coronavirus-mediated cell fusion or syncytia formation, comprising: (a) contacting cultured cells expressing a coronavirus Spike protein with the agent; and (b) detecting cell fusion or syncytia formation by automated microscopy; thereby identifying an agent that modulates coronavirus-mediated cell fusion or syncytia formation. 2. A method according to paragraph 1, wherein the coronavirus is SARS-CoV-2. 3. A method according to paragraph 1 or paragraph 2, wherein step (b) is performed using automated high content microscopy. 4. A method according to any preceding paragraph, wherein cell fusion or syncytia formation is detected by quantifying cytoplasmic and/or nuclear area of cells. 5. A method according to any preceding paragraph, wherein cell fusion or syncytia formation is detected by quantifying a number of nuclei per cytoplasm. 6. A method according to any preceding paragraph, wherein the method comprises: (i) preparing donor cultured cells expressing the coronavirus Spike protein and acceptor cultured cells that are capable of fusing with the donor cultured cells; (ii) contacting the donor and acceptor cultured cells with the agent; and (iii) detecting fusion of the donor and acceptor cultured cells. 7. A method according to paragraph 6, wherein the donor cultured cells comprise nanoparticles that emit light at a first wavelength and the acceptor cultured cells comprise nanoparticles that emit light at a second wavelength; and fusion of the donor and acceptor cultured cells is detected by quantifying cells that emit light at the first wavelength and second wavelength. 8. A method according to paragraph 7, wherein the nanoparticles are quantum dots. 9. A method according to any preceding paragraph, wherein the agent is an antibody or fragment thereof. 10. A method according to paragraph 9, wherein the agent is a neutralising antibody against SARS-CoV-2. 11. A method according to any of paragraphs 1 to 10, wherein the agent is a small organic molecule. 12. A method according to any preceding paragraph, wherein the agent inhibits coronavirus-mediated disease in a subject. 13. A method according to paragraph 12, wherein the subject is a mammal. 14. A method according to paragraph 13, wherein the mammal is a human. 15. A method for detecting an immune response against a coronavirus in a subject, comprising: (a) contacting cultured cells expressing a coronavirus Spike protein with a sample from the subject; and (b) detecting cell fusion or syncytia formation of the cultured cells by microscopy; wherein inhibition of coronavirus-mediated cell fusion or syncytia formation in the presence of sample is indicative of an immune response against a coronavirus in the subject. 16. A method according to paragraph 15, wherein the sample comprises serum, or a component thereof. 17. A method of treating or preventing a coronavirus infection or coronavirus-associated disease, comprising administering to a subject in need thereof a therapeutically effective amount of: (i) an anoctamin/transmembrane protein 16 (TMEM16) inhibitor; (ii) a cardiac glycoside; or (iii) an agent identified by the method of any of paragraphs 1 to 14, preferably clofazimine or salinomycin. 18. A pharmaceutical composition for use in treating or preventing a coronavirus infection or coronavirus-mediated disease, comprising: (i) an anoctamin (ANO)/transmembrane protein 16 (TMEM16) inhibitor; (ii) a cardiac glycoside; or (iii) an agent identified by the method of any of paragraphs 1 to 14; preferably clofazimine or salinomycin; and optionally one or more pharmaceutically acceptable excipients, diluents and/or carriers. 19. A method or pharmaceutical composition according to paragraph 17 or paragraph 18, wherein the coronavirus is SARS-CoV-2 or the disease is COVID-19. 20. A method or pharmaceutical composition according to any of paragraphs 17 to 19, wherein the TMEM16 inhibitor is a TMEM16F inhibitor, preferably wherein the TMEM16 inhibitor is selected from niclosamide, nitazoxanide, hexachlorophene, dichlorophen and gefitinib. 21. A method or pharmaceutical composition according to any of paragraphs 17 to 19, wherein the cardiac glycoside is selected from digitoxin, ouabain, lanatoside C and digitoxigenin. 22. A method or pharmaceutical composition according to any of paragraphs 17 to 21, wherein the method is used to treat or prevent cell fusion or syncytia formation associated with a coronavirus infection. SEQUENCES SEQ ID NO: 7 - Severe acute respiratory syndrome coronavirus 2 protein-coding sequence (Wuhan-Hu-1) (NCBI accession number NC_045512.2, position 21563-25384)

5

SEQ ID NO: 8 - Severe acute respiratory syndrome coronavirus 2 protein-coding sequence (D614G variant)

5

SEQ ID NO: 9 - Severe acute respiratory syndrome coronavirus 2 protein-coding sequence (Alpha variant/UK lineage B.1.1.7)

5

SEQ ID NO: 10 - Severe acute respiratory syndrome coronavirus 2 protein-coding sequence (Beta variant/South African lineage B.1.351)

5

SEQ ID NO: 11 - Severe acute respiratory syndrome coronavirus 2 protein-coding sequence (Gamma variant/Brazil lineage B.1.1.248.1)

5

SEQ ID NO: 12 - Severe acute respiratory syndrome coronavirus 2 protein-coding sequence (P2 spike variant, vaccine)

5

SEQ ID NO: 13 – Severe acute respiratory syndrome coronavirus 2 surface (Spike) glycoprotein (delta variant) (GenPept: QXP08802.1)

5

SEQ ID NO: 14 – Severe acute respiratory syndrome coronavirus 2 surface (Spike) glycoprotein (beta variant/South African lineage B.1.351)

SEQ ID NO: 15 - Severe acute respiratory syndrome coronavirus 2 S protein (Alpha variant/UK lineage B.1.1.7)

SEQ ID NO: 16 - Severe acute respiratory syndrome coronavirus 2 S protein (Gamma variant/Brazil lineage B.1.1.248.1)

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All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

CLAIMS 1. A pharmaceutical composition for use in treating or preventing cell fusion or syncytia formation associated with a coronavirus infection or coronavirus-mediated disease, comprising: (i) an anoctamin (ANO)/transmembrane protein 16 (TMEM16) inhibitor, preferably niclosamide; or (ii) clofazimine; or (iii) salinomycin; and optionally one or more pharmaceutically acceptable excipients, diluents and/or carriers.

2. A pharmaceutical composition for use according to claim 1, wherein the coronavirus is SARS-CoV-2 or the disease is COVID-19.

3. A pharmaceutical composition for use according to claim 1 or claim 2, wherein the TMEM16 inhibitor is a TMEM16F inhibitor.

4. A pharmaceutical composition for use according to any preceding claim, wherein the TMEM16 inhibitor is selected from niclosamide, nitazoxanide, hexachlorophene, dichlorophen and gefitinib.

5. A pharmaceutical composition for use according to claim 4, wherein the TMEM16 inhibitor is niclosamide.

6. A pharmaceutical composition for use according to any of claims 1 to 4, wherein the TMEM16 inhibitor is an agent other than niclosamide.

7. A pharmaceutical composition for use according to any of claims 1 to 4 or 6, wherein the TMEM16 inhibitor is nitazoxanide.

8. A pharmaceutical composition for use according to any preceding claim, wherein the composition is used to block phosphatidylserine externalization in the treatment of infected subjects.

9. A pharmaceutical composition for use according to any preceding claim, wherein the composition is used to treat a subject suffering from severe COVID-19 disease.

10. A pharmaceutical composition for use according to any preceding claim, wherein the composition is used to treat a subject suffering from severe COVID-19-associated lung pathology.

11. A pharmaceutical composition for use according to any preceding claim, wherein the composition is used to treat a subject suffering from COVID-19-associated pneumonia, acute respiratory distress syndrome (ARDS), systemic inflammation, multiple organ failure and/or lung thrombosis.

12. A pharmaceutical composition for use according to any preceding claim, wherein the composition is administered to a subject 1-90 days, 5-60 days or 10-30 days after coronavirus (preferably SARS-CoV-2) infection.

13. A method for identifying an agent that modulates coronavirus-mediated cell fusion or syncytia formation, comprising: (a) contacting cultured cells expressing a coronavirus Spike protein with the agent; and (b) detecting cell fusion or syncytia formation by automated microscopy; thereby identifying an agent that modulates coronavirus-mediated cell fusion or syncytia formation.

14. A method according to claim 13, wherein the coronavirus is SARS-CoV-2.

15. A method according to claim 13 or claim 14, wherein step (b) is performed using automated high content microscopy.

16. A method according to any of claims 13 to 15, wherein cell fusion or syncytia formation is detected by quantifying cytoplasmic and/or nuclear area of cells.

17. A method according to any of claims 13 to 16, wherein cell fusion or syncytia formation is detected by quantifying a number of nuclei per cytoplasm.

18. A method according to any of claims 13 to 17, wherein the method comprises: (i) preparing donor cultured cells expressing the coronavirus Spike protein and acceptor cultured cells that are capable of fusing with the donor cultured cells; (ii) contacting the donor and acceptor cultured cells with the agent; and (iii) detecting fusion of the donor and acceptor cultured cells.

19. A method according to claim 18, wherein the donor cultured cells comprise nanoparticles that emit light at a first wavelength and the acceptor cultured cells comprise nanoparticles that emit light at a second wavelength; and fusion of the donor and acceptor cultured cells is detected by quantifying cells that emit light at the first wavelength and second wavelength.

20. A method according to claim 19, wherein the nanoparticles are quantum dots.

21. A method according to any of claims 13 to 20, wherein the agent is an antibody or fragment thereof.

22. A method according to claim 21, wherein the agent is a neutralising antibody against SARS-CoV-2.

23. A method according to any of claims 13 to 22, wherein the agent is a small organic molecule.

24. A method according to any of claims 13 to 23, wherein the agent inhibits coronavirus- mediated disease in a subject.

25. A method according to claim 24, wherein the subject is a mammal.

26. A method according to claim 25, wherein the mammal is a human.

27. A method for detecting an immune response against a coronavirus in a subject, comprising: (a) contacting cultured cells expressing a coronavirus Spike protein with a sample from the subject; and (b) detecting cell fusion or syncytia formation of the cultured cells by microscopy; wherein inhibition of coronavirus-mediated cell fusion or syncytia formation in the presence of sample is indicative of an immune response against a coronavirus in the subject.

28. A method according to claim 27, wherein the sample comprises serum, or a component thereof.

29. A method of treating or preventing a coronavirus infection or coronavirus-associated disease, comprising administering to a subject in need thereof a therapeutically effective amount of: (i) an anoctamin/transmembrane protein 16 (TMEM16) inhibitor; (ii) a cardiac glycoside; or (iii) an agent identified by the method of any of claims 13 to 28, preferably clofazimine or salinomycin.

30. A pharmaceutical composition for use or method according to any preceding claim, wherein the coronavirus is a SARS-CoV-2 variant selected from Wuhan-Hu-1, D614G, alpha (B.1.1.7), beta (B.1.351), gamma (B.1.1.248.1) and delta (B.1.617.2), preferably wherein the coronavirus comprises a spike (S) protein sequence as defined in any one of SEQ ID NOs: 1, 13, 14, 15 or 16, or a sequence having at least 85%, 90%, 95% or 99% sequence identity thereto.