A composition with anti-inflammatory and antiviral effects of extracellular vesicles derived from placenta

Abstract

The present invention relates to types and effects of various substances derived from the placenta, namely, extracellular vesicles (EVs) derived from the placenta, umbilical cord, umbilical cord blood, and placenta, and mesenchymal stem cells (pMSCs) derived from the placenta, umbilical cord, umbilical cord blood, and placenta, as well as types and effects of various biomolecules including miRNA and cargo contained in the EVs. The present invention has confirmed that the miRNA contained in the EVs can not only effectively degrade the RNA genome of the coronavirus, thereby having a direct antiviral effect, but also the anti-inflammatory effects of various biomolecules possessed by the EVs themselves, thereby indirectly preventing and treating various viral infections.

Description

Technical Field

[0001] The present invention relates to types and effects of various substances derived from the placenta, namely, extracellular vesicles (EVs) derived from the placenta, umbilical cord, umbilical cord blood, and placenta, and mesenchymal stem cells (pMSCs) derived from the placenta, umbilical cord, umbilical cord blood, and placenta, as well as types and effects of various biomolecules including miRNA and cargo contained in the EVs. The present invention has confirmed that the miRNA contained in the EVs can effectively degrade the RNA genome of the coronavirus, thereby not only having a direct antiviral effect, but also the anti-inflammatory effects of various biomolecules possessed by the EVs themselves, thereby indirectly preventing and treating various viral infections. Background Art

[0002] The placenta, umbilical cord, and umbilical cord blood are fetal-derived tissues and stem cells that can be obtained during childbirth. The placenta, in particular, is a byproduct of its shedding from the mother's uterus and serves to connect the fetus and mother.

[0003] The placenta, umbilical cord, and umbilical cord blood have long been considered temporary organs discarded after childbirth. However, researchers have recently discovered that various cell types exist in different parts of the placenta, including mesenchymal cells, decidua cells, amnion cells, and endothelial cells.

[0004] It is reported that the placenta, umbilical cord and umbilical cord blood contain essential amino acids, melatonin, nucleic acid components such as RNA and DNA, antioxidant enzyme SOD (superoxide dismutase), hyaluronic acid (hyaluronic acid), antioxidants, cytokines, insulin-like growth factor (insulin-like-growth factor), epidermal growth factor (EGF) and senescent cell activating factor (SCAF), etc.

[0005] It is reported that the placenta, umbilical cord and umbilical cord blood are rich in nutrients that are transferred to the fetus, and have immunosuppressive functions that prevent the mother's immune system from attacking the fetus.

[0006] The placenta, umbilical cord, and umbilical cord blood are usually discarded after being used for maternal or fetal diagnosis. However, research is currently underway from various perspectives to utilize the placenta's properties in the medical field.

[0007] In the medical field, various products have been developed that utilize the nutritional components of the placenta, such as pharmaceuticals produced by hydrolyzing human placenta and sold in the form of injectable ampoules. However, research on the placenta from the perspective of placental extracellular vesicles (EVs) and the various biomolecules they contain, particularly miRNA, is relatively limited.

[0008] In addition, in clinical trials conducted so far, previous viral therapeutics have strong side effects, making it difficult to prescribe them to patients with mild symptoms, or to use high-volume antiviral drugs for treatment to achieve the same effect as antiviral drugs.

[0009] Therefore, it is necessary to develop more virus-specific drugs to overcome the side effects of existing antiviral drugs. Summary of the Invention

[0010] Problems to be solved by the invention

[0011] The purpose of the present invention is to solve the above-mentioned problems of the prior art and provide a composition with anti-inflammatory and antiviral effects, which specifically includes extracellular vesicles (EVs) derived from the placenta, umbilical cord, umbilical cord blood, and placenta, mesenchymal stem cells (pMSCs) derived from the placenta, umbilical cord, umbilical cord blood, and placenta, and extracellular vesicles (EVs) derived therefrom, as well as various biomolecules (cargos) including miRNA and cargo contained in the EVs, and analyzes them.

[0012] Another object of the present invention is to provide a composition for preventing or treating coronaviruses that frequently mutate, wherein the composition binds to a 3'UTR site with fewer mutations in the genome of the coronavirus.

[0013] Methods used to solve problems

[0014] One aspect of the present invention provides a cell-free composition comprising at least one carrier, excipient or diluent, and a biomolecule extracted from placenta, umbilical cord or umbilical cord blood.

[0015] In the present invention, the biomolecules are selected from any one or more of proteins, peptides, antigens, antibodies, protein fragments, DNA, RNA, cells, microvesicles and other biological particles, preferably including extracellular vesicles and miRNA, but not limited thereto.

[0016] In the present invention, the carrier, excipient or diluent can be lactose, dextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, tricalcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0017] The composition of the present invention is characterized by being cell-free. Intravenous injection of stem cells can cause embolism, blood coagulation, and immune reactions. Therefore, the present invention has advantages in this regard.

[0018] Specifically, the biological molecules can be EVs obtained directly from the placenta, umbilical cord, umbilical cord blood, and extracellular vesicles (EVs) derived from mesenchymal stem cells (pMSC) derived from the placenta, umbilical cord, and umbilical cord blood. The biological molecules are 77 miRNAs contained in tissue-derived EVs, tissue extract-derived EVs, and pMSC-EVs, and can be selected from one or more of the group consisting of miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p, and miR-181a-5p, but are not limited thereto.

[0019] Specifically, the 77 miRNAs are respectively hsa-miR-34a-5p, hsa-miR-197-3p, hsa-miR-92b-3p, hsa-miR-199a-3p, hsa-miR-181b-5p, hsa-miR-181a-5p, hsa-miR-1307-3p, hsa-miR-139-5p, hsa-miR-125b-5p, hsa-let-7a-5p, hsa-miR-100-5p, hsa-miR-26a-5p, hsa-let-7i-5p, hsa-miR-16-5p, hsa-miR-15a-5p, hsa-miR-92a-3p, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-7-5p, hsa-miR-484, hsa-miR-328-3p, hsa-miR-22-3p, hsa-miR-324-3p, hsa-miR-744-5p, hsa-miR-451a, hsa-miR-423-5p, hsa-miR-423-3p, hsa-miR-193a-5p, hsa-miR-10a-5p, hsa-miR-21-5p, hsa-miR-3615, hsa-miR-320c, hsa-miR-122-5p, hsa-miR-24-3p, hsa-miR-27a-3p, hsa-miR-23a-3p, hsa-miR-150-5p, hsa-miR-99b-5p, hsa-miR-125a-5p, hsa-miR-26b-5p, hsa-miR-149-5p, hsa-miR-103a-3p, hsa-miR-99a-5p, hsa-let-7c-5p, hsa-miR-155-5p, hsa-miR-1,85-5p, hsa-miR-1249-3p, hsa-let-7b-5p, hsa-miR-425-5p, hsa-miR-425-3p, hsa-miR-191-5p, hsa-let-7g-5p, hsa-miR-15b-5p, hsa-miR-574-3p, hsa-miR-143-3p, hsa-miR-378a-3p, hsa-miR-146a-5p, hsa-miR-196b-5p, hsa-miR-25-3p, hsa-miR-93-5p, hsa-miR-29a-3p, hsa-miR-320a-3p, hsa-miR-30d-5p, hsa-miR-204-5p, hsa-let-7f-5p, hsa-let-7d-5phsa-let-7d-3p, hsa-miR-23b-3p, hsa-miR-27b-3p, hsa-miR-199b-3p, hsa-miR-126-3p, hsa-miR- 221-3p, hsa-miR-222-3p, hsa-miR-532-5p, hsa-miR-223-3p, hsa-miR-652-3p and hsa-miR-424-3p. ,

[0020] In addition, the cell-free composition of the present invention may include, as the biomolecule, extracellular vesicles and / or one or more of the 77 types of miRNAs, but is not limited thereto.

[0021] The present invention demonstrates that microRNA (miRNA) is a small non-coding RNA composed of 18 to 25 nucleotides (Nucleotide) in base sequence length. MiRNA inhibits the virus by effectively degrading the RNA genome of SARS-CoV-2.

[0022] The present inventors confirmed that miRNAs directly interact with the 3'UTR of SARS-CoV-2, and among the 77 miRNAs present in tissue-EVs, tissue extract EVs and MSC-EVs, 18 miRNAs that bind to the 3'UTR of SARS-CoV-2 were selected, and the top 5 miRNAs with higher expression levels and stronger binding were selected among the 18 miRNAs.

[0023] Specifically, the 18 miRNAs are hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-miR-181a-5p, hsa-miR-26a-5p, hsa-miR-34a-5p, hsa-miR-23a-3p, hsa-miR-125b-5p, hsa-miR-125a-5p, hsa-miR-103a-3p, hsa-miR-223-3p, hsa-miR-25 -3p, hsa-miR-26b-5p, hsa-miR-193a-5p, hsa-miR-1307-3p, hsa-miR-155-5p, hsa-miR-185-5p, hsa-miR-424-3p and hsa-miR-23b-3p, and the five miRNAs are miR-92a-3p, miR-103a-3p, miR-181a-5p, miR-26a-5p and miR-23a-3p respectively.

[0024] In the examples of the present invention, it was confirmed that the above five miRNAs were all expressed from tissue-EVs and tissue extract EVs, thereby successfully inhibiting SARS-CoV-2.

[0025] In addition, mesoderm-derived extracellular vesicles (pMSC-EVs) present in tissue-EVs, tissue extract EVs, and MSC-EVs contain many anti-inflammatory factors known to prevent fatal cytokine storms and have good regenerative effects. Experimental results confirmed that EVs regulate the inflammatory environment in several host cells known to express ACE2 receptors to inhibit inflammatory responses.

[0026] Another aspect of the present invention provides an antiviral composition containing the cell-free composition.

[0027] Specifically, the virus can be selected from one or more of the group consisting of coronavirus, HIV, influenza, enterovirus, porcine reproductive and respiratory syndrome virus, hepatitis C virus and bovine viral diarrhea virus, more specifically, it can be a new coronavirus (NMC-nCoV02) or SARS-CoV-2, but is not limited thereto.

[0028] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating viral infection, which contains an antiviral composition.

[0029] Specifically, the virus may be a coronavirus, but is not limited thereto.

[0030] The pharmaceutical composition may further comprise carriers, excipients and diluents conventionally used for the formulation of pharmaceutical compositions.

[0031] The carrier, excipient or diluent may include, but is not limited to, lactose, dextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, tricalcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate or mineral oil.

[0032] In addition, the composition can be used in the form of oral preparations such as acid agents, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or non-oral preparations in the form of injections, nasal sprays, inhalation liquids, creams, gels, ointments, suppositories, or lotions.

[0033] Solid preparations for oral administration include tablets, pills, granules, capsules, and the like. These solid preparations can be prepared by mixing the miRNA composition and its fractions with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to the excipients, lubricants such as magnesium stearate and talc can also be used.

[0034] Liquid preparations for oral administration may be suspensions, liquid solutions, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to simple diluents such as water and liquid paraffin.

[0035] Preparations for parenteral administration can use sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. The non-aqueous solvents and suspensions can use vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, as well as injectable esters such as ethyl oleate. As the mechanism of the suppository, witepsol, polyethylene glycol, polysorbate 61, cocoa butter, laurel fat, and glycerin gelatin can be used.

[0036] A pharmaceutical composition containing the antiviral composition can be administered to a subject at a pharmacologically effective dose. The term "pharmacologically effective dose" refers to a sufficient amount necessary to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The effective dose can be determined based on factors such as the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, route of administration, excretion rate, duration of treatment, concomitant medications, and other factors known in the medical field.

[0037] The pharmaceutical composition can be administered as a single therapeutic agent, in combination with other therapeutic agents, or sequentially or simultaneously with currently used therapeutic agents. The administration may be single or multiple. In view of the various factors described above, it is preferred that the dosage be administered in the minimum amount to achieve the maximum effect, thereby minimizing side effects. This dosage can be readily determined by one of ordinary skill in the art.

[0038] The preferred dosage of the composition for treatment of the present invention may vary depending on the patient's condition and weight, the severity of the disease, the type of drug, the route of administration, and the course of treatment. Even so, a person skilled in the art can select an appropriate dosage. Preferably, the dosage of the composition for treatment of the present invention is such that the number of EVs reaches 10 after daily administration, based on the amount of EVs. 2 to 10 16 More preferably, the number of EVs reaches 10 5 to 10 12 , but not limited to this.

[0039] In addition, based on the amount of miRNA, the daily dosage is 0.0001 to 1000 mg / kg. To achieve a more effective effect, it is preferably administered at 0.01 to 100 mg / kg. When administered, it can be administered once a day or multiple times a day. The dosage and frequency of administration are not intended to limit the scope of the present invention in any way.

[0040] Effects of the Invention

[0041] The present invention provides a composition comprising EVs derived from the placenta, umbilical cord, umbilical cord blood, and placenta, and mesenchymal stem cells (pMSCs) derived from the placenta, umbilical cord, umbilical cord blood, and placenta, and extracellular vesicles (EVs) derived therefrom, as well as miRNA contained in the EVs and a composition comprising these. When the composition is used to develop a therapeutic agent for preventing or treating coronavirus, the following effects are achieved: 1) the miRNA of the present invention binds to the 3'UTR of the coronavirus genome with high specificity, and is therefore expected to have almost no side effects on normal tissues and internal organs; 2) the 3'UTR of the viral gene is a highly conserved site with almost no mutations, and is therefore rarely affected by mutations even in RNA viruses that frequently mutate, and is therefore generally applicable to RNA viruses; 3) the various biomolecules present in EVs also have the effect of regenerating damaged tissues and immunomodulatory effects, and therefore, the use of a therapeutic agent using the composition will make the effect more significant.

[0042] In addition, in addition to the direct antiviral effect described above, the composition of the present invention has an indirect effect of alleviating symptoms caused by viral infection because EVs can contain various biological substances and miRNAs that inhibit inflammation by interacting with genes related to inflammation.

[0043] That is, the composition of the present invention is characterized by having, in addition to a direct antiviral effect, an indirect therapeutic effect of suppressing inflammation and cytokine storm caused by viral infection, and therefore can be used as a preventive agent for viral infection.

[0044] The effects of the present invention are not limited to the above-mentioned effects, and should be understood to include all effects that can be inferred from the constitution of the invention described in the specification or claims of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This study involves an antiviral mechanism of action of tissue-EVs, tissue extract EVs, MSC-EVs, and the miRNAs they contain against SARS-CoV-2. The direct antiviral effect is that miRNAs directly bind to the 3'UTR, 5'UTR, or coding sequence of SARS-CoV-2, thereby downregulating SARS-CoV-2 RNA. The indirect antiviral effect is that miRNAs and proteins contained in EVs regulate the expression of inflammatory mRNAs, which regenerates damaged tissues through this regulation, and their miRNAs and proteins modulate the pro-inflammatory environment to suppress cytokine storms.

[0046] Figure 2 The results of observation of pMSC-EV characteristics (A: pMSC-EVs were CD63 positive as detected by fluorescence microscopy. B: The average size of pMSC-EVs was 121.8 nm in diameter in 1 ml of extract. C: Representative TEM images. D: Western blotting results of pMSC-EVs for CD81, CD9, annexin A2, and HSP70);

[0047] Figure 3 It is a potential binding site for the miRNA of the present invention to bind to the 3′UTR of SARS-CoV-2;

[0048] Figure 4 The results of qPCR analysis were performed to confirm whether the miRNA of the present invention was present in EVs;

[0049] Figure 5 Luciferase assay results were performed to confirm whether the EV of the present invention has a direct antiviral effect (A: PCR fragments were cloned into a luciferase reporter plasmid between the luciferase ORF and the synthetic poly(A) sequence to prepare a recombinant plasmid. B: Relative luciferase activity in SK-N-BE(2)C cells transfected with the recombinant plasmid);

[0050] Figure 6 The results show that the miRNA of the present invention has an effect on multiple coronaviruses (A: 3'UTR of five coronaviruses and the binding site of the miRNA. B: Interaction between the miRNA of the present invention and other viruses);

[0051] Figure 7 Schematic diagram of the experimental method used to confirm the indirect antiviral effects of EVs and miRNAs;

[0052] Figure 8 Results confirming the effects of EV and miRNA treatment on BEAS-2B (gastrointestinal epithelial cells) (A: EV, B: miRNA);

[0053] Figure 9 The results show the protective and repair effects of EV and miRNA treatment on LL-24 (A: EV, B: miRNA);

[0054] Figure 10 The results of the protection and repair effects of MEF (fibroblasts) and BV2 (microglial cell line) were confirmed. Figure 10 A and Figure 10 As shown in B;

[0055] Figure 11 This is the result of confirming the expression levels of inflammatory-related factors after treating M2 neural cells with EVs;

[0056] Figure 12 The images show the cell lines before and after treatment with PKH26-labeled EVs.

[0057] Figure 13 This is a conceptual diagram of the process by which the EVs and miRNAs of the present invention use all direct and indirect pathways to treat or improve viruses and symptoms of viral infection.

[0058] Best Practice

[0059] The present invention will be described in detail below by way of examples. However, the following examples are only provided to illustrate the present invention and therefore, the present invention should not be limited by the following examples.

[0060] Experimental methods and materials

[0061] Obtaining placental extracts and EVs

[0062] Human placentas without medical, obstetric, or surgical complications were obtained. Each placenta was carefully dissected and washed several times with PBS, then mechanically pulverized and digested with 0.5% collagenase IV (Sigma, St. Louis, MO, USA) at 37°C for 30 seconds to obtain placental extracts and obtain EVs.

[0063] Cell lines and viruses

[0064] Vero cells, a cell line derived from African green monkey kidney epithelial cells, were used and cultured in DMEM (Dulbecco's Modified Eagle's medium, Gibco, USA).

[0065] In early February 2020, green monkey kidney E6 cells were infected with the novel coronavirus (NMC-nCoV02) and SARS-CoV-2, both isolated from respiratory specimens of patients infected with the novel coronavirus in China. Viral titers were confirmed by 50% tissue culture infectious capacity (TCID50) based on cytopathic effect (CPE). All experiments involving viruses were conducted in a biosafety level 3 (BLS-3) laboratory.

[0066] Cytotoxicity assay

[0067] The cytotoxic effect of EVs on green monkey kidney cells was evaluated by MTT-assay.

[0068] Specifically, monolayers of green monkey kidney cells were washed with phosphate-buffered saline (PBS) in 96-well plates and treated with serial amounts of EVs. 1x104 green monkey kidney cells were incubated in 96-well plates at 5% CO2 and 37°C for 24 hours, and absorbance was measured at wavelengths of 500 to 600 nm using a plate reader.

[0069] Cytopathic effect (CPE) inhibition analysis

[0070] 1x104 green monkey kidney cells were injected into each 96-well plate and treated with 50μL of culture medium containing EVs and 50μL of SARS-CoV-2 virus at 37°C for 72 hours. Cells inoculated with the virus served as a control. Infected cells were observed under a microscope using 100% CPE. Cells were stained with 1% crystal violet. The percentage of CPE in EV-treated cells was calculated (positive wells / total wells) and the results were photographed.

[0071] Nanoparticle tracking analysis

[0072] EVs were diluted to a final volume of 1 ml in fetal bovine serum (PBS) and examined in a ZetaView nanoparticle tracking imaging microscope (ParticelMetrix, Inning, Germany). The software manufacturer's basic settings for EVs or nanoparticles were selected, and three scans were performed for each measurement.

[0073] Fluorescence imaging of EVs

[0074] EVs were biotinylated using EZ-Link Sulfo-NHS-LC LC-Biotin (Thermo Fisher Scientific, Waltham, MA, USA).

[0075] Biotinylated EVs were loaded onto Zeba spin desalting columns 7K MWCO (ThermoScientific) to remove remaining free biotin.

[0076] For staining, 20 μL of biotinylated EVs were added to a circle drawn on a streptavidin-coated glass slide (Arrayit Corporation). After 30 minutes, EVs were fixed with BD Fix Perm (BD Biosciences) and blocked with 0.2% BSA-PBS.

[0077] Immunofluorescence staining of EVs was performed using anti-human CD63 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) (SC-5275) for 2 h at room temperature, followed by incubation with Alexa Fluor 488-conjugated secondary antibodies for 1 h at room temperature.

[0078] Transmission electron microscopy (TEM) imaging method

[0079] A 5 ml aliquot of the diluted sample containing 0.5 μg of protein was dropped onto a hydrophilic grid. After a few minutes, the grid was washed with distilled water and exposed to 2% uranyl acetate for 20 seconds. Images were acquired using a JEM-1010 microscope (JEOL) operating at 80 kV.

[0080] Western blotting

[0081] EVs were solubilized in 10× RIPA buffer containing a protease cocktail tablet (Roche) and phosphatase inhibitors II and III (Sigma). Protein concentration was measured using the BCA assay (Thermo Fisher Scientific). A 30 μg aliquot of each sample was subjected to 10% SDS-PAGE followed by Western blotting.

[0082] The cells were blocked with 10% skim milk in TBS-T for 1 hour. Antibodies against the following proteins were obtained from the following suppliers: CD81 (1:1,000, Santa Cruz Biotechnology), Alix (1:1,000, Santa Cruz Biotechnology), CD9 (1:500, Santa Cruz), and GAPDH (1:10,000, sc-32233, Santa Cruz Biotechnology).

[0083] Primary antibodies were diluted in TBS-T and incubated in the blots overnight at 4°C, followed by incubation with secondary antibodies for 1 hour. Immunoreactivity was detected using enhanced chemiluminescent HRP substrate (Millipore).

[0084] Cultured cells

[0085] The human hepatocellular carcinoma cell line HepG2 and the mouse microglial cell line BV2 were purchased from the American Type Culture Collection (ATCC, Manassas, CA, USA). Both cell lines were cultured in growth medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin / streptomycin (P / S) at 37°C and 5% CO2. Both cell lines were passaged every 2 to 4 days.

[0086] Normal lung cell line LL24 was also obtained from ATCC. LL24 cells were grown in RPMI-1640 medium (Gibco / Life Technologies, USA, NY, USA) supplemented with 10% FBS and 1% P / S at 37°C in a humidified atmosphere of 5% CO2. Passage was performed every three days.

[0087] MEF cells were isolated from mouse embryos at day -13.5. MEF cells were cultured in DMEM supplemented with 10% FBS, 1% antibiotic-antimycotic (250 ng / mL amphotericin, 100 U / mL penicillin, and 100 μg / mL streptomycin), 8 μg / mL tyrosine (Sigma), and 15 μg / mL gentamicin (Gibco) at 37°C and 5% CO2. MEF cells were passaged every 3 days and used at passages 2 to 5.

[0088] Transformed human bronchial epithelial cell line BEAS-2B was cultured in 5 μg / mL gentamicin (Gibco) and serum-free 1X Keratinocyte SFM (Gibco) at 37°C and 5% CO2. The medium was changed every 2-3 days, and the cells were passaged every 4-5 days.

[0089] Neurons were isolated from human fetal tissue with the consent of the pregnant mothers at gestational weeks 10, 12, and 14. Neurons were cultured in DMEM / F12 (Gibco) medium supplemented with 50 μg / mL of B27 supplement (Gibco), gentamicin (Gibco), human bFGF, human EGF 20 ng / mL (Peprotech), tocopherol, and tocopherol acetate (Sigmaaldrich) at 1 μg / mL at 37°C and 5% CO2 and 3% CO2.

[0090] PKH26 marks processing in EVs

[0091] EVs were labeled with the PKH26 Red Fluorescent Cell Linker Kit for General Cell Membranes (Sigma-Aldrich) to label general cell membranes. Specifically, EV particles were resuspended in diluent C at 200 μg / mL and then mixed with a dye solution (2 μL of PKH26 ethanol dye and 500 μL of diluent C) at a 1:1 ratio for 5 minutes. Next, an equal volume of 1% bovine serum albumin (BSA) was added to bind excess dye, and the resulting solution was desalted using a 7K MWCO Zeba Spin Desalting Column (7K MWCO Zeba Spin Desalting Columns, Thermo Fisher Scientific).

[0092] Subsequently, the samples were stored at -80°C.

[0093] LPS and EV processing

[0094] For the experiments, cells were seeded at equal density in wells of 96-well or 6-well plates (Nunc, Roskilde, Denmark). To demonstrate repair effects, cells were stimulated with LPS (0.5-2 μg / mL for MEFs and BV2 cells, 2 or 4 μg / mL for HepG2 cells and LL24 cells) and then treated with EVs for 24 h.

[0095] To confirm the protective effect, cells were pretreated with EVs for 24 h and then stimulated with LPS for 24 h. After 24 h of incubation, cells were subjected to MTT analysis or RNA extraction.

[0096] miRNA transfection

[0097] For transfection of miRNA, 6 × 10 4 to 2×10 5 The cells were seeded into 24-well plates and infected with 20 nM miRNA (hsa-miR-92a-3p, hsa-miR-26a-5p, hsa-miR-23a-3p, hsa-miR-103-3p, and hsa-miR-181-5p) (organisms). The pre-miR miRNA negative control group was replaced with Lipofectamine 3000.

[0098] MTT assay

[0099] The cytotoxicity of EVs was evaluated using the MTT assay. Briefly, cells were seeded into 96-well plates, treated with the indicated concentrations of EVs, and stimulated with LPS for 24 hours. After the culture supernatant was removed, the resulting dark blue crystals were dissolved in DMSO. Absorbance was measured at 570 nm.

[0100] RNA extraction and quantitative PCR (qPCR)

[0101] Total RNA was extracted from BV2, MEF, HepG2, LL24, BEAS-2B, and neural cells for qPCR, and miRNA was isolated from EVs. Total RNA was extracted using TRIzol reagent (Invitrogen).

[0102] cDNA was synthesized from 1 μg of total RNA using a cDNA kit (iNtRON). miRNA was extracted using a total exosomal RNA and protein isolation kit (Thermo Fisher Scientific) and reverse transcribed using miScript II RT (Qigen).

[0103] For PCR and qPCR, primers for genes associated with inflammation were designed. The reaction mixture (total volume of 20 μL) contained 10 μM primer mix, SYBR-Green with low ROX (Enzynomics), nuclease-free water (dark, non-ionic) and cDNA 2 μL. The conditions were as follows: denaturation / activation step at 95°C for 10 minutes, followed by 95°C for 15 seconds, 56°C for 30 seconds, and 72°C for 20 seconds, and this cycle was repeated 40 times. Reactions were performed in a StepOne Real-Time PCR instrument (Applied Biosystems). Quantification of gene expression was based on the C T value.

[0104] Transfection and reporter assays

[0105] A 208 bp fragment of the 3'UTR of the SARS-CoV-2 genome was synthesized using a biomimetic method. The 3'UTR fragment was degraded with XbaI and inserted downstream of the luciferase gene in pGL3-control (Promega) to obtain pGL3covi-3UTR_Luc.

[0106] All constructs were confirmed by sequencing. Human neuroblastoma SK-N-BE(2)C cells were maintained in DMEM supplemented with 10% FBS. All media contained 100 U / mL penicillin and 100 mg / mL streptomycin. For transfection, cells were plated at 1.2 x 10 cells / mL in DMEM without antibiotics 1 day prior to transfection. 5 Individual cells / well were plated into 24-well plates. Transfection was performed using Lipofectamine 2000 (Invitrogen). The total amount of DNA was 0.5 μg per well, which was composed of pGL3covi-3UTR_Luc 0.2 μg and pRSVβgal 0.3 μg as an internal control group. Each transfection also contained a labeled miRNA. After 24 hours after transfection, the cells derived from each well were dissolved with 100 μL of a lysis buffer (25 mM triphosphate [pH 7.8], 2 mM DTT, 2 mM CDTA [1,2-diaminocyclohexane tetraacetic acid], 10% glycerol, and 1% triketone X-100). The same volume of firefly luciferase substrate was added, and luciferase activity was determined using a luminometer plate reader, and β-galactosidase activity was standardized.

[0107] RNA sequencing and data processing

[0108] Small RNA sequencing analysis was performed using extracellular vesicles (EVs) obtained from eight different hpMSC culture media and two placental tissue extracts. Sequencing was performed on a BGISeq-500 sequencer at the Beijing Genomics Institute (BGI, Shenzhen, China). Primers were aligned to the human reference genome (GRCh38) using subread aligner, and the featureCounts tool was used to obtain miRNA primer counts. The number of miRNA reads was normalized, and low-expressing miRNAs were filtered out using the edgeRR package. The expression of each miRNA was converted to CPM (counts per million). Read quality control was performed using the qrqcR package, and the distribution of small RNA frequencies was obtained using sRNAtoolbox.

[0109] miRNA target prediction and functional analysis

[0110] To search for 3'UTR and miRNA binding sites, the PITA tool (Kertesz et al., 2007) was used.

[0111] The SARS-CoV-2 full genome sequence (NC_045512.2) was obtained from the NCBI sequence database (O'Leary O'Leary et al, 2016), and the 3'UTR sequence was extracted from the SARS-CoV-2 full genome.

[0112] We used the PITA tool with basic settings to predict miRNA binding sites.

[0113] Novel miRNA binding sites were predicted using the miRDB custom prediction tool ( Chen and Xiaowei, 2020 ).

[0114] To confirm the biological functions of miRNAs, the targets of experimentally verified miRNAs were obtained from miRTarBase (Huang et al. 2020), and miRNAs with a proportion below 1% were excluded from the analysis.

[0115] To confirm the biological functions of miRNAs, GO term and KEGG pathway analyses were performed using DAVID Bioinformatics Resource 6.8 (Huang et al., 2009).

[0116] Functional analysis results were visualized using Cytoscape 3.8 and Pathview R packages.

[0117] About ACE2 expression analysis

[0118] Human tissue and cell line expression data were obtained from the Human Protein Atlas database (Uhlén et al., 2015). ACE2 expression in each cell or tissue type was analyzed using this expression data.

[0119] Analysis of conserved regions of miRNA binding sites

[0120] To analyze the conserved regions of miRNA binding sites in SARS-CoV-2, 3'UTR sequences were obtained from the NCBI reference sequence database, and 3'UTR sequences of viruses related to SARS-CoV-2 were used.

[0121] Specifically, SARS coronavirus (NC_004718.3), SARS-CoV-2 (NC_045512.2), SARS coronavirus BJ01 (AY278488.2), bat SARS coronavirus HKU3-1 (DQ022305.2), bat SARS-like coronavirus SL-CoVZC45 (MG772933.1) and bat SARS-like coronavirus SL-CoVZC2129.1 (MG) were used.

[0122] The MUSCLE tool (Edgar et al., 2004) was used to align the 3'UTR sequences of coronaviruses, and basic values ​​were used for muscle tissue (MUSCLE) analysis.

[0123] Statistical analysis

[0124] Statistical analysis was performed using analysis of variance (ANOVA) followed by Tukey HSD post hoc tests in R. Heatmaps are plotted as log2-CPM of miRNA expression in hpMSC-EVs and generated using the gplots R package (Warnes et al., 2015). P values ​​for GO term analysis and KEGG pathway analysis were modified for multiple comparisons using the Benjamini-Hochberg method. Data are presented as mean ± standard error of the mean, and p values ​​or adjusted p values ​​< 0.05 were considered significant.

[0125] Hereinafter, the effects of the present invention confirmed by the above-mentioned experimental method will be described in detail with reference to the accompanying drawings. However, the following examples are only for the purpose of helping to understand the present invention, and therefore, the present invention should not be limited by the following examples.

[0126] Example 1 Viral Inhibition Mechanism of Placenta-EV miRNA on SARS-CoV-2 Virus

[0127] Figure 1 This is the mechanism of antiviral action of placental EV miRNA against SARS-CoV-2. The direct antiviral mechanism against SARS-CoV-2 is achieved through direct binding of miRNA to the 3'UTR, 5'UTR, or coding region of the virus, inducing downregulation of SARS-CoV-2 RNA. Furthermore, indirect therapeutic effects include modulating the expression of inflammatory mRNAs, thereby suppressing the cytokine storm.

[0128] That is, the present invention demonstrates that EVs derived from mesenchymal stem cells (MSCs) and the miRNA and protein molecules they contain are capable of regenerating damaged tissues and regulating miRNAs and anti-inflammatory environments.

[0129] EVs isolated from pMSCs were positive for the common EV marker CD63 ( Figure 2 A), EV size was analyzed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM).

[0130] The hydrodynamic diameter of exosomes measured by nanoparticle tracking analysis (NTA) was 121.8 nm ( Figure 2 B), Representative TEM images of Figure 2 C. Additional characterization of EVs isolated by Western blotting of EV markers showed typical EV markers such as CD81, CD9, annexinA2, and HSP70 ( Figure 2 D).

[0131] Example 2 Screening for miRNAs that bind to SARS-CoV-2 virus

[0132] Based on the fact that the viral genome can be targeted by human miRNAs, the present inventors confirmed whether miRNAs in EVs can interact with the 3'UTR of SARS-CoV-2.

[0133] The 3'UTR of SARS-CoV2 contains binding sites targeting miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p, and miR-181a-5p.

[0134] Specifically, the 3'UTR sequencing of SARS-Cov-2 was aligned, and sequence analysis was performed using MicroInspector software, and the results were predicted as follows: a total of 18 miRNAs targeted the 3'UTR of SARS-Cov-2 using PITA software (Table 1), and 27 miRNAs bound to the entire genomic sites of the SARS-CoV-2 virus (Table 2).

[0135]

Table 1

[0136]

[0137] Table 1 is a list of 18 miRNAs targeting the 3'UTR of SARS-Cov-2.

[0138]

Table 2

[0139]

[0140] Table 2 is a list of 27 miRNAs predicted to bind to the entire genome of the SARS-CoV-2 virus. The expression (expression%) in Table 2 is calculated by the corresponding miRNA CPM / total miRNA CPM. The higher the score, the greater the possibility of binding. According to the reference, a score above 80 means that binding is possible.

[0141] Table 3 below shows the binding site information and binding prediction results within the SARS-CoV-2 genome. Five miRNAs (miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p, and miR-181a-5p) were selected based on their thermodynamic energy scores and high scores using the PITA and miRDB prediction tools.

[0142]

Table 3

[0143]

[0144] PITA was used to predict the potential binding sites of the five miRNAs finally selected, miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p, and miR-181a-5p, that may bind to the 3′UTR of SARS-CoV-2 ( Figure 3 A, 3B). Using PITA, we calculated the total free energy for each of these miRNAs. For example, the binding energy for miR-181a-5p targeting the 3′UTR was -microRNA target hybridization energy, or 18.7 kcal / mol, suggesting that binding to the miRNA 3′UTR occurs spontaneously. Lower binding thermodynamic energies (kcal / mol) indicate stronger binding.

[0145] Then, to confirm whether the above five miRNAs were present in EVs, qPCR analysis was performed ( Figure 4The results confirmed that miRNAs in EVs were highly expressed in the order miR-92a-3p, miR-181a-5p, miR-26a-5p, miR-23a-3p, and miR-103a-3p. Specifically, miR-92a-3p and miR-181a-5p were significantly more expressed than the others, and their order was consistent with the predicted values. These results suggest that since EVs contain five miRNAs, they themselves could be used as therapeutic agents against SARS-CoV-2.

[0146] Example 3 confirms the direct antiviral effects of EV and miRNA

[0147] To confirm whether each unique signature miRNA or five miRNAs selected in Example 2 above can directly interact with the SARS-CoV-2 genome, a luciferase assay was performed.

[0148] Luciferase-reporter assays were performed to confirm whether individual specific miRNAs or five miRNAs directly interacted with the SARS-CoV-2 genome.

[0149] The PCR fragment was cloned into the luciferase reporter plasmid between the luciferase ORF and the synthetic poly(A) sequence. The recombinant plasmid was named pGL3 SARS-CoV-3'-UTR_Luc( Figure 5 A). The recombinant plasmid was then transfected into human neuroblastoma SK-N-BE(2)C cells, and luciferase activity was measured 48 hours after transfection.

[0150] like Figure 5 As shown in B, the relative luciferase activity in SK-N-BE(2)C cells transfected with the recombinant plasmid was significantly reduced compared with that in cells transfected with the psi-control empty vector (ps < 0.0001).

[0151] Specifically, when the 3'UTR of coronavirus was co-transfected with five miRNAs or five miRNAs were co-transfected into DF-1 cells with their respective expression vectors, the relative luciferase activity was downregulated, and this result confirmed that the luciferase activity of psi-59UTR was significantly decreased.

[0152] The above experimental results mean that miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p and miR-181a-5p bind to the 3'UTR of SARS-CoV-2, thereby inducing significant silencing.

[0153] Based on this result, it was confirmed that all five miRNAs directly bound to the 3'UTR of SARS-CoV-2, and it was confirmed that one or more miRNAs selected from the group consisting of miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p and miR-181a-5p can degrade the SARS-CoV-2 viral genome, thereby inhibiting SARS-CoV-2 infection.

[0154] Example 4 demonstrates direct antiviral activity against a variety of coronaviruses and other viruses

[0155] To confirm the hypothesis that 3′UTR sequences are conserved in the coronavirus family and that miRNA target sites are conserved in all coronaviruses, five coronaviruses were randomly selected and the sequencing of the 3′UTR regions was compared using the MUSLCE tool.

[0156] like Figure 6 As shown in A, it is confirmed that the 3'UTRs of the five coronaviruses are mostly conserved, and therefore, their degree of variation is not large, especially the binding sites predicted to bind to the five miRNAs (marked with red boxes) are also well conserved.

[0157] In other words, given that the results of the 3'UTR verification of various coronaviruses showed that there were almost no mutations in the 3'UTR and the binding rate of miRNA was high, it can be predicted that the five selected miRNAs may have universal antiviral capabilities and thus be applicable to new viruses produced by mutations.

[0158] It is known that multiple miRNAs of EVs interact with other viruses ( Figure 6 B).

[0159] Specifically, we demonstrated that miR-150-5p, miR-223-3p, and miR-29-3p interact with HIV, thereby inhibiting viral translation and ultimately latent T cells.

[0160] It is reported that miR-23b-3p blocks the translation of enterovirus 71, while Let-7c-5p can reduce the expression of matrix proteins important for influenza virus.

[0161] miR-181a-5p, miR-181b-5p, miR-23a-3p, miR-23b-3p, and miR-378a-3p degrade porcine reproductive and respiratory syndrome virus.

[0162] miR-122-5p and let-7c-5p inhibit hepatitis C virus and bovine viral diarrhea virus, respectively.

[0163] That is, EVs and the various miRNAs contained in EVs exert inhibitory effects by binding to a variety of viruses and 3'UTRs with low mutation frequencies. This result suggests that EVs can be used as common therapeutic and preventive therapeutic agents against various viral infections.

[0164] Example 5 Confirmation of the indirect antiviral effect of EV and miRNA

[0165] Viruses that infect the respiratory system, including coronaviruses, penetrate cells expressing the ACE2 receptor, leading to cell damage.

[0166] Because ACE2 receptors are primarily found in the liver, kidneys, male reproductive organs, muscle tissue, and gastrointestinal tract, these target organs can be damaged by the virus. This view is supported by the fact that a large number of patients developed liver damage or organ damage in recent clinical studies.

[0167] Among eight COVID-19 patients admitted to the intensive care unit of Wuhan Children's Hospital in China, six had elevated levels of C-reactive protein, procalcitonin, and dehydrogenase. Furthermore, four of the eight had abnormal liver function tests. Three of these patients developed a cytokine storm, which was particularly severe in critically ill patients.

[0168] Another study demonstrated that 74 of 651 patients (11.4%, mean age 46.14 years) experienced at least one gastrointestinal (GI) symptom, including nausea, vomiting, or diarrhea, and 10.8% of the patients had liver disease.

[0169] In addition, among the 74 patients with COVID-19, 29 patients (39.19%) had high fever (38.5°C), 23 patients (31.08%) reported fatigue, 8 patients (10.81%) reported difficulty breathing, and 16 patients (21.62%) reported headaches.

[0170] These results suggest that ACE2 receptors are expressed in LL24 (lung fibroblasts), BEAS-2B cells (GI epithelial cells), and hepatocytes. Furthermore, as confirmed by the BrainAtlas database, ACE2 expression levels in the brain have not been fully investigated. However, even if actual expression levels are low, given the clinical findings above, we predict that the brain is likely a target organ for SARS-CoV-2. Similarly, ACE2 is highly expressed in brain tumor cells, suggesting that brain cells may be targets of the virus.

[0171] Therefore, to confirm the indirect antiviral effects of various EV-derived miRNAs, experiments were conducted on cells that may be hypersensitive to SARS-COV2, such as hepatocytes, GI cells, and brain cells.

[0172] The specific experimental methods and cell culture methods are as described above. In short, the protective effect of EVs and miRNAs was confirmed by treating the cell lines with EVs and miRNAs for 24 hours and then with LPS. Conversely, the repair effect was confirmed by treating the cell lines with LPS for 24 hours and then with EVs and miRNAs. Figure 7 ).

[0173] Example 6 Anti-inflammatory and cytokine storm inhibitory effects of EV and miRNA

[0174] Using BEAS-2B (gastrointestinal epithelial cells), LL24 (lung cell line), HepG2 (liver cancer cell line), MEF (fibroblasts), BV2 (microglial cell line) and M2 (neuronal cells) as the subjects, the anti-inflammatory and cytokine storm inhibitory effects of EVs and miRNAs were tested by different LPS and EV treatments. The results are as follows: Figures 8 to 12 shown.

[0175] The protective effect was confirmed by treating BEAS-2B (gastrointestinal epithelial cells) with EV and then with LPS. Figure 8 As shown in A, the results confirmed that the relative expression of inflammatory factors IL-1, IL-6, IL-8 and TNF-a was significantly reduced compared with the case where none of them were treated with EVs.

[0176] In addition, if Figure 8 As shown in B, the protective effect was confirmed by treating five miRNAs separately and then treating with LPS. The results showed that the expression of IL-1, IL-6 and IL-8 was significantly reduced when miRNA181a was treated.

[0177] When LL-24 was treated with EV, the protective and repair effects were confirmed, and the results were as follows Figure 9 As shown in A, the results confirmed that when treated with EV, the expression of inflammatory factors IL-1, IL-6 and IL-8 were significantly reduced, and the protection and repair effects were excellent, especially the repair effect was excellent.

[0178] In addition, if Figure 9 As shown in B, the five miRNAs were treated with LL-24 and then with LPS to confirm the protective effect. The results showed that when miRNA181a was treated, the expression of IL-6 was significantly reduced to the same level as the control group.

[0179] The protective and repair effects of MEF (fibroblasts) and BV2 (microglial cell line) were confirmed, and the results were respectively Figure 10 As shown in A and 10B, the results confirmed that the inflammatory factors IL-1, IL-6 and TNF-a were significantly reduced in both MEFs and BV2 after EV treatment.

[0180] The expression levels of inflammatory factors in neuronal cells M2 after EV treatment were confirmed, and the results were shown as follows Figure 11 As shown, the results confirmed that only IL-1 showed a significant effect.

[0181] Figure 12 The images are taken before and after treatment of each of the above cell lines with PKH26-labeled EVs, confirming the successful binding (fusion) of EVs to each cell.

[0182] In other words, the above results can be combined to confirm that the EVs of the present invention and the miRNA contained in the EVs individually or as a whole exhibit the effect of inhibiting the expression of inflammatory factors, and therefore not only have anti-inflammatory effects, but also have the effect of inhibiting the cytokine storm of hypersensitivity reactions.

[0183] Example 7: The therapeutic effects of EV and miRNA on viral infections

[0184] The EV and miRNA of the present invention use all direct and indirect pathways to treat or improve viruses and viral infection symptoms ( Figure 13 ).

[0185] Through the above-mentioned various experiments, the inventors have confirmed that EVs derived from the placenta, umbilical cord, umbilical cord blood, placenta, stem cells (pMSC) obtained from these tissues, and EVs derived therefrom can not only improve pathological damage to the lungs, but also significantly improve other types of pneumonia and viral infections.

[0186] In particular, EVs contain stem cell regenerative factors, so when treating pneumonia, they exert a therapeutic mechanism similar to that of mesenchymal stem cells, but are considered to be safer.

[0187] Specifically, intravenous injection of mesenchymal stem cells can cause embolism and blood coagulation, while EVs are safer than stem cells because their size ranges from 200nm to 1 billion nanometers. EVs themselves are more stable than stem cells, are easier to store, do not transform into malignant or harmful cells, and are less likely to produce immune responses.

[0188] In addition, the experimental results of the present invention confirm that the miRNA in tissue-EV, tissue extract EV and pMSC-EV can directly bind to the viral genome, inhibit the transcription of RNA viruses, and thus inhibit the proliferation of the virus.

[0189] As other important functions of EV miRNAs, the 77 miRNAs present in various EVs target mRNAs that stimulate immune responses, and therefore can remove virally transcribed RNAs (viral RNAs), thus having not only direct antiviral effects but also very powerful indirect effects.

[0190] In addition, the miRNA of the present invention not only binds to Viral RNA, but also interacts with pro-inflammatory genes to significantly inhibit the expression of inflammatory factors, thereby resolving cytokine storms.

[0191] In addition, since the miRNA of the present invention has the effect of upregulating IFN-α / β levels, it can inhibit viral infection that evades the host immune response by inhibiting the IFN-α / β signaling pathway.

[0192] It was also confirmed that 18 miRNAs from various EVs directly interacted with the 3'UTR of SARS-CoV-2, and 5 major miRNAs derived from EVs were able to successfully inhibit SARS-CoV-2.

[0193] Several patients infected with the coronavirus have experienced liver and gastrointestinal damage in addition to primary respiratory symptoms. Of the 99 patients admitted to Wuhan Jinyintan Hospital, 43 had impaired liver function, one of whom had severe liver damage. While the extent of liver damage in mild cases is unclear, severe cases have shown deterioration in key liver function indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH).

[0194] Therefore, not only can it exert direct viral inhibition ability, but it can also synergistically exert the anti-inflammatory and other effects of EVs derived from the placenta, umbilical cord, and umbilical cord blood. Therefore, if EVs derived from the placenta are used as a therapeutic agent for coronavirus, it is expected to have a significant and excellent therapeutic effect.

[0195] The foregoing description of the present invention is merely illustrative. Therefore, it should be understood that a person skilled in the art could readily implement the present invention in other specific forms without altering the technical principles or essential features of the present invention. Therefore, the embodiments described above are, in all respects, merely illustrative and should not be construed as limiting. For example, components described in a single form may be implemented separately, and similarly, components described in a separate form may also be implemented in combination.

[0196] Therefore, the scope of the present invention should be determined by the following claims, and all modifications and variations derived from the meaning and scope of the claims and equivalent concepts should be included in the scope of the present invention.

Claims

1. Use of an antiviral composition in the preparation of an antiviral drug, wherein: The antiviral composition comprises: at least one carrier, excipient or diluent, and also a biomolecule extracted from the placenta, The biomolecule is miRNA contained in extracellular vesicles of placenta-derived mesenchymal stem cells, The miRNA is selected from one or more of the group consisting of hsa-miR-92a-3p, hsa-miR-181a-5p, hsa-miR-26a-5p, hsa-miR-23a-3p, and hsa-miR-103a-3p, The virus is the SARS-CoV-2 coronavirus.

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