Viral growth inhibitor

Abstract

To provide a composition with antiviral activity against viruses such as influenza virus and SARS-CoV-2 virus.SOLUTION: The present invention provides a composition containing a bile acid receptor agonist, specifically a TGR5 agonist, PPARα agonist, FXR agonist, or PXR agonist, more specifically, cholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, or the like.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention is directed to providing compositions having antiviral activity comprising bile acid receptor agonists. [Background technology]

[0002] When viruses such as influenza virus and the new coronavirus (SARS-CoV-2 virus) infect a host organism, they invade cells through the mucosal surfaces of tissues in the oral, respiratory, digestive and urinary systems, and establish infection. Influenza virus and SARS-CoV-2 virus are known to infect through the mucous membranes of the respiratory tract.

[0003] In response to such viral infections, the body responds to pathogen infections through "mucosal immunity," which protects the body from invading pathogens such as viruses, and "systemic immunity," which works to eliminate foreign substances once pathogens have entered the body. The intestinal flora plays a major role in this "mucosal immunity."

[0004] Intestinal bacteria are thought to affect immune development and function in the body. It has been suggested that intestinal bacteria themselves and substances metabolized by intestinal bacteria are deeply involved in "innate immunity" against viruses, bacteria, etc.

[0005] Regarding influenza virus, it is known that when mice whose intestinal bacterial flora has been imbalanced by taking antibiotics for a long period of time are infected intranasally with a non-lethal dose of influenza virus and the immune response to the influenza virus is analyzed two weeks after infection, the virus-specific IgG antibody titer in the blood, the IgA antibody titer in the nasal wash, the virus-specific CD4T and CD8T cell responses in the spleen, and the number of CTLs in the lungs are reduced (Non-Patent Document 1).

[0006] In addition, in the case of novel coronavirus disease (COVID-19) caused by SARS-CoV-2, it has been suggested that intestinal bacteria may have a significant impact on the immune status of COVID-19 patients with respect to the difference in severity, and there is interest in evaluating SARS-CoV-2 infection, nutrition, and gastrointestinal function. In addition, since it has been revealed that more than 60% of SARS-CoV-2 virus-infected patients show gastrointestinal symptoms such as diarrhea, nausea, and vomiting, and that this group of patients tends to have a high tendency for the disease to be severe, it has also been suggested that the severity of COVID-19 may be predicted by analyzing the composition of the human intestinal microbiota (Non-Patent Document 2).

[0007] Thus, although a relationship between viral infection and the intestinal microbiota has been suggested, the relationship has not yet been elucidated. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Ichinohe T, et al., Proc Natl Acad Sci USA 108: 5354-5359, 2011 [Non-Patent Document 2] https: / / www.medrxiv.org / content / 10.1101 / 2020.04.22.20076091v1 Summary of the Invention [Problem to be solved by the invention]

[0009] The objective of the present invention is to develop a composition with antiviral activity by utilizing the effects of intestinal bacteria on immunity and on infection with viruses, including influenza viruses and SARS-CoV-2 viruses. [Means for solving the problem]

[0010] As a result of intensive research, the inventors of the present invention found that animals administered with a bile acid receptor agonist have significantly improved resistance to viral infection, and thus completed the present invention. Specifically, the inventors of the present invention demonstrated that the above-mentioned problems can be solved by providing a composition having antiviral activity containing a bile acid receptor agonist.

[0011] More specifically, in order to solve the above-mentioned problems, the present application provides the following aspects: [1]: A composition having antiviral activity comprising a bile acid receptor agonist; [2]: The composition according to [1], wherein the virus is an enveloped virus; [3]: The composition according to [2], wherein the virus is an influenza virus or a coronavirus; [4]: The composition according to any one of [1] to [3], wherein the bile acid receptor is selected from the group consisting of TGR5, PPARα, FXR, and PXR; [5]: The composition according to any one of [1] to [4], wherein the bile acid receptor agonist is a bile acid or a derivative thereof, a TGR5 agonist, a PPARα agonist, an FXR agonist, or a PXR agonist; [6]: Bile acid receptor agonists include cholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, hyocholic acid, glycohyocholic acid, taurohyocholic acid, muricholic acid, glycomylic acid, tauromylic acid 5α-cyprinol, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, lithocholic acid, glycolithocholic acid, taurolithocholic acid, hyodeoxycholic acid, glycohyodeoxycholic acid, taurohyodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, CCDC (HY-14229), INT-777, Hyodeoxycholic acid, TC-G 1005, INT-767, fenofibrate, pemafibrate, GW4064, Colforsin (Forskolin, HL 362), Turofexorate Isopropyl (XL335), Chenodeoxycholic Acid, T0901317, Sevelamer HCl, Lithocholic acid, Nidufexor (LMB-763), fexaramine, LY2562175, Obeticholic Acid, Vonafexor (EYP001), Guggulsterone E&Z, Tropifexor (LJN452), Cilofexor, SR-12813, and Rifampicin. Effect of the Invention

[0012] The composition of the present invention can be used to reduce the titer of the virus even when an animal is infected with the virus by inoculating the composition, and can therefore be used to prevent the symptoms from worsening. In addition, some of the substances that can be used as active ingredients are known as food ingredients, and can also be used as food compositions having antiviral activity. [Brief description of the drawings]

[0013] [Figure 1]FIG. 1 shows that mice fed a low-fiber diet and given antibiotics have reduced resistance to influenza virus and reduced survival rates. [Diagram 2] Figure 2 shows that when mice were fed a low-fiber diet or given antibiotics and then infected with influenza virus, the ratio of neutrophils in the alveoli, which is an indicator of viral infection, increased (Figure 2(a-1)(a-2)), and the level of CXCL1 produced in the blood by macrophages, neutrophils, and epithelial cells, which is an indicator of an inflammatory response, increased (Figure 2(b-1)(b-2)). [Figure 3-1] FIG. 3-1 shows that application of deoxycholic acid, a bile acid, reduced the titer of influenza virus infecting mice in a concentration-dependent manner (FIGS. 3(a-1) and (a-2)). [Figure 3-2] Figure 3-2 shows that administration of bile acids, deoxycholic acid or ursodeoxycholic acid, to mice improves resistance to influenza virus and survival rate (Figures 3(b-1)(b-2)(b-3)). [Figure 4] FIG. 4 shows that in cells to which various bile acids have been applied, the expression of influenza virus genes in cells after infection with influenza virus is reduced. [Diagram 5] FIG. 5 shows that application of a bile acid receptor agonist reduces influenza virus titer (a) and improves resistance to influenza virus and survival rate (b). [Figure 6] FIG. 6 shows that intracellular influenza virus gene expression after influenza virus infection is reduced in cells treated with a bile acid receptor agonist. [Figure 7] FIG. 7 shows that antibiotic treatment reduces resistance to SARS-CoV2 virus and reduces survival in Syrian hamsters. [Figure 8]FIG. 8 shows that application of the bile acid deoxycholic acid reduces the titer of SARS-CoV2 virus infecting Syrian hamsters. [Figure 9] FIG. 9 shows that administration of the bile acid cholic acid to Syrian hamsters improves their resistance to the SARS-CoV2 virus and improves their survival rate. [Figure 10] FIG. 10 shows that in cells treated with various bile acids, the expression of SARS-CoV2 viral genes in cells after infection with the SARS-CoV2 virus is reduced. [Figure 11] FIG. 11 shows that SARS-CoV2 viral titers are reduced when bile acid receptor agonists are applied. [Figure 12] FIG. 12 shows that application of a bile acid receptor agonist reduces the expression of SARS-CoV2 viral genes in cells after SARS-CoV2 virus infection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present inventors found that animals administered antibiotics or fed a low-fiber diet had significantly reduced resistance to viral infection and increased mortality. It is believed that the administration of antibiotics or the application of a low-fiber diet disrupts the balance of intestinal bacteria, and that disruption of the intestinal balance causes a reduction in resistance to viral infection.

[0015] The inventors also speculated that an imbalance in intestinal bacteria caused abnormalities in the metabolism of bile acids in the intestine, and demonstrated that application of bile acids (primary bile acids or secondary bile acids) to cells reduced the infectious titer of the virus in the cells, and administration of bile acids to animals increased resistance to viral infection and reduced mortality.

[0016] Furthermore, assuming that bile acids exert their effects by sending signals into cells, they demonstrated that application of a bile acid receptor agonist to cells reduced the infectious titer of the virus in the cells, and that administration of a bile acid receptor agonist to animals increased resistance to viral infection and reduced mortality.

[0017] Based on these findings, the inventors of the present invention found that animals administered with a bile acid receptor agonist have significantly improved resistance to viral infection, and have completed the present invention. Specifically, the inventors of the present invention demonstrated that the above-mentioned problems can be solved by providing a composition having antiviral activity containing a bile acid receptor agonist.

[0018] The composition of the present invention can be provided as a pharmaceutical composition, but when the active ingredient is one of the active ingredients described below that can be used as a food (for example, a primary bile acid produced in the body or a secondary bile acid produced by the action of bacteria in the intestinal tract), it can also be provided as a food composition.

[0019] <Target of treatment> Here, the target virus in the present invention is preferably an enveloped virus for which the antiviral activity of the composition in the present invention has been confirmed. Known enveloped viruses include influenza virus, RS virus, parainfluenza virus, coronavirus, herpes simplex virus, varicella-zoster virus, measles virus, rubella virus coronavirus, influenza virus, herpes virus, rubella virus, etc. The composition of the present invention can be used as a composition having antiviral activity against infection with any of these viruses. Of these viruses, the virus in the present invention is preferably influenza virus or coronavirus.

[0020] In the present invention, antiviral activity can include inhibition of virus attachment to cells, inhibition of virus entry into cells, reduction of viral protein production in virus-infected cells, inhibition of any step in the viral replication cycle in virus-infected cells, reduction of the infectious titer of the virus produced from virus-infected cells, resistance of an animal infected with the virus to the virus, non-fatal infection of an animal infected with the virus, and the like.

[0021] <Active ingredient> In the present invention, the bile acid receptor agonist used as an active ingredient refers to a substance that binds to a bile acid receptor expressed on the cell surface and sends an activation signal to the cell. Substances known in the art as bile acid receptors include TGR5, PPARα, FXR, and PXR, and in the present invention, any of these bile acid receptors may send a signal into the cell.

[0022] In the art, bile acid receptors include TGR5, PPARα, farnesoid X receptor (FXR), and pregnane X receptor (PXR). Bile acids synthesized in the liver are secreted into the intestine via the bile duct. Most bile acids are reabsorbed in the ileum in the lower part of the small intestine and return to the liver via the portal vein. Bile acids are recycled through this "enterohepatic circulation". Bile acid receptors are receptors of cells involved in this "enterohepatic circulation".

[0023] Here, TGR5 is a bile acid receptor expressed on the surface of the cell membrane of L cells present in the human digestive tract. It is a receptor classified as a G protein-coupled receptor, and when it detects bile acids on the cell surface, it increases the concentration of cAMP within the cell. When TGR5 is activated, it acts to suppress the function of nearby macrophages, while at the same time regulating the body's energy consumption, and is known to have anti-obesity and anti-diabetic effects.

[0024] PPARα is a nuclear receptor for bile acids that is distributed in the liver, kidney, heart, and skeletal muscles, and is particularly abundant in the liver. PPARα forms a heterodimer with retinoid X receptor (RXR) in the nucleus and controls gene transcription by binding to a ligand. It is activated by free fatty acids and other physiological ligands (signal transmitters), and plays an important role in fatty acid metabolism.

[0025] Farnesoid X receptor (FXR) is a nuclear receptor that is highly expressed in the liver, small intestine, kidney, and adrenal gland. It forms a heterodimer with retinoid X receptor (RXR) in the nucleus and binds to the FXR responsive element (FXRE) of target genes, promoting or suppressing their transcription. Intermediates of bile acid biosynthesis also act as strong physiological ligands for FXR. These tissues are the sites where cholesterol is metabolized and decomposed into bile acids or steroid hormones and excreted from the body, and it has been revealed that FXR functions to maintain bile acid / cholesterol homeostasis.

[0026] Pregnane X receptor (PXR) is a nuclear receptor expressed in tissues such as the liver and intestinal tract, and is involved in the expression and induction of important proteins that determine pharmacokinetics, such as CYP3A and P-glycoprotein. When a ligand binds to PXR, PXR is activated and forms a dimer with retinoid X receptor (RXR), which then migrates into the nucleus and functions as a transcription factor. It has been reported that activation of PXR results in an inflammation-reducing effect.

[0027] The bile acid receptor agonist in the present invention refers to the substance that binds with the above-mentioned bile acid receptor and sends activation signal to cells.For example, the bile acid receptor agonist that can be used in the present invention can include bile acid or its derivative, TGR5 agonist, PPARα agonist, FXR agonist, or PXR agonist.

[0028] Bile acids that can be used as bile acid receptor agonists in the present invention include primary bile acids that are biosynthesized in the liver of animals, and secondary bile acids that are produced as metabolites by intestinal bacteria when a portion of the primary bile acids is metabolized by the intestinal bacteria in the intestinal tract.

[0029] Examples of primary bile acids include cholic acid, chenodeoxycholic acid, hyocholic acid, muricholic acid, and 5α-cyprinol. Here, conjugated bile acids in which these primary bile acids are bound to glycine or taurine, i.e., glycocholic acid, which is the glycine conjugated form of cholic acid, and taurocholic acid, which is the taurine conjugated form, glycochenodeoxycholic acid, which is the glycine conjugated form of chenodeoxycholic acid, and taurochenodeoxycholic acid, which is the taurine conjugated form, glycohyocholic acid, which is the glycine conjugated form of hyocholic acid, and taurohyocholic acid, which is the taurine conjugated form, glycomyuricholic acid, which is the glycine conjugated form of muricholic acid, and tauromyuricholic acid, which is the taurine conjugated form, are also included in primary bile acids. In the present invention, derivative compounds of these primary bile acids can also be used.

[0030] Examples of secondary bile acids include deoxycholic acid, lithocholic acid, hyodeoxycholic acid, and ursodeoxycholic acid. Here, conjugated bile acids that exist in combination with glycine or taurine, that is, glycodeoxycholic acid, which is the glycine conjugated form of deoxycholic acid, and taurodeoxycholic acid, which is the taurine conjugated form of taurideoxycholic acid, which is the glycine conjugated form of lithocholic acid, and taurideoxycholic acid, which is the taurine conjugated form of taurolithocholic acid, which is the glycine conjugated form of hyodeoxycholic acid, and taurideoxycholic acid, which is the taurine conjugated form of hyodeoxycholic acid, and glycoursodeoxycholic acid, which is the glycine conjugated form of ursodeoxycholic acid, and tauroursodeoxycholic acid, which is the taurine conjugated form of ursodeoxycholic acid, are also included in secondary bile acids. In the present invention, derivative compounds of these secondary bile acids can also be used.

[0031] Examples of TGR5 agonists that can be used in the present invention include, but are not limited to, CCDC (HY-14229) having the following chemical structure, INT-777, hyodeoxycholic acid, TC-G 1005, and INT-767.

[0032] [ka]

[0033] [ka]

[0034] PPARα agonists that can be used in the present invention include, but are not limited to, fenofibrate and pemafibrate, which have the following chemical structures:

[0035] [ka]

[0036] FXR agonists that can be used in the present invention include, but are not limited to, GW4064, having the following chemical structure, Colforsin (Forskolin, HL 362), Turofexorate Isopropyl (XL335), Chenodeoxycholic Acid, T0901317, Sevelamer HCl, Lithocholic acid, Nidufexor (LMB-763), fexaramine, LY2562175, Obeticholic Acid, Vonafexor (EYP001), Guggulsterone E&Z, Tropifexor (LJN452), and Cilofexor.

[0037] [ka]

[0038] [ka]

[0039] [ka]

[0040] [ka]

[0041] PXR agonists that can be used in the present invention include, but are not limited to, SR-12813, which has the following chemical structure, and rifampicin.

[0042] [ka]

[0043] The composition having antiviral activity of the present invention can be used to prevent viral infection, treat viral infection, or accelerate recovery from viral infection. That is, the present invention can provide a method for preventing viral infection, a method for treating viral infection, or a method for accelerating recovery from viral infection, comprising administering a bile acid receptor agonist. The bile acid receptor agonist used here is as described above.

[0044] This method can be effectively used against infections caused by enveloped viruses, particularly influenza viruses and coronaviruses.

[0045] In the present invention, the method of administration of the bile acid receptor agonist can be appropriately determined depending on whether it is a method for preventing viral infection, a method for treating viral infection, or a method for hastening recovery from viral infection. For example, viral infection can be prevented by continuously administering a bile acid receptor agonist to animals, including humans, on a daily basis, and when a viral infection of an animal, including humans, is confirmed, the viral infection can be treated or recovery from the viral infection can be hastened by administering the bile acid receptor agonist until the virus can no longer be detected in a biological sample.

[0046] The present invention will be described in more detail below with reference to examples, but the following examples are not intended to limit the present invention in any way. EXAMPLES

[0047] Example 1: Changes in influenza virus resistance by diet or antibiotic administration In this example, it was confirmed whether a low-fiber diet or administration of antibiotics would affect resistance to influenza viruses in mice.

[0048] For the experiment, 7 to 10 female C57BL / 6 (wild type) mice were assigned to each of three groups: a control group that was given a normal diet (CMF, Oriental Yeast Co., Ltd.) and water, a low-fiber diet group (LF group), and a normal diet and an eight-drug antibiotic mixture group (Abx).They were kept under SPF conditions with free access to food and water, with a 12-hour light / dark cycle, for 4 weeks.

[0049] Mice in the antibiotic-treated group (Abx) were housed for 4 weeks with free access to water containing ampicillin (1 g / L; Nacalai Tesque), vancomycin (500 mg / L; Duchefa Biochemie), neomycin sulfate (1 g / L; Nacalai Tesque), metronidazole (1 g / L; Nacalai Tesque), gentamicin (10 mg / L; Nacalai Tesque), penicillin (100 U / ml; Nacalai Tesque), streptomycin (100 U / ml; Nacalai Tesque), and amphotericin B (0.25 mg / L; Nacalai Tesque) in their drinking water.

[0050] Thereafter, the mice were intranasally infected with influenza A virus (A / PR8 strain, provided by the National Institute of Infectious Diseases) by intranasally administering 30 μL of a virus suspension (1,000 pfu of influenza virus in PBS) to the mice under complete anesthesia by intraperitoneal injection of sodium pentobarbital (the day of infection was designated as Day 0).

[0051] These mice were kept for 14 days under the same conditions as before viral infection, and the results of confirming survival and death are shown in Figure 1. As shown in these results, when fed a normal diet, all mice survived (survival rate 100%), whereas in both the LF group fed a low-fiber diet and the Abx group administered antibiotics, some mice died 7 or 8 days after viral infection, and after 14 days the survival rate had fallen to 20% or less.

[0052] Lung tissues from mice 1–7 days after viral infection were harvested by perfusing 10 ml of PBS through the right ventricle, cutting into pieces, and incubating in HBSS containing 2.5 mM Hepes and 1.3 mM EDTA at 37°C for 30 min. Cells were then resuspended in RPMI containing 5% FBS, 1 mM CaCl2, 1 mM MgCl2, 2.5 mM Hepes, and 0.5 mg / ml collagenase D (Roche) and incubated at 37°C for 60 min. After RBC lysis, the resulting cells were filtered through a 70 μm cell strainer (BD) to prepare single cell suspensions.

[0053] The cells were then incubated with neutrophil-specific eFluor450-labeled anti-Ly6G and APC-labeled anti-Ly6C antibodies. Flow cytometry analysis was performed using a FACSVerse flow cytometer (BD Biosciences). Analysis was performed using FlowJo software (Tree Star, Inc.).

[0054] We also measured CXCL1, a type of chemokine that is induced to be produced in the blood by macrophages, neutrophils, and epithelial cells activated by inflammatory mediators. CXCL1 in 100 μl of serum was measured using Mouse CXCL1 ELISA Kit (#KE10019).

[0055] The percentage of neutrophils in lung cells in each experimental group is shown in Figures 2(a-1) and (a-2). In these figures, the percentage of neutrophils was higher in the LF and Abx groups compared to the control group, suggesting that the inflammatory response due to viral infection was enhanced.

[0056] The amount of CXCL1 in the serum of each experimental group (pg / ml) is shown in Figure 2(b-1) and (b-2). In these figures, the amount of CXCL1 in the serum was higher in the LF and Abx groups compared to the control group, suggesting that the viral infection caused a stronger inflammatory response in the body than in the control group.

[0057] Example 2: Changes in influenza virus resistance by application of bile acids In this example, it was confirmed whether administration of bile acid changes resistance to influenza virus in mice.

[0058] First, we confirmed whether the titer of influenza virus was changed by the addition of bile acids. Influenza A virus (A / PR8 strain, provided by the National Institute of Infectious Diseases) was infected at MOI=0.01, and deoxycholic acid (Nacalai Tesque, Inc.) dissolved in DMSO was added as a bile acid to the culture medium of Madin-Darby canine kidney (MDCK) cells (JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition) at 0 mM, 0.3 mM, 0.6 mM, 1.25 mM, and 2.5 mM, and the titer of influenza virus was measured after 24 hours. The virus titer was quantified by a standard plaque assay using Madin-Darby canine kidney (MDCK) cells.

[0059] As a result, it was shown that when 0.6 mM, 1.25 mM, or 2.5 mM deoxycholic acid was added, the influenza virus titer was reduced in a concentration-dependent manner (Figure 3(a-1)).

[0060] Next, the change in influenza virus titer when 1.25 mM deoxycholic acid was added was quantified by a standard plaque assay using Madin-Darby canine kidney (MDCK) cells 24 hours or 48 hours after infection. As a result, in the group without deoxycholic acid (only DMSO added), the virus titer increased over time, whereas in the group with deoxycholic acid, the virus titer did not increase over time (Figure 3(a-2)).

[0061] Next, we investigated whether bile acids affect the survival rate of mice infected with influenza virus. For the experiment, 16 to 22 female C57BL / 6 (wild type) mice were assigned to each of the following groups: a low-fiber diet (LF (water) group), a low-fiber diet + 2% cholesterol group (LF (w / 2% cholesterol) group) (cholesterol is a raw material for bile acids), a low-fiber diet + 0.5 mM deoxycholic acid group (LF (w / 0.5 mM DCA) group), and a group supplemented with 0.5 mM ursodeoxycholic acid (LF (w / 0.5 mM UDCA) group). They were kept under SPF conditions with free access to food and water, and a 12-hour light / dark cycle for 4 weeks.

[0062] Thereafter, the mice were intranasally infected with influenza A virus (A / PR8 strain, provided by the National Institute of Infectious Diseases) by intranasally administering 30 μL of a virus suspension (1,000 pfu of influenza virus in PBS) to the mice under complete anesthesia by intraperitoneal injection of sodium pentobarbital (the day of infection was designated as Day 0).

[0063] These mice were kept for 14 days under the same conditions as before virus infection, and the survival and death were confirmed. The results are shown in Figure 3 (b-1), (b-2), and (B-3). As shown by these results, compared with the LF (water) group fed a low-fiber diet, the survival rate was improved in the group to which cholesterol, a raw material for bile acids, was added, and the survival rate was further improved in the group to which deoxycholic acid (secondary bile acid) or ursodeoxycholic acid (secondary bile acid) was added, which are bile acids.

[0064] Example 3: Intracellular changes in response to influenza virus by application of bile acids In this example, it was confirmed what changes occurred in influenza virus proliferation in cells infected with influenza virus by application of bile acids.

[0065] At the same time as infection with influenza A virus (A / PR8 strain, provided by the National Institute of Infectious Diseases) MOI=0.01, cholic acid (CA) (Nacalai Tesque, Inc.), deoxycholic acid (DCA) (Nacalai Tesque, Inc.), taurocholic acid (TCA) (Nacalai Tesque, Inc.), taurodeoxycholic acid (TDCA) (Nacalai Tesque, Inc.), or ursodeoxycholic acid (UDCA) (Chemodex) dissolved in DMSO were added to the culture medium of Madin-Darby canine kidney (MDCK) cells to a concentration of 0.125 mM as bile acids. The cells were harvested 9 or 24 hours after virus infection.

[0066] The cell extracts were collected at each time point, and the expression levels of tubulin and influenza virus proteins (NS1, M2) were analyzed by Western blotting. As a result, at 9 hours after virus infection, the expression levels of both NS1 and M2 were decreased in deoxycholic acid, taurocholic acid, taurodeoxycholic acid, and ursodeoxycholic acid, and the expression levels of NS1 and M2 were particularly decreased in deoxycholic acid and ursodeoxycholic acid. This tendency was almost the same at 24 hours after virus infection (Figure 4). In other words, it was suggested that secondary bile acids suppress influenza virus translation in cells.

[0067] Example 4: Changes in influenza virus resistance by application of bile acid receptor agonists In this example, it was confirmed whether administration of a bile acid receptor agonist altered resistance to influenza virus in mice.

[0068] First, we confirmed whether the titer of influenza virus was changed by the addition of bile acid receptor agonists. After infection with influenza A virus (A / PR8 strain, provided by the National Institute of Infectious Diseases) at MOI = 0.01, FXR agonist GW4064 (Selleck) and TGR5 agonist HY-14229 (Namiki Shoji) dissolved in DMSO were added to the culture medium of Madin-Darby canine kidney (MDCK) cells (JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition) at 1 μM as bile acid receptor agonists, and the titer of influenza virus was measured after 24 hours. The virus titer was quantified by a standard plaque assay using Madin-Darby canine kidney (MDCK) cells.

[0069] The results showed that the addition of the TGR5 agonist HY-14229 reduced the influenza virus titer (FIG. 5(a)).

[0070] Next, we investigated whether bile acid receptor agonists affect the survival rate of mice infected with influenza virus. The experiment was performed only for the TGR5 agonist HY-14229, which was shown to reduce influenza virus titers in this example.

[0071] Female C57BL / 6 mice (wild type) were divided into two groups (25 mice and 73 mice) and housed under specific pathogen-free conditions with a low-fiber diet and free access to water for 4 weeks under a 12-h light / dark cycle. The 73 mice group had 100 μM of the TGR5 agonist HY-14229 added to their drinking water (100 μM TGR5 group), while the other group (25 mice) did not (water group).

[0072] Thereafter, the mice were intranasally infected with influenza A virus (A / PR8 strain, provided by the National Institute of Infectious Diseases) by intranasally administering 30 μL of a virus suspension (1,000 pfu of influenza virus in PBS) to the mice under complete anesthesia by intraperitoneal injection of sodium pentobarbital (the day of infection was designated as Day 0).

[0073] These mice were kept under the same conditions as before the virus infection for 14 days, and the survival and death were confirmed. The results are shown in Figure 5(b). As shown in the results, the survival rate was improved in the group to which the TRG5 agonist was added, compared to the group given only water (water group).

[0074] Example 5: Intracellular changes in response to influenza virus by application of bile acid receptor agonists In this example, it was confirmed what changes occurred in influenza virus proliferation in cells infected with influenza virus by application of a bile acid receptor agonist.

[0075] The effects of bile acid receptor agonist FXR agonist GW4064 and TGR5 agonist HY-14229 in influenza-infected cells were examined. After infection with influenza A virus (A / PR8 strain, National Institute of Infectious Diseases) at MOI=0.01, Madin-Darby canine kidney (MDCK) cells were cultured with GW4064 or HY-14229 dissolved in DMSO at 0.1μM, 1μM, 10μM, or 100μM, and incubated for 24 hours. Cells died when 10μM or 100μM GW4064 was added, so they were excluded from the experiment.

[0076] 24 hours after virus infection, cell lysates were collected and the expression levels of tubulin and influenza virus proteins (NS1, M2) were analyzed by Western blotting.

[0077] As a result, 11 hours after viral infection, the expression level of SARS-Cov-2 nucleocapsid was reduced in all cases of cholic acid, deoxycholic acid, taurocholic acid, taurodeoxycholic acid, and ursodeoxycholic acid, with the expression level being significantly reduced when deoxycholic acid and ursodeoxycholic acid were applied (Figure 10).

[0078] Example 6: Changes in SARS-CoV-2 virus resistance with antibiotic application In this example, we confirmed whether the application of antibiotics altered resistance to the SARS-CoV-2 virus in Syrian hamsters.

[0079] In the experiment, 5-6 one-month-old female Syrian hamsters (Japan SLC Inc.) were assigned to each of the control group (Control group) that was given water together with a normal diet (CMF, Oriental Yeast Co., Ltd.) and the group (Abx) that was given a seven-drug antibiotic mixture (see Example 1 above) together with a normal diet. They were kept under SPF conditions with free access to food and water, with a 12-hour light / dark cycle, for 4 weeks. The antibiotic group (Abx) was administered antibiotics in the free drinking water as in Example 1.

[0080] The Syrian hamsters were then infected intranasally with SARS-CoV-2 / UT-NCGM02 / Human / 2020 / Tokyo (provided by Professor Yoshihiro Kawaoka, Institute of Medical Science, University of Tokyo). The virus was then infected intranasally with 2 × 10 6 This was done by intranasally administering pfu of SARS-CoV-2 (the day of infection was designated Day 0).

[0081] All experiments with SARS-CoV-2 were carried out in the enhanced biosafety level 3 (BSL-3) containment research facility at the University of Tokyo, following the institute's biosafety operating procedures.

[0082] These Syrian hamsters were kept under the same conditions as before the virus infection for 14 days, and the survival and death were confirmed. The results are shown in Figure 7. As shown in the results, when fed a normal diet, all of the hamsters survived (survival rate 100%), whereas in the Abx group, which received antibiotics, some individuals died 3 days after the virus infection, and after 14 days the survival rate had dropped to 20% or less.

[0083] Example 7: Changes in SARS-CoV-2 viral resistance by application of bile acids In this example, we confirmed whether the administration of bile acids altered resistance to the SARS-CoV-2 virus in Syrian hamsters.

[0084] First, we examined whether the titer of SARS-CoV-2 virus changed with the addition of bile acids. SARS-CoV-2 virus (SARS-CoV-2 / UT-NCGM02 / Human / 2020 / Tokyo (provided by Professor Yoshihiro Kawaoka, Institute of Medical Science, University of Tokyo) ~10 7 Deoxycholic acid (in DMSO) was added as a secondary bile acid to the pfu / ml virus solution at final concentrations of 0 mM, 0.3 mM, 0.6 mM, 1.25 mM, and 2.5 mM. After 60 min of incubation at 37°C, SARS-CoV-2 virus titers were quantified by standard plaque assay using Madin-Darby canine kidney (MDCK) cells.

[0085] The results showed that the titer of the SARS-CoV-2 virus decreased when 1.25 mM deoxycholic acid was added, and that the titer of the SARS-CoV-2 virus was significantly reduced when 2.5 mM deoxycholic acid was added (Figure 8).

[0086] Next, we investigated whether bile acids affect the survival rate of Syrian hamsters when they are infected with the SARS-CoV-2 virus. One-month-old female Syrian hamsters (Japan SLC Inc.) were divided into two groups of 5 to 6 animals each. One group had 0.5 mM of the bile acid cholic acid (Nacalai Tesque, Inc.) added to their drinking water (CA (0.5 mM) group), while the other group did not (Control group). They were kept under SPF conditions with free access to the same diet as in Example 6 and free access to water, with a 12-hour light / dark cycle, for 4 weeks.

[0087] The Syrian hamsters were then infected intranasally with the highly virulent (British) SARS-CoV-2 virus mutant strain QK002 (lineage B.1.1.7) (provided by the National Institute of Infectious Diseases). The virus was infected by intranasal route at 2 × 10 6 This was done by intranasally administering pfu of SARS-CoV-2 (the day of infection was designated Day 0).

[0088] These Syrian hamsters were kept under the same conditions as before the virus infection for 14 days, and the survival and death results were confirmed, as shown in Figure 9. As shown by these results, the survival rate was improved in the group to which cholic acid was added (CA (0.5 mM) group) compared to the group to which only water was given (Control group).

[0089] Example 8: Intracellular changes in response to SARS-CoV-2 virus by application of bile acids In this example, we examined what changes the application of bile acids had on the proliferation of SARS-CoV-2 virus in cells infected with the SARS-CoV-2 virus.

[0090] As bile acids, cholic acid (CA) (Nacalai Tesque, Inc.), deoxycholic acid (DCA) (Nacalai Tesque, Inc.), taurocholic acid (TCA) (Nacalai Tesque, Inc.), taurodeoxycholic acid (TDCA) (Nacalai Tesque, Inc.), or ursodeoxycholic acid (UDCA) (Chemodex) dissolved in DMSO was added to the culture medium of VeroE6 / TMPRSS2 cells to a concentration of 0.125 mM. At that time, the cells were infected with the SARS-CoV-2 virus QK002 mutant strain (strain B.1.1.7) (provided by the National Institute of Infectious Diseases) at an MOI of 0.01, and the cells were harvested after 11 and 14 hours.

[0091] Eleven and 14 hours after virus infection, cell lysates were collected and the expression levels of tubulin and SARS-CoV-2 nucleocapsid were analyzed by Western blotting.

[0092] As a result, at 11 hours after virus infection, the expression level of nucleocapsid was decreased in each of cholic acid, deoxycholic acid, taurocholic acid, taurodeoxycholic acid, and ursodeoxycholic acid, but the expression level of nucleocapsid was significantly decreased when deoxycholic acid and ursodeoxycholic acid were added (Figure 10). This tendency was almost the same at 14 hours after virus infection (Figure 10).

[0093] Example 9: Changes in SARS-CoV-2 virus resistance by application of bile acid receptor agonists In this example, we examined whether the administration of a bile acid receptor agonist altered resistance to the SARS-CoV-2 virus in Syrian hamsters.

[0094] We determined whether SARS-CoV-2 virus titers were altered by the addition of a bile acid receptor agonist. After infection with SARS-CoV-2 virus (SARS-CoV-2 / UT-NCGM02 / Human / 2020 / Tokyo (kindly provided by Professor Yoshihiro Kawaoka, Institute of Medical Science, University of Tokyo), 100 μM of bile acid receptor agonists, FXR agonist GW4064 (Selleck), TGR5 agonist HY-14229 (Namiki Shoji), and the negative control anti-influenza drug amantadine (Sigma-Aldrich), were added to the culture medium of Madin-Darby canine kidney (MDCK) cells (JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition) as bile acid receptor agonists. The SARS-CoV-2 virus titer was measured after 24 hours. The virus titer was quantified by standard plaque assay using Madin-Darby canine kidney (MDCK) cells.

[0095] The results showed that the addition of the FXR agonist GW4064 and the TGR5 agonist HY-14229 reduced the titer of the SARS-CoV-2 virus, and that the addition of the FXR agonist GW4064 significantly reduced the titer of the SARS-CoV-2 virus (Figure 11).

[0096] Example 10: Changes in intracellular response to SARS-Cov-2 virus by application of bile acid receptor agonists In this example, we examined whether the application of a bile acid receptor agonist had any effect on the proliferation of SARS-Cov-2 virus in cells infected with the SARS-Cov-2 virus.

[0097] The effects of the bile acid receptor agonist FXR agonist GW4064 and the TGR5 agonist HY-14229 in cells infected with the SARS-CoV-2 virus were examined. GW4064 or HY-14229 dissolved in DMSO was added to the culture medium of VeroE6 / TMPRSS2 cells at 10μM or 100μM, and the cells were infected with the SARS-CoV-2 virus (SARS-CoV-2 / UT-NCGM02 / Human / 2020 / Tokyo (provided by Professor Yoshihiro Kawaoka, Institute of Medical Science, University of Tokyo) at MOI=0.01. After 24 hours, the cells were harvested.

[0098] Twenty-four hours after virus infection, cell lysates were collected and the expression levels of tubulin and SARS-CoV-2 nucleocapsid were analyzed by Western blotting.

[0099] As a result, 24 hours after virus infection, both 100 μM GW4064 and HY-14229 reduced the expression of SARS-Cov-2 nucleocapsid (Figure 12). [Industrial Applicability]

[0100] The composition of the present invention can be used to reduce the titer of the virus even when an animal is infected with the virus by inoculating the composition, and can therefore be used to prevent the symptoms from worsening. In addition, some of the substances that can be used as active ingredients are known as food ingredients, and can also be used as food compositions having antiviral activity.

Claims

1. A composition having antiviral activity comprising a bile acid receptor agonist.

2. The composition of claim 1 , wherein the virus is an enveloped virus.

3. The composition of claim 2, wherein the virus is an influenza virus or a coronavirus.

4. The composition according to any one of claims 1 to 3, wherein the bile acid receptor is selected from the group consisting of TGR5, PPARα, FXR, and PXR.

5. The composition of any one of claims 1 to 4, wherein the bile acid receptor agonist is a bile acid or a derivative thereof, a TGR5 agonist, a PPARα agonist, an FXR agonist, or a PXR agonist.

6. Bile acid receptor agonists include cholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, hyocholic acid, glycohyocholic acid, taurohyocholic acid, muricholic acid, glycomylic acid, tauromylic acid 5α-cyprinol, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, lithocholic acid, glycolithocholic acid, taurolithocholic acid, hyodeoxycholic acid, glycohyodeoxycholic acid, taurohyodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, CCDC (HY-14229), INT-777, Hyodeoxycholic acid, TC-G 1005, INT-767, Fenofibrate, Pemafibrate, GW4064, Colforsin (Forskolin, HL 362), Turofexorate Isopropyl (XL335), Chenodeoxycholic Acid, T0901317, Sevelamer HCl, Lithocholic acid, Nidufexor (LMB-763), fexaramine, LY2562175, Obeticholic Acid, Vonafexor (EYP001), Guggulsterone E&Z, Tropifexor (LJN452), Cilofexor, SR-12813, and Rifampicin.

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