Preventive or therapeutic agent against livestock infectious disease caused by foot-and-mouth disease virus

Abstract

In order to take measures against livestock infectious diseases such as foot-and-mouth disease, the present invention provides an infectious disease prevention agent that has high production efficiency and that can be easily administered to livestock. Specifically, the present invention relates to an antiviral agent against foot-and-mouth disease virus (FMDV), comprising, as an active ingredient, at least one antiviral compound selected from the group consisting of sangivamycin and salts thereof, pactamycin and salts thereof, isororidin E and salts thereof, tubercidin and salts thereof, and nafuredin and salts thereof.

Description

Prevention or treatment of livestock infectious diseases caused by foot-and-mouth disease virus

[0001] This invention relates to an antiviral agent against foot-and-mouth disease virus (FMDV), a pharmaceutical or feed composition containing the antiviral agent, and methods for producing the same. The invention also relates to an antiviral agent and method for the treatment or prevention of foot-and-mouth disease.

[0002] Foot-and-mouth disease (FMD) is a highly virulent livestock disease caused by the FMD virus (FMDV). It infects even-toed ungulates such as cattle, pigs, and sheep, as well as wild animals, and is extremely contagious. In the event of an outbreak in Japan, disease control measures such as restricting the movement of livestock around the affected area and culling infected animals will be taken to prevent its spread. If it is deemed difficult to prevent the spread of infection through these measures, vaccination will be administered to healthy, uninfected livestock. Currently, inactivated vaccines for FMD are imported and stockpiled as national disease control materials. However, in addition to seven serotypes of FMD that are ineffective against each other by the vaccine, there are diverse antigenic differences even among strains belonging to the same serotype. If the antigenicity of the strain that has entered Japan does not match that of the stockpiled vaccine strain, there is a risk that the expected infection-preventing effect will not be achieved. Furthermore, since it takes at least seven days for stockpiled vaccines to provide sufficient immunity to vaccinated animals, preventing the spread of the epidemic requires vaccinating all susceptible livestock in and around areas suspected of exposure to the virus, resulting in an enormous number of animals to be vaccinated. Moreover, while these stockpiled vaccines suppress the onset of the disease, they do not prevent infection itself, meaning that even asymptomatic animals could potentially become new sources of infection. For this reason, in Japan, all vaccinated animals are subject to culling, further exacerbating the damage to the livestock industry.

[0003] Nishi T. et al. (2022) Antiviral Res 208:105425

[0004] Therefore, fast-acting antiviral drugs against FMDV are useful as disease control measures and spread prevention technologies during outbreaks. To date, several synthetic compounds have been reported to show antiviral effects against FMD, but due to issues such as toxicity to the host and manufacturing costs, none have been put into practical use as anti-FMD drugs. In the inventor's previous research, it was revealed that pyrazinecarboxamide derivative T-1105 rapidly spreads throughout the body when administered orally to pigs and shows excellent antiviral effects against FMD (Non-Patent Literature 1), but as with the above, improvements in safety and manufacturing costs are desired.

[0005] The present invention aims to develop a disease prevention drug that is highly efficient to produce and easy to administer to livestock, for the purpose of combating infectious diseases of livestock such as FMD.

[0006] The inventors continued their search for substances that inhibit the proliferation of foot-and-mouth disease virus (FMDV) in microbial cultures. As a result, they discovered that sangibamycin, patamycin, isorolidine E, tubercidine, and nafrezin each possess FMDV inhibitory activity, and based on this finding, they completed the present invention.

[0007] That is, the present invention includes, for example, the following aspects and embodiments: [1] An antiviral agent against foot-and-mouth disease virus (FMDV), comprising as an active ingredient at least one antiviral compound selected from the group consisting of sangibamycin and its salts, patamycin and its salts, isorolidine E and its salts, tubercidine and its salts, and nafrezin and its salts. [2] The antiviral agent according to [1], wherein the antiviral compound is contained in a culture medium obtained by culturing at least one microorganism selected from the group consisting of Actinomycete OH-4711 strain (accession number NITE BP-04184), Actinomycete K04-0072 strain (accession number NITE BP-04180), Filamentous fungus FKI-10030 strain (accession number NITE BP-04177), Actinomycete KP-2324 strain (accession number NITE BP-04183), and Filamentous fungus FKI-12011 strain (receipt number NITE ABP-04468). [3] The antiviral agent according to [1], wherein the target of administration or ingestion is an animal belonging to the order Artiodactyla. [4] The antiviral agent according to [1], wherein it is a feed additive. [5] A pharmaceutical or feed composition containing the antiviral agent according to any one of [1] to [4]. [6] The composition according to [5] for use in the prevention or treatment of foot-and-mouth disease. [7] The composition according to [5], wherein the target of administration or ingestion is an animal belonging to the order Artiodactyla. [8] A method for producing a pharmaceutical or feed composition for the prevention or treatment of foot-and-mouth disease, comprising incorporating the antiviral agent according to any one of [1] to [4] into a pharmaceutical raw material or feed raw material. [9] A method for treating or preventing foot-and-mouth disease in a subject, comprising administering an effective amount of the antiviral agent according to any one of [1] to [4] to the subject.

[10] The method according to [9], wherein the subject is an animal belonging to the order Artiodactyla.

[0008]

[11] At least one microorganism or a derivative thereof selected from the group consisting of Actinomycete OH-4711 strain (accession number NITE BP-04184), Actinomycete K04-0072 strain (accession number NITE BP-04180), Filamentous fungus FKI-10030 strain (accession number NITE BP-04177), Actinomycete KP-2324 strain (accession number NITE BP-04183), and Filamentous fungus FKI-12011 strain (receipt number NITE ABP-04468), or a processed product thereof.

[12] A culture medium containing an antiviral compound obtained by culturing at least one microorganism selected from the group consisting of Actinomycete strain OH-4711 (accession number NITE BP-04184), Actinomycete strain K04-0072 (accession number NITE BP-04180), Filamentous fungus strain FKI-10030 (accession number NITE BP-04177), Actinomycete strain KP-2324 (accession number NITE BP-04183), and Filamentous fungus strain FKI-12011 (receipt number NITE ABP-04468).

[0009] The present invention provides an antiviral agent against foot-and-mouth disease virus (FMDV), a pharmaceutical or feed composition for use in the treatment or prevention of foot-and-mouth disease, and a method for producing the same. The antiviral compound used in the present invention effectively inhibits the proliferation of FMDV and is therefore useful for the treatment or prevention of foot-and-mouth disease.

[0010] This graph shows the antiviral effects of compounds in cells infected with foot-and-mouth disease virus (FMDV). It also shows the IC50 effect of antiviral compounds in vitro. ５０ This table summarizes the other characteristics. This table summarizes the effects of antiviral compounds on the survival rate of foot-and-mouth disease model mice.

[0011] This specification encompasses the disclosures of Japanese Patent Application No. 2024-186875, filed on 23 October 2024, which forms the basis of the priority of this application. The present invention is described in detail below. The following embodiments are illustrative for illustrating the present invention and are not intended to limit the present invention to these embodiments only. The present invention can be carried out in various forms without departing from its spirit.

[0012] The present invention is based on the discovery of compounds (hereinafter also referred to as "antiviral compounds") having antiviral activity against foot-and-mouth disease virus (FMDV) from among compounds obtained from various microbial cultures. Accordingly, the present invention relates to an antiviral agent against foot-and-mouth disease virus (FMDV) comprising at least one antiviral compound as an active ingredient.

[0013] The antiviral compounds that can be used in the present invention include the following: (1) Sangibamycin and its salt sangibamycin are compounds represented by the following formula I, and their production and purification methods are also known (e.g., J. Med. Chem., 11:939-941 (1968)).

[0014]

[0015] Sangibamycin can be produced, for example, using Actinomycete strain OH-4711 (accession number NITE BP-04184) (Example 1). Actinomycete strain OH-4711 was deposited on October 18, 2024, with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE, Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu, Chiba, Japan 292-0818, Japan) as the international depositary authority under the Budapest Convention for the Deposit of Patent Microorganisms (accession number NITE BP-04184). Alternatively, sangibamycin is also available commercially.

[0016] (2) Pactamycin and its salt pactamycin are compounds represented by the following formula II, and their production and purification methods are also known (e.g., J. Antibiot. Ser. A., 17:230-233 (1964)).

[0017]

[0018] Pactamycin can be produced, for example, using Actinomycete strain K04-0072 (accession number NITE BP-04180) (Example 2). Actinomycete strain K04-0072 was deposited on October 18, 2024, with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE, Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu, Chiba, Japan 292-0818, Japan) as the international depositary authority under the Budapest Convention for the Deposit of Patent Microorganisms (accession number NITE BP-04180). Alternatively, pactamycin is also available commercially.

[0019] (3) Isorolidine E and its salt isorolidine E are compounds represented by the following formula III, and their production and purification methods are also known (e.g., Tetrahedron Let., 47, 4903 (1977)).

[0020]

[0021] Isorolidine E can be produced, for example, using the filamentous fungus strain FKI-10030 (accession number NITE BP-04177) (Example 3). The filamentous fungus strain FKI-10030 was deposited on October 18, 2024, with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE, Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu, Chiba, Japan 292-0818, Japan) as the international depositary authority under the Budapest Convention for the Deposit of Patent Microorganisms (accession number NITE BP-04177). Alternatively, isorolidine E is also available as a commercially available product.

[0022] (4) Tubercidine and its salt tubercidine are compounds represented by the following formula IV, and their production and purification methods are also known (e.g., J. Antibiot. Ser. A, 10:201-204 (1957)).

[0023]

[0024] Tubercidine can be produced, for example, using Actinomycetes strain KP-2324 (accession number NITE BP-04183) (Example 4). Actinomycetes strain KP-2324 was deposited on October 18, 2024, with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE, Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu, Chiba, Japan 292-0818, Japan) as the international depositary authority under the Budapest Convention for the Deposit of Patent Microorganisms (accession number NITE BP-04183). Alternatively, tubercidine is also available commercially.

[0025] (5) Nafrezin and its salts are compounds represented by the following formula V, and their production and purification methods are also known (e.g., J. Antibiot., 54:234-238 (2001)).

[0026]

[0027] Nafrezin can be produced, for example, using the filamentous fungus strain FKI-12011 (receipt number NITE ABP-04468) (Example 5). The filamentous fungus strain FKI-12011 was sent for deposit to the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE, Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu, Chiba, Japan 292-0818, Japan), which is the international depositary authority under the provisions of the Budapest Convention for the Deposit of Patent Microorganisms, and was received on October 7, 2025 (receipt number NITE ABP-04468). Alternatively, nafrezin is also available as a commercially available product.

[0028] Accordingly, in one embodiment, the present invention provides an antiviral agent comprising at least one antiviral compound selected from the group consisting of sangibamycin and its salts, patamycin and its salts, isorolidine E and its salts, tubercidine and its salts, and nafrezin and its salts as an active ingredient.

[0029] The salts of the compounds shown in the above formulas I to V can also be used as antiviral compounds. Such salts are not particularly limited, and examples include addition salts with inorganic acids such as hydrochloric acid and sulfuric acid, organic acids such as acetic acid, citric acid, tartaric acid, and maleic acid, salts with alkali metals such as potassium and sodium, salts with alkaline earth metals such as calcium and magnesium, ammonium salts, salts with organic bases such as ethylamine salts and arginine salts, and the like.

[0030] In the present specification, the antiviral compounds include not only the compounds represented by the above formulas, but also hydrates, solvates, derivatives, precursors, prodrugs, complexes, radioisotopes, etc. of the above compounds as long as their antiviral activity is maintained. For example, precursors or prodrugs that are converted into the above compounds in the body after being administered to a subject, complexes bound to other components (polymers, polyethylene glycol, etc.), deuterium forms of the above compounds, etc. can also be used as antiviral compounds in the present invention.

[0031] Whether the compound or complex to be used has the desired antiviral activity can be evaluated in vitro or in vivo. For example, by infecting test cells with FMDV and determining the survival rate of the infected cells treated with the compound to be evaluated compared with controls (non-infected cells, untreated infected cells, etc.), it is possible to evaluate whether the compound, etc. has antiviral activity against FMDV (for example, Example 6). Also, by administering the compound to be evaluated to a model animal infected with FMDV and observing the changes in the survival rate and symptoms compared with controls (non-infected animals, untreated infected model animals, etc.), it is possible to evaluate whether the compound, etc. has antiviral activity against FMDV (for example, Example 7).

[0032] The antiviral compound may be obtained as a commercial product, may be produced by chemical synthesis, or may be isolated from the above-mentioned microorganisms, or the culture solution of the microorganisms may be used as it is. The culture solution may be diluted or concentrated as necessary, and may also be subjected to sterilization treatment, freeze-drying, spray-drying treatment, etc. known in the art.

[0033] The antiviral agent according to the present invention may contain, as an active ingredient, one antiviral compound or a salt thereof, or may contain a combination of two or more antiviral compounds or salts thereof. When using two or more compounds, they may be provided as a mixture or administered separately.

[0034] The antiviral agent according to the present invention can be formulated into a pharmaceutical composition or a feed composition. Therefore, in one aspect, the present invention provides a pharmaceutical or feed composition containing the antiviral agent according to the present invention.

[0035] When the antiviral agent according to the present invention is formulated into a pharmaceutical composition, in addition to the active ingredient, it may also contain a veterinarily acceptable carrier or additive. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, mannitol, sorbitol, lactose, and the like. The additives used are appropriately selected or combined according to the dosage form.

[0036] The dosage form of the antiviral agent according to the present invention varies depending on the administration method. The administration method is not particularly limited and can be carried out by oral administration or parenteral administration, such as subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, transdermal administration, rectal administration, and the like.

[0037] When the antiviral agent according to the present invention is administered orally, it may be in any form such as tablets, capsules (hard capsules, soft capsules, microcapsules, etc.), granules, powders, pills, lozenges, internal aqueous solutions, liquids, suspensions, emulsions, syrups, etc., or it may be a dried product that is redissolved before use. When the antiviral agent according to the present invention is administered parenterally, a formulation form such as an injectable preparation for intravenous injection (including drip infusion), intramuscular injection, intraperitoneal injection, and subcutaneous injection (e.g., solution, emulsion, suspension), suppositories, inhalants, aerosols, and other topical preparations can be selected, and in the case of an injectable preparation, it may be provided in the form of a unit dose ampoule or a multi-dose container.

[0038] These various formulations can be manufactured by conventional methods by appropriately selecting excipients, bulking agents, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, pH adjusters, preservatives, solubilizers, antiseptics, flavoring and odor-masking agents, absorption enhancers, analgesics, stabilizers, isotonic agents, etc., which are commonly used in pharmaceuticals (veterinary drugs).

[0039] The amount of antiviral compound to be incorporated into an antiviral agent varies depending on its use, dosage form, and route of administration, but for example, it can be 0.1 to 99% by weight, preferably 1 to 90% by weight, based on the total weight.

[0040] Furthermore, the effective amount (dosage or intake) of the antiviral agent according to the present invention varies depending on the type of antiviral compound contained, the purpose of administration (prevention or treatment), the type and weight of the recipient, the route of administration, and the number of administrations, and can be changed over a wide range of periods. For example, it can be administered to the recipient once a day or in several divided doses, or once every 2 to 10 days, over a period of about 1 week to about 1 year.

[0041] The antiviral agent according to the present invention is not limited to use as a pharmaceutical composition, but may also be incorporated into, for example, a feed composition. That is, the antiviral agent according to the present invention can be used as a feed additive. "Feed composition" or "feed" refers to natural products and their processed products containing one or more nutrients, and includes all food and beverages. A feed composition containing the antiviral agent according to the present invention is useful as a feed for the prevention or treatment of foot-and-mouth disease.

[0042] When the antiviral agent according to the present invention is incorporated into a feed composition, it can be added to various forms of feed, such as solid feed, liquid feed, and gel feed.

[0043] Solid feeds can take the form of granules, pellets, blocks, etc. When the antiviral agent according to the present invention is incorporated into a solid feed composition, it may contain not only the active ingredient but also feed ingredients, additives, etc. Such feed ingredients are not limited to, but include whole cottonseed, cottonseed hulls, cottonseed meal, soybean meal, soybean hulls, corn gluten feed, hominy feed, dried distilled grain residue, and rice milling by-products. Possible additives include silage, nutritional supplements, vitamins, minerals, salt, grains (wheat, barley, oats, corn), fiber, hay, alfalfa, ryegrass, beets, sugar syrup, blood meal, bone meal, yeast, bromgrass, canarygrass, tomatoes, carrots, peas, pea vine hay, safflower, wormwood, sorghum, cheatgrass, clover, fat, grapes, hominy, hops, pasture hay, Sudan grass, sunflower, timothy grass hay, meat meal, Milo, oranges, timothy grass, potatoes, white beans, peanuts, grassland hay, rapeseed flour, soybeans, and protein.

[0044] When the antiviral agent according to the present invention is incorporated into a liquid feed composition, it may contain not only the active ingredient but also feed ingredients, additives, etc. Examples of such feed ingredients, though not limited to them, include water, sugar solution, microbial culture solution, milk, or milk replacer. Examples of additives that can be incorporated include ammonium sulfate, calcium carbonate, sodium chloride, defluorinated phosphate, diammonium phosphate, dicalcium phosphate, limestone, monoammonium phosphate, monocalcium phosphate, sodium tripolyphosphate, and urea.

[0045] When the antiviral agent according to the present invention is added to a feed composition, the amount added can be such that the antiviral compound content is 0.1 to 99% by weight of the total feed. The effective intake amount is determined appropriately on a case-by-case basis, taking into consideration the type of animal, its weight, the severity of its symptoms, etc. Intake can be divided into several doses per day, in which case the amount can be divided according to the number of doses. Furthermore, it can be taken continuously over a long period of time.

[0046] The antiviral agent according to the present invention may, if necessary, be coated with a single or multiple layers of enteric-coated material to become enteric-coated. Enteric-coated materials are those that do not dissolve in acidic pH ranges such as gastric juice, but dissolve in neutral pH ranges, and examples include hypromellose phthalate, shellac, zein, lactoferrin, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethyl ethylcellulose, and cellulose acetate phthalate.

[0047] The antiviral agent according to the present invention can effectively inhibit the proliferation of foot-and-mouth disease virus (FMDV). FMDV is classified as a Picornaviridae, Aftovirus genus, and is serologically classified into seven serotypes: O, A, C, Asia1, SAT1, SAT2, and SAT3. The antiviral agent according to the present invention may be effective against at least one, two, three, four, five, six, or seven serotypes of FMDV. Furthermore, because it can effectively inhibit the proliferation of FMDV, the antiviral agent according to the present invention can be used for the prevention or treatment of foot-and-mouth disease. Foot-and-mouth disease (FMD) is an acute febrile infectious disease of artiodactyls caused by foot-and-mouth disease virus (FMDV), and FMDV-infected animals exhibit clinical symptoms such as fever, blister formation around the mouth and on the hooves, salivation, and lameness.

[0048] In this specification, “treatment” means improvement of at least some of the symptoms of foot-and-mouth disease, cessation of disease progression or worsening, or complete recovery. In this specification, “prevention” means preventing a person who is not infected with foot-and-mouth disease from becoming infected with foot-and-mouth disease, or preventing the recurrence of foot-and-mouth disease.

[0049] The subjects to whom the antiviral agent according to the present invention is administered or ingested are not particularly limited as long as they are animals infected with or that transmit foot-and-mouth disease virus (FMDV). For example, it can be administered or ingested by animals belonging to the order Artiodactyla, such as domestic animals (cattle, pigs, goats, etc.), wild animals (wild boars, hippos, camels, deer, water buffalo, giraffes, etc.), and laboratory animals (model mice, model rats, etc.).

[0050] In one embodiment, the present invention provides a method for treating or preventing foot-and-mouth disease in a subject, comprising administering an effective amount of the antiviral agent according to the present invention to the subject. “Effective amount,” as used herein, refers to an amount of antiviral agent sufficient to provide treatment or prevention against foot-and-mouth disease when administered to a subject for the treatment or prevention of the disease. The effective amount is determined as appropriate depending on the type of antiviral compound used, the purpose of administration (prevention or treatment), the type of subject, body weight, etc.

[0051] The antiviral agent according to the present invention may be administered in combination with other pharmaceuticals. Examples of such other pharmaceuticals include other therapeutic or preventive agents for foot-and-mouth disease (such as vaccines), therapeutic or preventive agents for other diseases or symptoms, anti-inflammatory agents, intestinal regulators, immunomodulators, and health promoters. The other pharmaceuticals may be administered simultaneously with the antiviral agent or sequentially.

[0052] As shown in the examples described below, the antiviral agent according to the present invention has low cytotoxicity and high safety. Therefore, it is effective in treating or preventing foot-and-mouth disease over the long term while avoiding side effects, genotoxicity, and environmental problems.

[0053] As described above, the antiviral agent according to the present invention has the effect of inhibiting the proliferation of FMDV. Therefore, the antiviral agent according to the present invention can be used in the manufacture of pharmaceutical or feed compositions for the treatment or prevention of foot-and-mouth disease.

[0054] The method for producing a pharmaceutical or feed composition for the treatment or prevention of foot-and-mouth disease according to the present invention includes incorporating the antiviral agent according to the present invention into a pharmaceutical or feed ingredient. The pharmaceutical or feed ingredient that can be used in the production method according to the present invention is not particularly limited as long as it can be used for administration or feeding to the target artiodactyl animals, and can be incorporated into commercially available pharmaceutical ingredients, artificial feeds, or natural feeds. Furthermore, the amount to be incorporated is not particularly limited as long as it is an amount that can effectively inhibit the proliferation of FMDV, and a person skilled in the art can appropriately set it considering the type of antiviral compound, the purpose of administration (prevention or treatment), the target of administration, body weight, etc.

[0055] In another embodiment, the present invention provides a novel microbial strain, specifically, at least one microorganism or a derivative thereof selected from the group consisting of Actinomycete OH-4711 strain (accession number NITE BP-04184), Actinomycete K04-0072 strain (accession number NITE BP-04180), Filamentous fungus FKI-10030 strain (accession number NITE BP-04177), Actinomycete KP-2324 strain (accession number NITE BP-04183), and Filamentous fungus FKI-12011 strain (acceptance number NITE ABP-04468), or a processed product thereof.

[0056] Actinomycete strain OH-4711 (accession number NITE BP-04184), Actinomycete strain K04-0072 (accession number NITE BP-04180), Filamentous fungus strain FKI-10030 (accession number NITE BP-04177), and Actinomycete strain KP-2324 (accession number NITE BP-04183) are deposited with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture) as described above. Filamentous fungus strain FKI-12011 (receipt number NITE ABP-04468) has been received for deposit with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture) as described above.

[0057] Derived strains from the Actinomycete strain OH-4711 (accession number NITE BP-04184), Actinomycete strain K04-0072 (accession number NITE BP-04180), Filamentous fungus strain FKI-10030 (accession number NITE BP-04177), Actinomycete strain KP-2324 (accession number NITE BP-04183), and Filamentous fungus strain FKI-12011 (receipt number NITE ABP-04468) can also be obtained as strains distinct from the parent strains, possessing antiviral activity equal to or greater than that of the parent strains. Such derived strains can be produced by placing the parent strain under unconventional conditions for the parent strain, such as irradiating the parent strain with ultraviolet light, culturing the parent strain in the presence of mutagenic substances, culturing at temperatures other than the optimal temperature, or culturing in the presence of organic solvents. The target derivative strain can be selected based on the indicator that it has antiviral activity equivalent to or greater than that of, for example, Actinomycete strain OH-4711 (accession number NITE BP-04184), Actinomycete strain K04-0072 (accession number NITE BP-04180), Filamentous fungus strain FKI-10030 (accession number NITE BP-04177), Actinomycete strain KP-2324 (accession number NITE BP-04183), or Filamentous fungus strain FKI-12011 (receipt number NITE ABP-04468).

[0058] The above-mentioned microbial strains and their derivatives can be prepared by culturing them under appropriate conditions using a culture medium commonly used for culturing actinomycetes or filamentous fungi. The culture medium used can be any medium, such as a natural medium, synthetic medium, liquid medium, or solid medium, as long as it contains a carbon source, nitrogen source, inorganic salts, etc., and is capable of efficiently culturing actinomycetes or filamentous fungi. Those skilled in the art can appropriately select a known medium suitable for the strain being used. The above-mentioned microbial strains and their derivatives are cultured at 20°C to 40°C, preferably 25°C to 30°C. The temperature conditions can be adjusted using a constant temperature bath, mantle heater, jacket, etc. The culture can be carried out using any culture method, such as static culture, shaking culture, or tank culture. The culture time can be 3 hours to 30 days or longer, preferably about 10 hours to 20 days. The pH of the culture medium at the start of cultivation may be adjusted to 3.5 to 8.0, or it may be left unadjusted.

[0059] Microbial strains can be obtained as live cells or as a culture medium containing live cells, separated from the culture medium by separation techniques such as filtration and centrifugation after culturing. Furthermore, sterilized cells can be obtained by subjecting these live cells or their culture medium to known sterilization treatments, such as autoclaving. Alternatively, treated products can be prepared from live cells or live cell culture medium and subjected to sterilization treatment as needed.

[0060] The processed microbial strain is obtained by subjecting the microbial cells of the strain to physical, chemical, or biological treatments such as drying, destruction or crushing, or enzymatic treatment. Therefore, examples of the processed products according to the present invention include dried cells, destroyed cells, and crushed cells.

[0061] Dried bacterial cells can be obtained by subjecting the bacterial cells isolated from the culture medium, which have been sterilized if necessary, to a drying process. Examples of drying processes include drum drying, spray drying, vacuum drying, and freeze-drying.

[0062] Destroyed or lysated bacterial cells can be obtained by treating bacterial cells by crushing, grinding, enzymatic treatment, chemical treatment, lysis, etc. Destroyed or lysated bacterial cells essentially consist of all the solid and soluble components of the destroyed or crushed bacterial cells and can be obtained, for example, by freeze-drying the destroyed or crushed material as is. Destroying or crushing can be carried out by known methods, such as physical crushing, enzymatic lysis, chemical treatment, etc. Physical crushing can be carried out wet or dry and can be done by stirring using a homogenizer, ball mill, bead mill, dyno mill, planetary mill, etc., or by using a jet mill, French press, cell disruptor, etc. Enzymatic lysis can be carried out by destroying the cellular structure of the bacteria using an enzyme such as lysozyme. Chemical treatment can be carried out by destroying the cellular structure of the bacteria using a surfactant such as glycerol fatty acid ester or soybean phospholipid.

[0063] The above-mentioned bacterial cells and bacterial-treated materials can be used as is, or they can be subjected to sterilization treatment to become sterilized. Sterilization treatments include known treatments such as sterilization by high-energy irradiation, heat sterilization, pressure sterilization, and autoclave sterilization.

[0064] In another embodiment, the present invention provides a culture medium containing an antiviral compound obtained by culturing at least one microorganism selected from the group consisting of Actinomycete OH-4711 strain (accession number NITE BP-04184), Actinomycete K04-0072 strain (accession number NITE BP-04180), Filamentous fungus FKI-10030 strain (accession number NITE BP-04177), Actinomycete KP-2324 strain (accession number NITE BP-04183), and Filamentous fungus FKI-12011 strain (acceptance number NITE ABP-04468).

[0065] Since antiviral compounds are produced in the culture medium by culturing the above-mentioned microorganisms, the present invention also relates to a culture medium containing such antiviral compounds. The culture medium according to the present invention may contain antiviral compounds and, optionally, microbial cells or lysates of microbial cells.

[0066] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0067] [Example 1] Production of Sangibamycin The production of sangibamycin and sangibamycin-containing extract was carried out using the Actinomycete strain OH-4711 (a strain deposited on October 18, 2024, at the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) (accession number NITE BP-04184)), referring to a previously reported method (J. Med. Chem., 11:939-941 (1968)). Specifically, maltose (Hayashibara Co., Ltd.) 5%, dry yeast (Nichifutsu Shoji Co., Ltd.) 1.5%, Ebios (Asahi Group Foods Co., Ltd.) 2.5%, KBr (Fujifilm Wako Pure Chemical Industries Co., Ltd.) 1.0%, KH ２ PO ４ (Manufactured by Kanto Chemical Co., Ltd.) 0.05%, MgSO ４ 7H ２Ten 500 mL Erlenmeyer flasks, each containing 100 mL of liquid culture medium (unadjusted pH) consisting of 0.05% O (manufactured by Kanto Chemical), were inoculated with 1 mL of actinomycete strain OH-4711, cultured in the liquid medium, and incubated with shaking at 27°C for 7 days.

[0068] 400 mL of ethanol was added to 1 L of culture medium and centrifuged at 3000 rpm for 15 minutes. Ethanol was removed from the supernatant to obtain a sangibamycin-containing extract. Subsequently, the extract was passed through an HP20 resin (φ35 x 50 mm) open column chromatography column to obtain the clear fraction. The obtained clear fraction was eluted stepwise using an ODS (φ35 x 50 mm) open column chromatography system with water-acetonitrile solvents (clear, 100:0, 60:40, 0:100), and the acetonitrile-water (60:40) fraction was dried under reduced pressure to obtain the crude substance (61 mg). The crude substance was dissolved in methanol and injected into a reverse-phase column (DEVELOSIL C30, φ20 x 250 mm, Nomura Chemical Co., Ltd., Japan) by high-performance liquid chromatography. Elution was performed under conditions of acetonitrile-water (5:95), flow rate of 7 mL / min, and UV 210 nm detection. The peak at a retention time of 45 minutes was isolated and dried under reduced pressure to obtain sangibamycin (2 mg).

[0069] [Example 2] Production of patamycin The production of patamycin and patamycin-containing extract was carried out using Actinomycete strain K04-0072 (a strain deposited on October 18, 2024, at the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) (accession number NITE BP-04180)), referring to a previously reported method (J. Antibiot. Ser. A., 17:230-233 (1964)). Specifically, 10 Erlenmeyer flasks, each containing 100 mL of a liquid culture medium (unadjusted pH) consisting of 2.4% starch (manufactured by Asahi Foods), 0.1% glucose (manufactured by Fujifilm Wako Pure Chemical Industries), 0.3% peptone (manufactured by Kyokuto Pharmaceutical Industries), 0.3% meat extract (manufactured by Kyokuto Pharmaceutical Industries), 0.5% yeast extract (manufactured by Oriental Yeast), and 0.4% CaCO3 (manufactured by Kanto Chemical), were inoculated with 1 mL of Actinomycete strain K04-0072, which had been cultured in the liquid culture medium, and incubated with shaking at 27°C for 6 days.

[0070] 1 L of culture medium was extracted twice with 1 L of ethyl acetate, and the ethyl acetate layer was dried under reduced pressure to obtain 1 g of crude material. The crude material was placed on a silica gel column (φ35 x 50 mm) packed with chloroform, and eluted in stages with a chloroform-methanol solvent system (100:0, 100:1, 100:2). After fine fractionation during chloroform-methanol (100:1) elution, the patamycin-containing fraction was collected and concentrated under reduced pressure to obtain patamycin (43 mg).

[0071] [Example 3] Production of Isorolidine E The production of isorolidine E and isorolidine E-containing extracts was carried out by referring to a previously reported method (Tetrahedron Let., 47, 4903 (1977)) using the filamentous fungus strain FKI-10030 (a strain deposited on October 18, 2024, at the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) (accession number NITE BP-04177)). Specifically, 25 mL of the filamentous fungus strain FKI-10030, cultured in liquid medium, was inoculated into six sterilized Erlenmeyer flask bags containing a solid medium consisting of 500 g of rice (manufactured by Hanasho) and 5 g of kelp tea (manufactured by Itoen), and the samples were incubated at 25°C for 14 days.

[0072] 3 kg of culture was mixed with 2.5 L of methanol and then filtered under reduced pressure. The methanol was removed from the resulting filtrate to obtain an isorolidine E-containing extract. Subsequently, using an ODS (φ35 x 50 mm) open column chromatography, stepwise elution was performed in a water-methanol solvent system (open pass, 100:0, 80:20, 60:40, 40:60, 20:80, 100:0), and the water-methanol (20:80) fraction was dried under reduced pressure to obtain the crude substance (2.9 g). 40 mg was dissolved in methanol and injected into a reverse-phase column (PEGASIL ODS SP100, φ20 x 250 mm, manufactured by Senshu Kagaku Co., Ltd., Japan) using high-performance liquid chromatography. Elution was performed under water-methanol (40:60), flow rate of 7 mL / min, and UV 210 nm detection. The peak at a retention time of 18 minutes was isolated and dried under reduced pressure to obtain isorolidine E (20 mg).

[0073] [Example 4] Production of Tubercidine The production of tubercidine and tubercidine-containing extract was carried out by referring to a previously reported method (J. Antibiot. Ser. A, 10:201-204 (1957)) using Actinomycete strain KP-2324 (a strain deposited on October 18, 2024, at the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) (accession number NITE BP-04183)). Specifically, glucose (manufactured by Fujifilm Wako Pure Chemical Industries) 1.5%, kinako (manufactured by Noko Tanaka Ryu Shoten) 1.0%, peptone (manufactured by Kyokuto Pharmaceutical Industry) 0.5%, meat extract (manufactured by Kyokuto Pharmaceutical Industry) 0.5%, NaCl (manufactured by Kanto Chemical) 0.5%, K ２ HPO ４ Ten 500 mL Erlenmeyer flasks, each containing 100 mL of a 0.05% liquid culture medium (unadjusted pH) (manufactured by Kanto Chemical Co., Ltd.), were inoculated with 1 mL of Actinomycete strain KP-2324, cultured in the liquid medium, and incubated with shaking at 27°C for 3 days.

[0074] 1 L of the culture medium was centrifuged at 3000 rpm for 15 minutes to obtain a tubercidine-containing extract. Subsequently, using HP20 resin (φ35 x 50 mm) open column chromatography, stepwise elution was performed in a water-acetone solvent system (straight through, 100:0, 10:90, 50:50, 0:100). The water-acetone (10:90) fraction and the (50:50) fraction were mixed and dried under reduced pressure to obtain the crude substance (520 mg). This was dissolved in methanol and injected into a reverse-phase column (CAPCEL PAK C18, φ20 x 250 mm, manufactured by Osaka Soda Co., Ltd., Japan) by high-performance liquid chromatography. Elution was performed under water-methanol (80:20), flow rate 7 mL / min, and UV 210 nm detection. The peak with a retention time of 11 minutes was separated and dried under reduced pressure to obtain tubercidine (0.5 mg).

[0075] [Example 5] Production of Nafrezin The production of nafrezin and nafrezin-containing extract was carried out by referring to a previously reported method (J. Antibiot., 54:234-238 (2001)) using the filamentous fungus strain FKI-12011 (a strain received for deposit on October 7, 2025, at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) (receipt number NITE ABP-04468)). Specifically, 50 mL of the filamentous fungus strain FKI-12011, cultured in liquid medium, was inoculated into two sterilized Erlenmeyer flask bags containing a solid medium consisting of 1000 g of rice (manufactured by Hanasho) and 10 g of kelp tea (manufactured by Itoen), and the cells were incubated at 25°C for 14 days.

[0076] 2 kg of culture was mixed with 2.0 L of methanol and stirred, then filtered under reduced pressure. The methanol was removed from the resulting filtrate to obtain a naprezin-containing extract. Subsequently, using ODS (φ55 x 55 mm) open column chromatography, stepwise elution was performed in a water-methanol solvent system (straight through, 80:20, 60:40, 40:60, 20:80, 0:100), and the water-methanol (100:0) fraction was dried under reduced pressure to obtain the crude substance (1.8 mg). Of this, 300 mg was used for stepwise elution using silica gel open column chromatography in a n-hexane-ethyl acetate solvent system (80:20, 80:20, 60:40, 60:40, 40:60, 20:80, 0:100), and the n-hexane-ethyl acetate (first elution 60:40) fraction was dried under reduced pressure to obtain the crude substance (8.4 mg). The obtained protomaterial was subjected to thin-layer chromatography three times using n-hexane-ethyl acetate (70:30) as the developing solvent. The thin layers with an Rf value of 0.4-0.5 were collected, eluted with methanol, and dried under reduced pressure to obtain naprezin (3.0 mg).

[0077] [Example 6] Verification of antiviral effect against FMDV - in vitro effect verification (1) Antiviral effect against FMDV and cytotoxicity in cultured cells In this example, the antiviral effect of the antiviral compound samples prepared in Examples 1 to 5 against FMDV was examined in vitro. The materials used were as follows: Cells used: Hamster kidney immortalized cells (BHK cells) Virus: Serotype O Turkey 2009 isolate Medium: Minimum Essential Media (MEM)

[0078] The antiviral compound samples generated in Examples 1 to 5 were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg / ml.

[0079] The cell survival rate (%) of cells infected with FMDV was determined by the following procedure. 1. Seed BHK cells in a 96-well plate (5,000 cells / well). 2. Incubate at 37°C for 24 hours. 3. Dilute the antiviral compound sample 100-fold with the medium. 4. Discard the supernatant of the 96-well plate of BHK cells and add the sample dilution at 50 μl / well. Prepare 2 plates (antiviral evaluation, toxicity evaluation) with 2 wells each for each sample. 5. Dilute the virus with the medium to adjust to 10 ２ TCID ５０ / 5 and make it 50 μl. 6. Add the virus dilution (add medium to the negative control) to the antiviral evaluation plate and add 50 μl / well of medium to all wells of the toxicity evaluation plate. 7. As a back-titration, perform titration of the virus dilution (10 wells / dilution). 8. Incubate at 37°C for 2 days. 9. Dispense 10 μl / well of CCK-8 solution (manufactured by Dojindo Laboratories). 10. Incubate at 37°C for 2 hours. 11. Determine the back-titration by the cytopathic effect (CPE). 12. Measure the absorbance at 450 nm with a plate reader. 13. Calculate the cell survival rate (%) of the sample well by the following formula: Formula: (Sample absorbance - absorbance of virus control) ÷ (Absorbance of negative control - absorbance of virus control) × 100

[0080] As target values, a cell survival rate of 25% or higher for antiviral evaluation and 50% or higher for toxicity evaluation will be judged as having antiviral effect. In addition, the conditions for the test to be valid are that the difference in absorbance between the negative control and the viral control is 0.8 or higher, and back titration is 10 １．６ ~10 ２．４ TICD ５０ The value was set to 50 μl.

[0081] The results are shown in Figure 1. From the results shown in Figure 1, it was found that the antiviral compound tested has an antiviral effect against FMDV at low concentrations in which no cytotoxicity was observed.

[0082] (2) Verification of the effective concentration of antiviral compounds against FMDV in cultured cells Next, the effective concentrations of the antiviral compound samples prepared in Examples 1 to 5 against FMDV were investigated. The materials used were as follows: Cells used: BHK cells Virus: Serotype O Turkey 2009 isolate Culture medium: MEM

[0083] The cell survival rate (%) of FMDV-infected cells was determined using the following procedure: 1. Seed BHK cells in a 96-well plate (5,000 cells / well). 2. Culture at 37°C for 24 hours. 3. Dilute the antiviral compound sample. 1) Dispense 150 μl / well of culture medium into all wells of a round-bottom plate. 2) Dilute the sample 100-fold with culture medium. 3) Dispense 150 μl of the diluted sample into the second row of 2 wells. 4) Perform 2-fold serial dilution using a multipipette. 4. Discard the supernatant from the cell plate and add 50 μl / well of the sample dilution. Prepare two plates for each sample (antiviral evaluation, toxicity evaluation). 5. Dilute the virus in culture medium and 10 ２ TCID ５０ / Adjust to 50 μl. 6. Add the virus diluent to the antiviral evaluation plate (add culture medium to the negative control), and add 50 μl / well of culture medium to all wells of the toxicity evaluation plate. 7. As back titration, perform titration of the virus diluent. 1) Place the virus in culture medium for 10 －１ from －４Dilute to 10. 2) Discard the supernatant from the 96-well plate of BHK cells and add 50 μl / well of culture medium to all wells. 3) Add 50 μl / well of virus diluent (dispense 10 wells per diluent). 8. Incubate at 37°C for 48 hours. 9. Dispense 10 μl / well of CCK-8 solution (manufactured by Dojin Chemical Laboratories). 10. Incubate at 37°C for 2 hours. 11. Determine back titration by CPE. The condition for a successful test is back titration of 10. １．６ ~10 ２．４ TICD ５０ The volume was set to 50 μl. 12. Measure the absorbance at 450 nm using a plate reader. 13. Calculate the cell survival rate (%) in the sample well using the following formula: Formula: (Sample absorbance - Absorbance of virus control) ÷ (Absorbance of negative control - Absorbance of virus control) × 100 14. Determine the 50% inhibitory concentration (IC) of each antiviral compound. ５０ The antiviral efficacy evaluation well is calculated by linear regression between the survival rate % and the drug concentration.

[0084] The results are shown in Figure 2. Figure 2 shows the IC obtained in this embodiment. ５０ Along with the previously reported LD ５０ The production microorganisms, lipid-soluble logD, PBS solubility, Caco2 membrane permeability, and liver Ms stability are also summarized. The effective concentration for FMDV is IC. ５０ Based on this information, the dosage and duration of administration to animals can be determined.

[0085] [Example 7] Efficacy evaluation test of antiviral compounds against FMDV using mice. In this example, the antiviral effect of the antiviral compound samples prepared in Examples 1 to 5 against FMDV was investigated in 6-week-old BALB / c mice. It should be noted that the use of mice as a model animal for foot-and-mouth disease (FMD) has been reported previously (Platt H. J Pathol Bacteriol 72: 299-312, 1956; Salguero FJ, et al., Virology 332:384-396, 2005; Skinner HH. Proc R Soc Med 44:1041-1044, 1951).

[0086] The mortality rate of mice infected with FMDV was determined using the following procedure: 1. A filtered antiviral compound was diluted with sterile PBS and administered 40 μg (100 μl) to 6 to 12 mice intraperitoneally or orally. 2. After 1 hour, the FMDV serotype O Turkey 2009 isolate (10 LD) diluted with sterile PBS was administered to each mouse. ５０ ) is administered intraperitoneally in 100 μl doses. Previous animal studies have confirmed that significant symptoms (depression, death) appear within 5 days of inoculation, and the 50% lethal infectious titer (LD50) is used. ５０ ) was calculated. 3. The same amount of antiviral compound was administered additionally 24 hours and 48 hours after the initial dose, and the mice were then reared for up to 6 days for clinical observation. Humane endpoints were defined as "(1) difficulty eating or drinking, (2) symptoms of distress (self-injurious behavior, abnormal posture, respiratory distress, vocalizations), (3) external abnormalities that show no signs of recovery (diarrhea, bleeding, soiling of the genitals), (4) rapid weight loss (20% in a few days), (5) appearance or significant growth of tumors," and if these conditions were reached, the mice would be euthanized immediately. Euthanasia was performed by placing Kimwipes soaked in isoflurane in an anesthesia box and exposing the mice to it, then rapidly dislocating the cervical vertebrae under deep anesthesia.

[0087] The results of this experiment are shown in Figure 3. In Figure 3, the values ​​shown in bold indicate improvement compared to the control group. For sangibamycin, a certain antiviral effect was confirmed with intraperitoneal and oral administration. For isoloridine E, a tendency for improvement in the final day survival rate was observed, and a certain antiviral effect was confirmed with intraperitoneal administration. For nafrezin, oral administration was observed to delay the decrease in mouse survival rate. For tubercidine, a certain antiviral effect was confirmed with intraperitoneal and oral administration. For patamycin, a certain antiviral effect was confirmed with intraperitoneal administration.

[0088] [Accession Number and Receipt Number] Accession Number NITE BP-04184 (Actinopropyl OH-4711 strain, deposited October 18, 2024) Accession Number NITE BP-04180 (Actinopropyl K04-0072 strain, deposited October 18, 2024) Accession Number NITE BP-04177 (Filamentous Fungus FKI-10030 strain, deposited October 18, 2024) Accession Number NITE BP-04183 (Actinopropyl KP-2324 strain, deposited October 18, 2024) Receipt Number NITE ABP-04468 (Filamentous Fungus FKI-12011 strain, received October 7, 2025)

Claims

1. An antiviral agent against foot-and-mouth disease virus (FMDV), comprising as an active ingredient at least one antiviral compound selected from the group consisting of sangibamycin and its salts, patamycin and its salts, isorolidine E and its salts, tubercidine and its salts, and nafrezin and its salts.

2. The antiviral agent according to claim 1, wherein the antiviral compound is contained in a culture medium obtained by culturing at least one microorganism selected from the group consisting of Actinomycete OH-4711 strain (accession number NITE BP-04184), Actinomycete K04-0072 strain (accession number NITE BP-04180), Filamentous fungus FKI-10030 strain (accession number NITE BP-04177), Actinomycete KP-2324 strain (accession number NITE BP-04183), and Filamentous fungus FKI-12011 strain (acceptance number NITE ABP-04468).

3. The antiviral agent according to claim 1, wherein the target of administration or ingestion is an animal belonging to the order Artiodactyla.

4. The antiviral agent according to claim 1, which is a feed additive.

5. A pharmaceutical or feed composition comprising the antiviral agent described in claim 1.

6. The composition according to claim 5, for use in the prevention or treatment of foot-and-mouth disease.

7. The composition according to claim 5, wherein the target of administration or ingestion is an animal belonging to the order Artiodactyla.

8. A method for producing a pharmaceutical or feed composition for the prevention or treatment of foot-and-mouth disease, comprising incorporating the antiviral agent described in claim 1 into a pharmaceutical raw material or feed raw material.

9. A method for treating or preventing foot-and-mouth disease in a subject, comprising administering an effective amount of the antiviral agent described in claim 1 to the subject.

10. The method according to claim 9, wherein the subject is an animal belonging to the order Artiodactyla.

11. At least one microorganism or a derivative thereof selected from the group consisting of Actinomycete strain OH-4711 (accession number NITE BP-04184), Actinomycete strain K04-0072 (accession number NITE BP-04180), Filamentous fungus strain FKI-10030 (accession number NITE BP-04177), Actinomycete strain KP-2324 (accession number NITE BP-04183), and Filamentous fungus strain FKI-12011 (receipt number NITE ABP-04468), or a processed product thereof.

12. A culture medium containing an antiviral compound obtained by culturing at least one microorganism selected from the group consisting of Actinomycete strain OH-4711 (accession number NITE BP-04184), Actinomycete strain K04-0072 (accession number NITE BP-04180), Filamentous fungus strain FKI-10030 (accession number NITE BP-04177), Actinomycete strain KP-2324 (accession number NITE BP-04183), and Filamentous fungus strain FKI-12011 (receipt number NITE ABP-04468).

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