Prophylactic or therapeutic drug for livestock infectious disease caused by african swine fever virus

Abstract

The present invention provides an infectious disease prophylactic drug as a countermeasure against livestock infectious diseases such as African swine fever, the prophylactic drug having high production efficiency and being easily administered to livestock. Specifically, the present invention pertains to an antiviral agent against African swine fever virus (ASFV) comprising, as an active ingredient, at least one antiviral compound selected from the group consisting of aspochalasin D and salts thereof, atpenin B and salts thereof, virantmycin and salts thereof, resistomycin and salts thereof, and anisomycin and salts thereof.

Description

Prevention or treatment of livestock infectious diseases caused by African swine fever virus

[0001] This invention relates to an antiviral agent against African swine fever virus (ASFV), 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 African swine fever.

[0002] African swine fever (ASF) is a viral infectious disease caused by the ASF virus, characterized by its high lethality to pigs and wild boars and its persistence in the external environment. If it enters Japan, it could cause immense damage to the pig farming industry. Considering the recent outbreaks in Asia, the risk is increasing year by year. However, there is still no practical vaccine for this disease, and the only way to prevent its spread is through the early detection and culling of infected pigs.

[0003] Special Publication No. 2022-545341

[0004] Therefore, fast-acting antiviral drugs against ASF virus (ASFV) are useful as disease control measures and techniques to prevent the spread of the disease during outbreaks. To date, several synthetic compounds have been reported to exhibit antiviral effects against ASF (for example, Patent Document 1). However, due to challenges such as toxicity to the host and manufacturing costs, none have been put into practical use as anti-ASF drugs.

[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 ASF.

[0006] The inventors continued their search for substances that inhibit the proliferation of African swine fever virus (ASFV) in microbial cultures. As a result, they discovered that aspochalasin D, atopenin B, vilantomycin, ristomycin, and anisomycin each possess ASFV 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 African swine fever virus (ASFV), comprising as an active ingredient at least one antiviral compound selected from the group consisting of aspochalasin D and its salts, atopenin B and its salts, vilantomycin and its salts, resistantycin and its salts, and anisomycin 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 the filamentous fungus FKI-6835 strain (accession number NITE BP-04176), the filamentous fungus FO-125 strain (accession number NITE BP-04178), the actinomycete AM-2722 strain (accession number NITE BP-04179), the actinomycete K05-0055 strain (accession number NITE BP-04181), and the actinomycete KP-1241 strain (accession number NITE BP-04182). [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 African swine fever. [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 African swine fever, comprising incorporating the antiviral agent according to any one of [1] to [4] into a pharmaceutical or feed ingredient. [9] A method for treating or preventing African swine fever 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 the filamentous fungus FKI-6835 strain (accession number NITE BP-04176), the filamentous fungus FO-125 strain (accession number NITE BP-04178), the actinomycete AM-2722 strain (accession number NITE BP-04179), the actinomycete K05-0055 strain (accession number NITE BP-04181), and the actinomycete KP-1241 strain (accession number NITE BP-04182), 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 the filamentous fungus strain FKI-6835 (accession number NITE BP-04176), the filamentous fungus strain FO-125 (accession number NITE BP-04178), the actinomycete strain AM-2722 (accession number NITE BP-04179), the actinomycete strain K05-0055 (accession number NITE BP-04181), and the actinomycete strain KP-1241 (accession number NITE BP-04182).

[0009] The present invention provides an antiviral agent against African swine fever virus (ASFV), a pharmaceutical or feed composition for use in the treatment or prevention of African swine fever, and a method for producing the same. The antiviral compounds used in the present invention effectively inhibit the proliferation of ASFV and are therefore useful for the treatment or prevention of African swine fever.

[0010] This graph shows the antiviral effects of compounds in cells infected with African swine fever virus (ASFV). ５０ This is a table summarizing the other characteristics.

[0011] This specification encompasses the disclosures of Japanese Patent Application No. 2024-186876, 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") that have antiviral activity against African swine fever virus (ASFV) from among compounds obtained from various microbial cultures. Accordingly, the present invention relates to an antiviral agent against African swine fever virus (ASFV) that contains 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) Aspochalasin D and its salt, aspochalasin D, are compounds represented by the following formula I, and their production and purification methods are also known (e.g., J. Nat. Prod., 67:328-332 (2004)).

[0014]

[0015] Aspochalasin D can be produced, for example, using the filamentous fungus strain FKI-6835 (accession number NITE BP-04176) (Example 1). The filamentous fungus strain FKI-6835 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-04176). Alternatively, aspochalasin D is also available commercially.

[0016] (2) Atopenine B and its salt, atopenine B, are compounds represented by the following formula II, and their production and purification methods are also known (e.g., J. Antibiot., 41:1769-1773 (1988)).

[0017]

[0018] Atopenin B can be produced, for example, using the filamentous fungus strain FO-125 (accession number NITE BP-04178) (Example 2). The filamentous fungus strain FO-125 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-04178). Alternatively, atopenin B is also available as a commercially available product.

[0019] (3) Virantomycin and its salt virantomycin are compounds represented by the following formula III, and their production and purification methods are also known (e.g., J. Antibiot., 33:1395-1396 (1980)).

[0020]

[0021] Virantomycin can be produced, for example, using Actinomycete strain AM-2722 (accession number NITE BP-04179) (Example 3). Actinomycete strain AM-2722 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-04179). Alternatively, virantomycin is also available commercially.

[0022] (4) Resistimycin and its salts are compounds represented by the following formula IV, and their production and purification methods are also known (e.g., J. Antibiot., 58:530-534 (2005)).

[0023]

[0024] Resistimycin can be produced, for example, using Actinomycete strain K05-0055 (accession number NITE BP-04181) (Example 4). Actinomycete strain K05-0055 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-04181). Alternatively, resistimycin is also available commercially.

[0025] (5) Anisomycin and its salt anisomycin are compounds represented by the following formula V, and their production and purification methods are also known (e.g., J. Antibiot., 46:1300-1302 (1993)).

[0026]

[0027] Anisomycin can be produced, for example, using Actinomycete strain KP-1241 (accession number NITE BP-04182) (Example 5). Actinomycete strain KP-1241 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-04182). Alternatively, anisomycin is also available commercially.

[0028] Accordingly, in one embodiment, the present invention provides an antiviral agent comprising at least one antiviral compound selected from the group consisting of aspochalasin D and its salts, atopenin B and its salts, vilantomycin and its salts, resistantycin and its salts, and anisomycin and its salts as an active ingredient.

[0029] The salts of the compounds represented by 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 ASFV 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 ASFV (for example, Example 6). Also, by administering the compound to be evaluated to a model animal infected with ASFV 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 ASFV.

[0032] The antiviral compound may be obtained as a commercial product, may be produced by chemical synthesis, may be isolated from the above-mentioned microorganisms, or the culture solution of the microorganism 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. Thus, 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, it may also contain, in addition to the active ingredient, a veterinary acceptable carrier or additive. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, 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 African swine fever.

[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 African swine fever virus (ASFV). ASFV is classified in the Asphaviridae family, Asphaviridae genus, Asphavirus. Furthermore, because it can effectively inhibit the proliferation of ASFV, the antiviral agent according to the present invention can be used for the prevention or treatment of African swine fever. African swine fever (ASF) is an acute infectious disease of even-toed ungulates caused by African swine fever virus (ASFV), and ASFV-infected animals exhibit clinical symptoms such as fever, lethargy, loss of appetite, leukopenia, subcutaneous hemorrhage, erythema, and diarrhea.

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

[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 African swine fever virus (ASFV) or vectors ASFV. For example, it can be administered or ingested by animals belonging to the order Artiodactyla, such as domestic animals (pigs, etc.), wild animals (wild boars, warthogs, etc.), and laboratory animals (model mice, model rats, etc.).

[0050] In one embodiment, the present invention provides a method for treating or preventing African swine fever 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 African swine fever when administered to a subject for the treatment or prevention of African swine fever. 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 prophylactic agents for African swine fever (such as vaccines), therapeutic or prophylactic 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 African swine fever 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 ASFV. 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 African swine fever.

[0054] The method for producing a pharmaceutical or feed composition for the treatment or prevention of African swine fever 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 ASFV, 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 the filamentous fungus FKI-6835 strain (accession number NITE BP-04176), the filamentous fungus FO-125 strain (accession number NITE BP-04178), the actinomycete AM-2722 strain (accession number NITE BP-04179), the actinomycete K05-0055 strain (accession number NITE BP-04181), and the actinomycete KP-1241 strain (accession number NITE BP-04182), or a processed product thereof.

[0056] As mentioned above, the filamentous fungus strain FKI-6835 (accession number NITE BP-04176), the filamentous fungus strain FO-125 (accession number NITE BP-04178), the actinomycete strain AM-2722 (accession number NITE BP-04179), the actinomycete strain K05-0055 (accession number NITE BP-04181), and the actinomycete strain KP-1241 (accession number NITE BP-04182) are deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture).

[0057] Derived strains from the filamentous fungus FKI-6835 (accession number NITE BP-04176), filamentous fungus FO-125 (accession number NITE BP-04178), actinomycete AM-2722 (accession number NITE BP-04179), actinomycete K05-0055 (accession number NITE AP-04181), and actinomycete KP-1241 (accession number NITE BP-04182) 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, 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, the filamentous fungus FKI-6835 strain (accession number NITE BP-04176), the filamentous fungus FO-125 strain (accession number NITE BP-04178), the actinomycete AM-2722 strain (accession number NITE BP-04179), the actinomycete K05-0055 strain (accession number NITE BP-04181), or the actinomycete KP-1241 strain (accession number NITE BP-04182).

[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 the filamentous fungus strain FKI-6835 (accession number NITE BP-04176), the filamentous fungus strain FO-125 (accession number NITE BP-04178), the actinomycete strain AM-2722 (accession number NITE BP-04179), the actinomycete strain K05-0055 (accession number NITE BP-04181), and the actinomycete strain KP-1241 (accession number NITE BP-04182).

[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 Aspochalasin D The production of aspochalasin D and aspochalasin D-containing extract was carried out by referring to a previously reported method (J. Nat. Prod., 67:328-332 (2004)) using the filamentous fungus strain FKI-6835 (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-04176). Specifically, 25 mL of the filamentous fungus strain FKI-6835, cultured in liquid medium, was inoculated into three sterile bags containing a solid medium consisting of 1.4 kg of rice (manufactured by Hanasho) and 9 g of kelp tea (manufactured by Itoen), and the bags were incubated at 25°C for 11 days.

[0068] 1.4 L of 80% ethanol aqueous solution was added to 1.4 kg of culture material, stirred, and then filtered under reduced pressure. Ethanol was removed from the resulting filtrate to obtain an aspochalasin D-containing extract. The obtained aqueous solution was separated twice with 700 mL of ethyl acetate, and the ethyl acetate layer was concentrated to dryness under reduced pressure. The ethyl acetate extract (2.2 g) was placed on a silica gel column (φ30 x 120 mm) packed with chloroform, and eluted in stages using a chloroform-methanol solvent system (200 mL each, 100:0, 100:1, 50:1, 10:1, 50:50, 0:100), and the chloroform-methanol (100:1) fraction was concentrated to dryness under reduced pressure. Crude material (943 mg) was dissolved in a small amount of methanol and injected into a reversed-phase column (PEGASIL ODS SP100, φ20 x 250 mm, manufactured by Senshu Chemical, Japan) by high-performance liquid chromatography. Elution was performed under conditions of a 30-minute gradient from water-acetonitrile-phosphate (30:70:0.05) to water-acetonitrile-phosphate (10:90:0.05), a flow rate of 8 mL / min, and UV 210 nm detection. The peak with a retention time of 13 minutes was isolated, acetonitrile was removed under reduced pressure, and then two liquid-liquid extractions were performed with 700 mL of ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure and allowed to dry to obtain aspochalasin D (135 mg).

[0069] [Example 2] Production of Atopenin B The production of atopenin B and atopenin B-containing extract was carried out by referring to a previously reported method (J. Antibiot., 41:1769-1773 (1988)), using the filamentous fungus strain FO-125 (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) The experiment was conducted using BP-04178. Specifically, 200 mL of the filamentous fungus strain FO-125, cultured in the liquid medium, was inoculated into one 90 L jar fermenter containing 60 L of liquid medium (unadjusted pH) consisting of 1.0% glucose (manufactured by Fujifilm Wako Pure Chemical Industries), 2.0% peptone (manufactured by Kyokuto Pharmaceutical Co., Ltd.), 0.3% yeast extract (manufactured by Oriental Yeast Co., Ltd.), and malt extract (manufactured by DIFUCO). The culture was then stirred and incubated at 27°C, 250 rpm, and aeration at 10 L / min for 6 days.

[0070] 60 L of the culture medium was centrifuged using a Sharpless method to obtain 60 L of an atopenin B-containing extract as the supernatant. Subsequently, using an HP20 resin (φ110 x 100 mm) open column chromatography, stepwise elution was performed in a water-methanol solvent system (6 L each, straight through, 100:0, 0:100), and methanol was removed from the water-methanol (0:100) fraction under reduced pressure. The resulting aqueous solution was subjected to four liquid-liquid extractions with 2.5 L of ethyl acetate, and the ethyl acetate layer was concentrated to dryness under reduced pressure. The ethyl acetate extract (7.4 g) was placed on a silica gel column (φ60 x 170 mm) packed with n-hexane, and stepwise elution was performed in a n-hexane-ethyl acetate solvent system (1.5 L each, 100:0, 90:10, 80:20, 70:30, 50:50, 30:70, 0:100), and the n-hexane-ethyl acetate (90:10) fraction was concentrated to dryness under reduced pressure. Crude material (656 mg) was dissolved in a small amount of methanol and injected into a reverse-phase column (PEGASIL ODS SP100, φ20 x 250 mm, manufactured by Senshu Chemical, Japan) by high-performance liquid chromatography. Elution was performed under the conditions of water-acetonitrile-trifluoroacetic acid (30:70:0.1), flow rate 7 mL / min, and UV 210 nm detection. The peak at a retention time of 20 minutes was isolated, and atopenin B (31 mg) was obtained by distilling off acetonitrile under reduced pressure and then freeze-drying.

[0071] [Example 3] Production of Virantomycin The production of vilantomycin and vilantomycin-containing extract was carried out by referring to a previously reported method (J. Antibiot., 33:1395-1396 (1980)), using Actinomycete strain AM-2722 (a strain deposited on October 18, 2024, at the Patent Microorganism Depository Center, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) (Accession number NITE) The experiment was conducted using BP-04179. Specifically, 200 mL of actinomycete strain AM-2722, cultured in the liquid medium, was inoculated into one 30 L jar fermenter containing 20 L of liquid medium (unadjusted pH) consisting of 1.0% glucose (manufactured by Fujifilm Wako Pure Chemical Industries), 2.0% maltose (manufactured by Hayashibara), 0.3% yeast extract (manufactured by Oriental Yeast Co., Ltd.), 0.5% gelatin (manufactured by Nitta Gelatin Co., Ltd.), and 1.0% NaCl (manufactured by Kanto Chemical Co., Ltd.). The culture was then stirred and incubated at 27°C, 150 rpm, and aeration at 10 L / min for 4 days.

[0072] 20 L of the culture medium was centrifuged at 3000 rpm for 5 minutes to obtain 20 L of a vilantomycin-containing extract as the supernatant. Subsequently, using HP20 resin (φ35 x 50 mm) open column chromatography, stepwise elution was performed in a water-methanol solvent system (2 L each, passed through, 100:0, 50:50, 0:100), and methanol was removed from the water-methanol (0:100) fraction under reduced pressure. The resulting aqueous solution was separated twice with 500 mL of ethyl acetate, and the ethyl acetate layer was concentrated to dryness under reduced pressure. The ethyl acetate extract (4 g) was placed on a silica gel column (φ60 x 600 mm) packed with n-hexane, and stepwise elution was performed in a n-hexane-ethyl acetate solvent system (200 mL each, 80:20, 0:100), and the n-hexane-ethyl acetate (80:20 and 0:100) fractions were concentrated to dryness under reduced pressure. Crude material (655 mg) was dissolved in a small amount of methanol and injected into a reverse-phase column (PEGASIL ODS SP100, φ20 x 250 mm, manufactured by Senshu Chemical, Japan) by high-performance liquid chromatography. Elution was performed under the conditions of water-acetonitrile (20:80), flow rate 7 mL / min, and UV 210 nm detection. The peak with a retention time of 15 minutes was isolated, and after removing acetonitrile under reduced pressure, vilantomycin (106 mg) was obtained by lyophilization.

[0073] [Example 4] Production of Resistimycin The production of resistimycin and resistimycin-containing extract was carried out by referring to a previously reported method (J. Antibiot., 58:530-534 (2005)) using Actinomycete strain K05-0055 (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-04181). Specifically, glucose (manufactured by Fujifilm Wako Pure Chemical Industries) 2.0%, corn steep liquor (manufactured by Iwaki) 0.5%, oatmeal (manufactured by Nippon Shokuhin Seizo Co., Ltd.) 1.0%, Pharmamedia (manufactured by ADM) 1.0%, KH ２ PO ４ (Manufactured by Kanto Chemical Co., Ltd.) 0.5%, MgSO ４ 7H ２Sixty 500 mL Erlenmeyer flasks, each containing 100 mL of liquid culture medium (unadjusted pH) consisting of 0.5% O (manufactured by Kanto Chemical Co., Ltd.), were inoculated with 1 mL of Actinomycete strain K05-0055, which had been cultured in the liquid culture medium, and incubated with shaking at 27°C for 7 days.

[0074] 6 L of acetone was added to 6 L of culture medium and extracted for 30 minutes. After extraction, the mixture was filtered under reduced pressure, and acetone was removed from the resulting filtrate to obtain 6 L of resistimycin-containing extract. Subsequently, three liquid-liquid extractions were performed with 6 L of ethyl acetate, and the ethyl acetate layer was concentrated to dryness under reduced pressure. The ethyl acetate extract was placed on a silica gel column (φ60 x 600 mm) packed with n-hexane, and eluted stepwise with 150 mL of n-hexane and 150 mL of chloroform. The chloroform-eluted fraction was concentrated to dryness under reduced pressure. The ethyl acetate extract was subjected to three solid-liquid extractions with 3 mL of n-hexane to obtain resistimycin (16 mg) as a n-hexane insoluble product.

[0075] [Example 5] Production of Anisomycin The production of anisomycin and anisomycin-containing extract was carried out by referring to a previously reported method (J. Antibiot., 46:1300-1302 (1993)) using the Actinomycete strain KP-1241 (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-04182). Specifically, one 30L jar fermenter contained 20L of liquid medium (unadjusted pH) consisting of 2.0% glucose (manufactured by Fujifilm Wako Pure Chemical Industries), 2.0% kinako (manufactured by Noko Tanaka Ryu Shoten), and 0.3% NaCl (manufactured by Kanto Chemical Co., Ltd.), and the Actinomycete strain KP-1241 (accession number NITE BP-04182) cultured in the liquid medium. BP-04182) was inoculated into 200 mL and cultured with stirring at 27°C, 150 rpm, and 10 L / min aeration for 4 days.

[0076] 20 L of the culture medium was centrifuged using a Sharpless centrifuge to obtain 20 L of anisomycin-containing extract as the supernatant. The obtained aqueous solution was separated once with 20 L of ethyl acetate, and the ethyl acetate layer was concentrated to dryness under reduced pressure. The ethyl acetate extract (4.1 g) was then eluted (50:50) using an ODS resin (φ35 x 50 mm) open column chromatography with a water-methanol solvent system to finely fractionate the fraction containing the target substance. The fraction containing the target substance was lyophilized after removing methanol under reduced pressure. The crude substance (1.5 g) was dissolved in a small amount of methanol and injected into a reverse-phase column (CAPCELL PAK C18, φ20 x 250 mm, Osaka Soda Co., Ltd., Japan) using high-performance liquid chromatography. Elution was performed under water-acetonitrile (97:3), flow rate 6 mL / min, and UV 210 nm detection. The peak containing the target substance was isolated, and anisomycin (151 mg) was obtained by lyophilizing after removing acetonitrile under reduced pressure.

[0077] [Example 6] Verification of antiviral effect against ASFV - In vitro efficacy verification (1) Antiviral effect and cytotoxicity against ASFV in cultured cells In this example, the antiviral effect against ASFV of the antiviral compound samples prepared in Examples 1 to 5 was investigated in vitro. The materials used were as follows: Cells used: Porcine kidney macrophage cell line (IPKM cells) Virus: Armenia 2007 isolate Medium: Dulbecco's modified eagle medium (with 5% bovine serum added) (5% DMEM FBS)

[0078] The antiviral compound samples produced 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 ASFv was determined by the following procedure. 1. Seed IPKM cells in a 96-well plate (cell count 10,000 / well). 2. Incubate at 37 °C for 48 hr. 3. Dilute the antiviral compound sample 100-fold with the medium. 4. Discard the supernatant of the 96-well plate of IPKM cells, and add the sample dilution at 50 μl / well. Prepare 2 plates (antiviral evaluation, toxicity evaluation) with 2 wells each. 5. Dilute the virus with the medium and adjust to 10 ２ TCID ５０ / 50 μl. 6. Add the virus dilution (medium for the negative control) to the antiviral evaluation plate, and add 50 μl / well of the 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 5 days. 9. Dispense 10 μl / well of the CCK-8 solution (manufactured by Dojindo Laboratories). 10. After spin-down, subject to vortexing. 11. Incubate at 37 °C for 2 hr. Determine the back-titration by CPE. 12. Measure the absorbance at 450 nm with a plate reader. 13. Calculate the cell survival rate (%) of the sample wells using the following formula: Formula: (Sample absorbance - Absorbance of virus control) ÷ (Absorbance of negative control - Absorbance of virus control) × 100

[0080] As a target value, those with a cell survival rate of 25% or more in the antiviral evaluation and 50% or more in the toxicity evaluation are determined to have an antiviral effect. Also, as a test establishment condition, the difference in absorbance between the negative control and the virus control is 0.8 or more, and in the back-titration, 10 １．６ ~10 ２．４ TICD ５０ / 50 μl was used.

[0081] The results are shown in Figure 1. From the results shown in Figure 1, it was found that the antiviral compound under test has an antiviral effect against ASFv at a low concentration without showing cytotoxicity.

[0082] (2) Verification of the effective concentration of antiviral compounds against ASFV in cultured cells Next, the effective concentrations of the antiviral compound samples prepared in Examples 1 to 5 against ASFV were investigated. The materials used were as follows: Cells used: IPKM cells Virus: Armenia 2007 isolate Medium: DMEM FBS 5%

[0083] The cell survival rate (%) of ASFV-infected cells was determined using the following procedure: 1. Seed IPKM cells in a 96-well plate (10,000 cells / well). 2. Culture at 37°C for 48 hours. 3. Dilute the antiviral compound. 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 antiviral evaluation and toxicity evaluation). 5. Dispense the virus into 10 ２ TCID ５０ Dilute and dispense into 50 μl portions. 6. Add the virus dilution at 50 μl / well to the antiviral evaluation plate (use culture medium for negative control). Add culture medium at 50 μl / well to all wells to the toxicity evaluation plate. 7. Back-titration of the virus dilution. 1) Place the virus in culture medium for 10 minutes. －１ from －４ Dilute to [value missing]. 2) Discard the supernatant from the 96-well plate of IPKM cells and add 50 μl / well of culture medium to all wells. 3) Add the virus diluent at 50 μl / well (dispense 10 wells per diluent). 8. Incubate at 37°C for 5 days. 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. 10 １．６ ~10 ２．４ TICD ５０ / The test is considered complete at 50 μl. 12. Measure the absorbance at 450 nm with 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. 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 stability in liver Ms were also summarized. The effective concentration for ASFV was determined as 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 ASFV using pigs The mortality rate of suckling piglets infected with ASFV was determined by the following procedure. 1. The filtration-sterilized antiviral compound was diluted with sterile PBS and administered 1.5 mg (1 ml) intraperitoneally or orally to four suckling piglets aged 4 to 6 days. 2. After 1 hour, the ASFV AQS-C-1-22 strain (1 × 10) diluted with sterile PBS was administered. ２ TCID ５０) is administered intramuscularly in 1 ml doses to the gluteal muscles. Previous experiments have shown that infection leads to clinical symptoms such as fever, loss of appetite, and decreased vitality, and death occurs within approximately 5 days. 3. The same amount of antiviral compound is administered additionally 24 hours and 48 hours after the initial dose, and the animals are then kept for 7 days for clinical observation. Humane endpoints are defined as "(1) depression, loss of appetite or inability to stand for 2 days, (2) symptoms of distress (self-injurious behavior, abnormal posture, respiratory distress, vocalizations), (3) visible abnormalities with no signs of recovery (vomiting, diarrhea, bleeding), (4) rapid weight loss (decreased subcutaneous fat, to the point where the vertebral and pelvic bone protrusions are visible through the skin), and (5) appearance or significant enlargement of a tumor." If these conditions are reached, the animals will be euthanized immediately. Euthanasia is performed by administering a triple anesthetic mixture (approximately 0.48 mL / head) consisting of 2 mg / kg (approximately 0.4 mL / head) of xylazine at 20 mg / mL, 10 mg / kg (approximately 0.8 mL / head) of ketamine at 50 mg / mL or 0.04 mg / kg (approximately 0.16 mL / head) of medetomidine hydrochloride at 1 mg / mL, 0.2 mg / kg (approximately 0.16 mL / head) of midazolam at 5 mg / mL, and 0.2 mg / kg (approximately 0.16 mL / head) of butorphanol tartrate at 5 mg / mL into the gluteal muscle to induce sedation, analgesia, and muscle relaxation. After confirming that deep anesthesia has been achieved by lying down and loss of reflexes, euthanasia is performed by bleeding through an axillary artery incision.

[0086] The results of this experiment confirmed that intraperitoneal and oral administration of aspochalasin D, atopenin B, vilantomycin, ristomycin, and anisomycin, respectively, resulted in antiviral effects that improved clinical symptoms and mortality rates.

[0087] [Accession number] Accession number NITE BP-04176 (filamentous fungus strain FKI-6835, deposited on October 18, 2024) Accession number NITE BP-04178 (filamentous fungus strain FO-125, deposited on October 18, 2024) Accession number NITE BP-04179 (Streptomyces strain AM-2722, deposited on October 18, 2024) Accession number NITE BP-04181 (Streptomyces strain K05-0055, deposited on October 18, 2024) Accession number NITE BP-04182 (Streptomyces KP-1241 strain, deposited on October 18, 2024)

Claims

1. An antiviral agent against African swine fever virus (ASFV), comprising as an active ingredient at least one antiviral compound selected from the group consisting of aspochalasin D and its salts, atopenin B and its salts, vilantomycin and its salts, resistimycin and its salts, and anisomycin 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 the filamentous fungus FKI-6835 strain (accession number NITE BP-04176), the filamentous fungus FO-125 strain (accession number NITE BP-04178), the actinomycete AM-2722 strain (accession number NITE BP-04179), the actinomycete K05-0055 strain (accession number NITE BP-04181), and the actinomycete KP-1241 strain (accession number NITE BP-04182).

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 African swine fever.

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 African swine fever, comprising incorporating the antiviral agent described in claim 1 into a pharmaceutical or feed ingredient.

9. A method for treating or preventing African swine fever 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 the filamentous fungus FKI-6835 strain (accession number NITE BP-04176), filamentous fungus FO-125 strain (accession number NITE BP-04178), actinomycete AM-2722 strain (accession number NITE BP-04179), actinomycete K05-0055 strain (accession number NITE BP-04181), and actinomycete KP-1241 strain (accession number NITE BP-04182), 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 the filamentous fungus strain FKI-6835 (accession number NITE BP-04176), the filamentous fungus strain FO-125 (accession number NITE BP-04178), the actinomycete strain AM-2722 (accession number NITE BP-04179), the actinomycete strain K05-0055 (accession number NITE BP-04181), and the actinomycete strain KP-1241 (accession number NITE BP-04182).

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