Norovirus inactivation agent

Abstract

To provide a norovirus inactivator useful for inactivation of norovirus, which can reliably inactivate norovirus attached to clothing, fiber products, cooking utensils or the like and can prevent or reduce the spread of norovirus infection.SOLUTION: A norovirus inactivator contains ethanol, and at least one selected from boric acid and a salt thereof as an active ingredient. Preferably, the composition has a pH of 8 or more, the content of ethanol is 40-80 mass%, and the content of at least one selected from boric acid and a salt thereof is 0.02-0.8 mass%.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a norovirus inactivating agent. [Background technology]

[0002] Norovirus is a non-enveloped (membrane-like structure) RNA virus classified in the Caliciviridae family and Norovirus genus. It is known to have strong resistance to acid (gastric acid) and to be infectious in small amounts (approximately 10 to 100 viruses). At present, there is no vaccine or treatment for norovirus, so it is important to prevent infection by removing or inactivating the virus by cleaning and disinfecting cooking utensils, clothing, hands, etc., on which the virus may be attached.

[0003] Norovirus has strong physicochemical resistance, so disinfectants containing ethanol and cationic surfactants, which are effective against many bacteria, may not be sufficiently effective against norovirus when used in a general manner. For this reason, chlorine-based disinfectants (sodium hypochlorite, etc.), iodine agents (povidone-iodine, etc.), aldehyde agents (glutaral, etc.), peracetic acid preparations, etc. are used to inactivate norovirus. However, chlorine-based disinfectants have the effect of bleaching the colors and patterns of textiles, which limits their use on textile products and clothing.

[0004] Incidentally, conventionally, since a laboratory culture system for human norovirus (HNV) that infects humans has not been established, feline calicivirus (FCV) and murine norovirus (MNV), which are also members of the Caliciviridae family, have been used as surrogate viruses to evaluate the inactivation effect of norovirus. However, FCV is a virus that causes respiratory infections rather than diarrheal infections, and is vulnerable to acids and alkalis, and many differences from HNV have been reported. It has also been reported that MNV is significantly more vulnerable to alcohol than other enveloped viruses, including HNV (Non-Patent Document 1). In fact, Non-Patent Document 2 reported that alcohol, which has been thought to be effective for some surrogate viruses, is insufficient to inactivate HNV in the recently established HNV culture system. Therefore, there is a need to find materials that can actually exert their full effect against HNV.

[0005] On the other hand, boric acid, which is an oxoacid of boron, and one of its salts, borax, have bactericidal and cleansing properties and are used as medicines (in the ophthalmic field) as well as in detergents and preservatives. However, it is not known that borax or borax have the ability to inactivate norovirus. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2015-78479 A [Non-patent literature]

[0007] [Non-Patent Document 1] Cromeans Theresa et al., Appl. Environ. Microbiol. 80.18 (2014): 5743-5751. [Non-Patent Document 2] Costantini, Veronica, et al. "Human norovirus replication in human intestinal enteroids as model to evaluate virus inactivation." Emerging infectious diseases 24.8 (2018): 1453. [Non-Patent Document 3] Seiichi Tobe et al., J. Oleo Sci. 61, (4) 211-216 (2012) [Non-Patent Document 4] Hirotaka Takagi and Tomoyoshi Sugiyama·Medicine and Pharmacology·Vol. 57, No. 3, 311-312·March 2007 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention relates to providing a norovirus inactivating agent that is effective in inactivating norovirus. [Means for solving the problem]

[0009] The inventors have discovered that the inactivation effect against norovirus is improved by combining ethanol with boric acid or a salt thereof, which was previously thought to be insufficient for inactivating norovirus, and that the combination of ethanol and boric acid or a salt thereof is effective for inactivating noroviruses including HNV.

[0010] That is, the present invention relates to the following 1) to 3). 1) A norovirus inactivation agent having as active ingredients one or more selected from ethanol, boric acid and its salts. 2) A norovirus inactivating agent comprising ethanol, and one or more members selected from boric acid and its salts, the composition having a pH of 8 or higher. 3) A method for inactivating norovirus, which comprises contacting a subject suspected of being contaminated with norovirus with the norovirus inactivating agent described in 1). Effect of the Invention

[0011] According to the present invention, norovirus attached to clothing, textile products, cooking utensils, etc. can be reliably inactivated, and the spread of norovirus infection can be prevented or reduced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The norovirus inactivating agent of the present invention contains, as active ingredients, one or more members selected from ethanol, and boric acid and its salts. One embodiment of the norovirus inactivating agent of the present invention is a composition containing ethanol, and one or more members selected from boric acid and salts thereof. Here, the pH of the composition at 25° C. is, from the viewpoint of HNV inactivation effect, preferably 8 or more, more preferably 9 or more, more preferably 10 or more, and preferably 13 or less, more preferably 12 or less. Also, the pH is preferably 8 to 13, more preferably 9 to 12, more preferably 10 to 12. The pH can be adjusted by changing the blending ratio of the following alkaline agent, acid agent, inorganic salt, etc.

[0013] The ethanol content in the composition of the norovirus inactivating agent of the present invention is, from the viewpoint of the norovirus inactivating effect, preferably 30% by mass or more, more preferably 35% by mass or more, more preferably 40% by mass or more, and preferably 80% by mass or less, more preferably 78% by mass or less, more preferably 75% by mass or less, and preferably 30 to 80% by mass, more preferably 35 to 78% by mass, more preferably 40 to 75% by mass.

[0014] As the raw material ethanol, ordinary ethanol such as fermented ethanol, synthetic ethanol, bioethanol, and refined ethanol can be used, but it is preferable to use ethanol of a quality that can ensure safety according to the purpose of disinfection and sterilization.

[0015] In the present invention, the boric acid may be any of orthoboric acid, metaboric acid, and tetraboric acid, and preferably orthoboric acid and tetraboric acid. These boric acids may be used alone or in combination of two or more.

[0016] Examples of the salt of boric acid include inorganic acid salts of boric acid, preferably alkali metal salts of boric acid (sodium salt, potassium salt, etc.), alkaline earth metal salts of boric acid (calcium salt, magnesium salt), etc. Boric acid or a salt thereof may be in the form of a hydrate, such as borax. The borate salt is preferably sodium borate, potassium borate, calcium borate, magnesium borate, manganese borate, etc., more preferably sodium borate, and more preferably borax. Note that borax means sodium tetraborate decahydrate (Na2B4O7.10H2O). Among boric acid and salts thereof, at least one of boric acid and borax is preferred, at least one of orthoboric acid and borax is more preferred, and borax is even more preferred.

[0017] The content of one or more selected from boric acid and salts thereof in the composition of the norovirus inactivating agent of the present invention is, from the viewpoint of the norovirus inactivating effect, preferably 0.02% by mass or more, more preferably 0.03% by mass or more, more preferably 0.05% by mass or more, and preferably 0.8% by mass or less, more preferably 0.5% by mass or less, more preferably 0.4% by mass or less, more preferably 0.2% by mass or less, and is 0.02 to 0.8% by mass, preferably 0.03 to 0.5% by mass, more preferably 0.05 to 0.4% by mass, more preferably 0.05 to 0.2% by mass.

[0018] In a preferred embodiment, the content of one or more selected from boric acid and its salts is preferably 0.7 to 0.8% by mass when the content of ethanol is less than 40% by mass. Furthermore, when the ethanol content is 40% by mass or more and less than 50% by mass, the content of one or more selected from boric acid and salts thereof is preferably 0.02 to 0.4% by mass, and when the ethanol content is 50% by mass or more and 80% by mass or less, the content of one or more selected from boric acid and salts thereof is preferably 0.02 to 0.4% by mass, more preferably 0.05 to 0.4% by mass, and more preferably 0.05 to 0.2% by mass.

[0019] The norovirus inactivating agent of the present invention may further contain an alkaline agent. The alkaline agent may be one or more selected from carbonates, hydrogen carbonates, dihydrogen phosphates, dihydrogen phosphates, triphosphates, hydroxides of alkali metals (e.g., sodium hydroxide, potassium hydroxide, etc.), hydroxides of alkaline earth metals (e.g., magnesium hydroxide, calcium hydroxide, etc.), alkali metal carboxylates (e.g., sodium acetate, potassium acetate, etc.), etc. Among these, one or more selected from carbonates, hydrogen carbonates, dihydrogen phosphates, dihydrogen phosphates, and triphosphates are preferred. Examples of the salt include sodium salts, potassium salts, and ammonium salts.

[0020] More specific examples of the alkaline agent include sodium carbonate, potassium carbonate, ammonium carbonate, sodium hydrogen carbonate, ammonium hydrogen carbonate, sodium dihydrogen phosphate, tripotassium phosphate, sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, and the like. Among these, one or more selected from sodium carbonate, potassium carbonate, sodium hydrogen carbonate, tripotassium phosphate, and sodium dihydrogen phosphate are preferred, and sodium hydrogen carbonate, sodium carbonate, and potassium carbonate are more preferred.

[0021] The norovirus inactivating agent of the present invention may further contain an acid agent. The acid agent may be one or more selected from organic acids such as citric acid, malic acid, lactic acid, tartaric acid, phytic acid, adipic acid, gluconic acid, succinic acid, etc., and inorganic acids such as phosphoric acid, hydrochloric acid, etc. By including an acid agent, the virus inactivation effect is improved and the pH adjustment of the virus inactivation agent is facilitated.

[0022] The content of the acid agent in the composition of the norovirus inactivating agent of the present invention is preferably 0.01% by mass or more, more preferably 0.03% by mass or more, more preferably 0.05% by mass or more, and is preferably 2% by mass or less, more preferably 1% by mass or less, more preferably 0.5% by mass or less, from the viewpoint of safety on the skin, and is preferably 0.01 to 2% by mass, more preferably 0.03 to 1% by mass, more preferably 0.05 to 0.5% by mass.

[0023] In addition, the norovirus inactivating agent of the present invention may contain various inorganic salts. Examples of inorganic salts include sodium chloride, potassium chloride, magnesium chloride, calcium chloride, ammonium chloride, sodium bromide, potassium bromide, calcium bromide, sodium sulfate, potassium sulfate, magnesium sulfate, ammonium sulfate, iron sulfate, copper sulfate, potassium aluminum sulfate, ammonium aluminum sulfate, sodium nitrate, potassium nitrate, ammonium nitrate, sodium nitrite, potassium nitrite, ammonium nitrite, sodium thiocyanate, potassium thiocyanate, ammonium thiocyanate, and sodium iodide.

[0024] Furthermore, in addition to the above-mentioned components, the norovirus inactivating agent of the present invention may contain additives such as chelating agents, silicones, thickeners, oils, natural extracts, colorants, and fragrances, as appropriate.

[0025] As shown in the examples below, a composition containing ethanol and one or more selected from boric acid and its salts exhibits an excellent virus inactivation effect against noroviruses (HNV, FCV). Therefore, the composition can be a norovirus inactivating agent for inactivating noroviruses including HNV. By applying the norovirus inactivating agent to an object where norovirus contamination is a concern, it becomes possible to inactivate norovirus in the object.

[0026] In the present invention, norovirus refers to a non-enveloped RNA virus classified in the Caliciviridae family and the Norovirus genus, and includes HNV (human norovirus), FCV (feline calicivirus), MNV (murine norovirus), and the like. Noroviruses are divided into seven genogroups (GI to GVII) based on homology of their genomic base sequences, and the HNVs that infect humans are nine GI species (GI.1, GI.2, GI.3, GI.4, GI.5, GI.6, GI.7, GI.8, GI.9), 19 GII species (GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.8, GII.9, GII.10, GII.12, GII.13, GII.14, GII.15, GII.16, GII.17, GII.20, GII.21, GII.22), and one GIV species (GIV.1); however, the HNV in the present invention may be of any of these genotypes. In the present invention, the HNV inactivation effect can be evaluated using a system in which human small intestinal organoids (hSIOs) are 3D cultured on an extracellular matrix, and then the intestinal epithelial cells obtained by inducing differentiation are infected with HNV (see Examples).

[0027] Norovirus can be inactivated using the norovirus inactivating agent of the present invention by contacting it with objects where norovirus contamination is a concern, such as kitchens (kitchen spaces such as kitchen floors and walls), sinks, refrigerators and other places where food is handled, cooking utensils such as knives and pots, tableware such as plates, refrigerators, living spaces such as toilets, bathrooms, washrooms, living rooms, bedrooms, textile products such as sofas, cushions, bedding, curtains, etc. Here, the contact is not particularly limited, and examples include directly spraying the norovirus inactivating agent of the present invention on the object, filling a spray device such as a trigger or aerosol and spraying it. The contact time is not limited, but is, for example, preferably 5 seconds or more, more preferably 10 seconds or more, and is preferably left for 60 minutes or less.

[0028] The form of the norovirus inactivating agent of the present invention for contact is not particularly limited, and it may be in the form of, for example, a liquid, gel, or paste. In the case of a liquid form, it can be used particularly as a spray, lotion, etc. For example, it can be filled into a known spray container such as a trigger spray container (direct pressure or pressure accumulation type) or a dispenser type pump spray container, and the spray amount can be adjusted so that it can be sprayed in a place where the virus is present or thought to be present.

[0029] It can also be used as a wet wipe (wet napkin, etc.). For example, the norovirus inactivating agent of the present invention, a solubilizing carrier, and various additives as necessary can be incorporated into a substrate containing a woven or nonwoven web of natural fibers, regenerated fibers such as rayon, synthetic fibers such as polyethylene, polypropylene, polyester, or a mixture of natural and synthetic fibers. Specifically, it can be used for sterilizing surfaces of pet grooming products, kitchens and bathrooms, exercise equipment, etc. It can also be used in addition to hand washing liquids, neutral detergents, deodorants, skin conditioners, etc.

[0030] When the composition is made into a gel or paste form using a water-soluble gel or oil-based gel, it can be used as a medical product by directly applying it to the human body, pets, etc., or it can be supported on paper, nonwoven fabric, etc. and used as a filter for an air purifier.

[0031] Furthermore, the norovirus inactivating agent of the present invention can be used by kneading it with a resin, such as natural, petroleum, or synthetic wax, rosin resin, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, polyester, polyolefin, or acrylic resin. The above kneaded product can be used as it is, or can be supported on a porous carrier, made into a sheet, or the sheet can be used as a laminate. Examples of the porous carrier include natural polymers such as cellulose and chitosan, the above synthetic resins, inorganic porous substances such as calcium silicate, and the like, which are made into any shape such as granules or sheets. The above kneaded product or laminate can be installed in, for example, air conditioning equipment, toilets, bathrooms, living rooms, hospital rooms, hospital waiting rooms, dust boxes, and the like, and can be used while gradually volatilizing the antiviral composition. EXAMPLES

[0032] Compositions of the present invention (Examples 1 to 15) and comparative compositions (Comparative Examples 1 to 4) were prepared by mixing ethanol, boric acid or a salt thereof, and other ingredients according to the formulations shown in Table 1, and the inactivation effects against HNV and FCV were evaluated by the methods described below. The results are also shown in Table 1. In the table, the solubility column indicates the solubility of boric acid and borax, and is defined as follows: ◯: completely dissolved, ×: presence of undissolved matter.

[0033] [Table 1]

[0034] <Evaluation method> 1. Evaluation of HNV inactivation effect (1) Culture of human small intestine organoids (hSIOs) hSIOs were embedded in Matrigel (Corning, 356231) and cultured in 3D on a 24-well plate. IntestiCult Organoid Growth Medium (Human) (STEMCELL Technologies, ST-06010) was used as the medium. The procedures for medium replacement, subculture, and monolayer formation using 96-well plates were performed according to the user manual. CultureSure Y-27632 (Fujifilm Wako Pure Chemical Industries, 036-24023), a ROCK (Rho-associated coiled-coil forming kinase) inhibitor, was added to the medium at a final concentration of 10 μM to inhibit anoikis for 2 days after trypsinization. A basal medium was prepared by adding 5 mL of GlutaMAX I (100x) (Gibco, 35050-061), 5 mL of 1M HEPES (Gibco, 15630080), and 5 mL of Penicillin-Streptomycin (Gibco, 15140122) to 500 mL of Advanced DMEM / F12 (Gibco, 12634010). A differentiation medium was prepared by mixing equal amounts of the basal medium and IntestiCult Organoid Growth Medium component A. Differentiation was induced for a total of 7 days by replacing the differentiation medium at 200 μL per well every 2 days in monolayered intestinal epithelial cells in a 96-well plate.

[0035] (2) Preparation of 10% emulsion of feces containing HNV (HuNoV) A 10% fecal emulsion was prepared from feces of a patient infected with HNV type GII.4. One tablet of cOmplete protease inhibitor cocktail tablets (Sigma-Aldrich, 11697498001), a protease inhibitor, was suspended in 50 mL of D-PBS(-). 1 g of feces was suspended in 10 mL of cOmplete-containing D-PBS(-) and mixed well with a test tube mixer. After standing at 4°C for 20 minutes, the mixture was centrifuged at 2,000×g for 10 minutes at 4°C. The supernatant was collected in a new tube and stored at -80°C until used in an infection experiment.

[0036] (3) Inactivation of HNV and infection of differentiated hSIO The 10% fecal emulsion containing HNV was diluted 10-fold with differentiation medium and filtered using a 1 mL syringe and a Millex HV Filter unit (Millipore, SLHVR04NL). 5 μL (2.8 × 10 6 The fecal solution was mixed with 45 μL of a suspension (treatment solution) of a given composition shown in Table 1 (corresponding to HNV genome copy) and reacted at room temperature for 30 seconds. Next, 1.45 mL of a basic medium containing 1 mg / mL bovine serum albumin (Sigma-Aldrich, A9647) was added to the drug-treated fecal solution. The centrifuge tube was placed in a fixed-angle rotor TLA-55 (Beckman Coulter) and ultracentrifuged for 1.5 hours at Rmax of 186047 × g (corresponding to 55,000 rpm) using an Optima MAX-TL (Beckman Coulter), and the supernatant was removed. The pellet was suspended in 100 μL of differentiation medium to prepare an infection solution for hSIO. The existing medium in the well was removed, and the infection solution prepared by the above method was applied to the hSIO after differentiation induction for 7 days. Incubation was performed at 37 ° C for 3 hours. After washing three times with 300 μL of basal medium, 250 μL of differentiation medium containing porcine bile extract (Sigma-Aldrich, B8631-100G) at a final concentration of 125 ppm was added, and the cells were cultured at 37°C under 5% CO2 until the time of sampling. 10 μL of the supernatant was collected immediately after the start of culture (day 0) and after 3 days of culture (day 3). The collected supernatant was stored at -80°C until it was subjected to RT-qPCR. In addition, the infected solution for hSIO was prepared by the same procedure using basal medium instead of the drug solution, and used as a non-drug control.

[0037] (4)RT-qPCR The number of HNV genome copies in the collected supernatant was quantified using the Norovirus Detection Kit G1 / G2 (Toyobo, FIK-273) according to the protocol. PCR amplification and data collection were performed using a LightCycler480II (Roche). Based on the measured viral load, the results were evaluated on a three-point scale: A: HNV was inactivated to the lower limit of detection; B: HNV was detected but at a lower level than the control (no drug treatment); and C: HNV proliferated at the same rate as the control (no drug treatment).

[0038] 2. Evaluation of FCV inactivation effect The study was conducted with reference to the "2015 Survey Report on Inactivation Conditions for Norovirus" (National Institute of Health Sciences) and JIS L 1922, which are generally known as standard methods for evaluating viral inactivation. In a 500-μL sample tube, 27 μL of a suspension (treatment solution) having a given composition shown in Table 1 and 1.0 × 10 7 3 μL of feline calicivirus solution (F-9 strain, ATCC VR-782) adjusted to PFU / mL was added and mixed well. After 30 seconds, the reaction was stopped by diluting 1 / 10 with quenching solution (D / E Neutralizing Broth). As a blank, the same operation was performed using 27 μL of PBS (phosphate buffered saline) instead of the treatment solution. After stopping the reaction, the mixture was further diluted 1 / 10 with DMEM medium (Dulbecco's modified Eagle medium), and 0.5 mL of the diluted mixture was used to infect CRFK cells (feline kidney-derived cell line) (ATCC CCL-94) washed with PBS (phosphate buffered saline), and after 1 hour, the medium was replaced with methylcellulose-containing medium. After 2 days, the cells of each sample at a specified time were stained with crystal violet, the number of plaques was counted, and the logarithmic reduction was calculated. Log reduction=log (number of blank plaques)−log (number of test sample plaques) In this specification, the log reduction is expressed as Δ4log, for example, when the log reduction is 4. The evaluation criteria are as follows, and the results are shown in the table. <Judgment criteria> 4: No plaques present (below detection limit: greater than Δ4log) 3:Δ2~3log 2:Δ1~2log 1: Less than Δ1 log

Claims

1. A norovirus inactivating agent comprising ethanol, and one or more members selected from boric acid and its salts, the composition having a pH of 8-10.

2. The norovirus inactivating agent according to claim 1, wherein the content of ethanol is 40 to 80 mass %, and the content of one or more selected from boric acid and salts thereof is 0.02 to 0.8 mass %.

3. The norovirus inactivating agent according to claim 1 or 2, wherein the norovirus is a human norovirus.

4. The norovirus inactivating agent according to any one of claims 1 to 3, wherein the salt of boric acid is borax.

5. A method for inactivating norovirus, comprising contacting a subject suspected of being contaminated with norovirus with the norovirus inactivating agent according to any one of claims 1 to 4.

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