JP7610364B2 - Norovirus inactivation agent - Google Patents

Description

The present invention relates to a norovirus inactivation agent.

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 acidity (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.

Norovirus has strong physicochemical resistance, so disinfectants containing ethanol or cationic surfactants, which are effective against many bacteria, may not be sufficiently effective against norovirus when used in the usual way. 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.

Incidentally, conventionally, since the culture system for human norovirus (HNV) that infects humans has not been established in the laboratory, feline calicivirus (FCV) and murine norovirus (MNV), which are also members of the Caliciviridae family, have been used as substitute viruses to evaluate the inactivation effect of norovirus. However, FCV is a virus that causes respiratory infections rather than diarrheal infections, and is weak against acids and alkalis, and many differences from HNV have been reported. In addition, it has been reported that MNV is significantly weaker against 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 substitute viruses in the recently established HNV culture system, is insufficient to inactivate HNV.
Therefore, there is a need to find a material that can actually exert its effect sufficiently against HNV.

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.

JP 2015-78479 A

Cromeans Theresa et al., Appl. Environ. Microbiol. 80.18 (2014): 5743-5751. Costantini, Veronica, et al. "Human norovirus replication in human intestinal enteroids as model to evaluate virus inactivation." Emerging infectious diseases 24.8 (2018): 1453. Seiichi Tobe et al., J. Oleo Sci. 61, (4) 211-216 (2012) Hirotaka Takagi, Tomoyoshi Sugiyama, Medicine and Pharmacology, Vol. 57, No. 3, 311-312, March 2007

The present invention relates to providing a norovirus inactivation agent that is effective in inactivating norovirus.

The inventors have discovered that the inactivation effect against norovirus is improved by using boric acid or a salt thereof in combination with ethanol, 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.

That is, the present invention relates to the following 1) to 3).
1) A norovirus inactivating agent having as active ingredients at least one selected from ethanol, and 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, comprising contacting a subject suspected of being contaminated with norovirus with the norovirus inactivating agent according to 1).

According to the present invention, norovirus attached to clothing, textiles, cooking utensils, etc. can be reliably inactivated, and the spread of norovirus infection can be prevented or reduced.

The norovirus inactivating agent of the present invention contains, as active ingredients, ethanol, and one or more members selected from boric acid and salts thereof.
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 the 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.

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 also preferably 80% by mass or less, more preferably 78% by mass or less, more preferably 75% by mass or less. Also, it is preferably 30 to 80% by mass, more preferably 35 to 78% by mass, more preferably 40 to 75% by mass.

Normal raw 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 guarantee safety for disinfection and sterilization applications.

In the present invention, the boric acid may be any of orthoboric acid, metaboric acid, and tetraboric acid, but orthoboric acid and tetraboric acid are preferred. These boric acids may be used alone or in combination of two or more.

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.) 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 (Na ₂ B ₄ O ₇ .10H ₂ O).
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.

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, in terms 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 also 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. Also, it 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.

In a preferred embodiment, the content of one or more selected from boric acid and its salts is 0.7 to 0.8% by mass when the content of ethanol is less than 40% by mass.
Furthermore, when the content of ethanol 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 content of ethanol 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.

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.

Specific examples of alkaline agents include sodium carbonate, potassium carbonate, ammonium carbonate, sodium bicarbonate, ammonium bicarbonate, sodium dihydrogen phosphate, tripotassium phosphate, sodium hydroxide, potassium hydroxide, sodium acetate, and potassium acetate. Of these, one or more selected from sodium carbonate, potassium carbonate, sodium bicarbonate, tripotassium phosphate, and sodium dihydrogen phosphate are preferred, and sodium bicarbonate, sodium carbonate, and potassium carbonate are more preferred.

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.

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, from the viewpoint of safety on the skin, and is also preferably 2% by mass or less, more preferably 1% by mass or less, more preferably 0.5% by mass or less. Also, it 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.

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.

In addition to the above-mentioned components, the norovirus inactivating agent of the present invention can also contain additives such as chelating agents, silicones, thickeners, oils, natural extracts, colorants, and fragrances, as appropriate.

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 inactivation agent for inactivating noroviruses including HNV. By applying the norovirus inactivation agent to an object where there is concern about norovirus contamination, it becomes possible to inactivate norovirus in the object.

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), etc.
Noroviruses are divided into seven genogroups (GI to GVII) based on the homology of their genome base sequences, and the HNV that infects 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). The HNV of 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 (hSIO) are 3D cultured on an extracellular matrix, and then the intestinal epithelial cells obtained by inducing differentiation are infected with HNV (see Examples).

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.

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.

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 added to 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 disinfecting 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.

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

Furthermore, the norovirus inactivating agent of the present invention can be used by kneading it with a resin, which can include synthetic resins such as natural, petroleum, and synthetic waxes, rosin resins, ethylene-vinyl acetate copolymers, ethylene-vinyl alcohol copolymers, polyesters, polyolefins, and acrylic resins.
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.

According to the formulations shown in Table 1, ethanol, boric acid or its salt, and other ingredients were mixed to prepare compositions of the present invention (Examples 1 to 15) and comparative compositions (Comparative Examples 1 to 4), and the inactivation effects against HNV and FCV were evaluated by the methods shown 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 ◯: completely dissolved, and ×: some undissolved material present.

<Evaluation method>
1. Evaluation of HNV inactivation effect (1) Culture of human small intestine organoid (hSIO) hSIO was embedded in Matrigel (Corning, 356231) on a 24-well plate and cultured in 3D. The medium used was IntestiCult Organoid Growth Medium (Human) (STEMCELL Technologies, ST-06010). The procedures for medium replacement, subculture, and monolayer formation using a 96-well plate were performed according to the user manual. For two days after trypsin treatment, CultureSure Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd., 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. A basal medium was prepared by adding 5 mL of GlutaMAX I (100x) (Gibco, 35050-061), 5 mL of 1 M 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 component A of IntestiCult Organoid Growth Medium. Differentiation was induced for a total of 7 days by replacing the differentiation medium at 200 μL per well at 2-day intervals with intestinal epithelial cells monolayered in a 96-well plate.

(2) Preparation of 10% emulsion of HNV (HuNoV)-containing feces A 10% emulsion of feces was prepared from feces of a patient 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 x 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.

(3) Inactivation of HNV and Infection of Differentiated hSIO 10% fecal emulsion containing HNV was diluted 10-fold with differentiation medium and filtered using a 1 mL syringe and Millex HV Filter unit (Millipore, SLHVR04NL). 5 μL of the filtered fecal solution (corresponding to 2.8×10 ⁶ HNV genome copies) was mixed with 45 μL of a suspension (treatment solution) of a given composition shown in Table 1 in a PA microcentrifuge tube (Beckman Coulter, 357448) and reacted at room temperature for 30 seconds. Next, 1.45 mL of a basal medium containing 1 mg/mL bovine serum albumin (Sigma-Aldrich, A9647) was added to the drug-treated fecal solution. The centrifuge tube was set in a fixed angle rotor TLA-55 (Beckman Coulter) and ultracentrifuged at Rmax for 1.5 hours at a centrifugal force of 186047×g (equivalent 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 infection solution prepared by the above method was applied to the hSIO after 7 days of differentiation induction in which the existing medium in the well had been removed. Incubation was carried out at 37 ° C for 3 hours. After washing three times with 300 μL of basic medium, 250 μL of differentiation medium containing porcine bile extract (Sigma-Aldrich, B8631-100G) was added to a final concentration of 125 ppm, and the cells were cultured under conditions of 37 ° C and 5% CO ₂ until the timing 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 infection solution for hSIO prepared by the same procedure using basal medium instead of the drug solution was used as a drug-free control.

(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). The procedure was performed according to the protocol. PCR amplification and data measurement were performed using a LightCycler 480II (Roche). there was.
Based on the measured viral load, A: HNV is inactivated to the lower limit of detection, B: HNV is detected but reduced compared to the control (no drug treatment), C: equivalent to the control (no drug treatment). The results were evaluated on a three-point scale: HNV proliferation, cytopenia, or cytopenia.

2. Evaluation of FCV inactivation effect Evaluation of FCV inactivation effect was performed with reference to the "2015 Report on Survey Conditions for Inactivation of 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) of a predetermined composition shown in Table 1 and 3 μL of a feline calicivirus solution (F-9 strain, ATCC VR-782) adjusted to 1.0×10 ⁷ PFU/mL were added and mixed well. After 30 seconds, the reaction was stopped by diluting 1/10 with a 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 solution was used to infect CRFK cells (feline kidney-derived cell line) (ATCC CCL-94) that had been washed with PBS (phosphate buffered saline), and after 1 hour, the medium was replaced with a methylcellulose-containing medium. After 2 days, the cells of each sample at a given time were stained with crystal violet, and the number of plaques was counted to calculate the logarithmic reduction.
Log reduction=log (number of blank plaques)−log (number of test sample plaques) In this specification, the log reduction is expressed as Δ4log when the log reduction is 4. The evaluation criteria are as follows, and the results are shown in the table.
<Criteria>
4: No plaques were present (below the detection limit: greater than Δ4 log)
3: Δ2 to 3 log
2: Δ1 to 2 log
1: Less than Δ1 log

Claims (5)

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 . 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 %. The norovirus inactivating agent according to claim 1 or 2 , wherein the norovirus is a human norovirus. The norovirus inactivating agent according to any one of claims 1 to 3 , wherein the salt of boric acid is borax. 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 .