JP5542682B2 - Calicivirus inactivation method - Google Patents

Description

Cross-reference of related applications

  This application claims the priority of Japanese Patent Application No. 2008-274103 filed on October 24, 2008 and Japanese Patent Application No. 2009-164896 filed on July 13, 2009, the entire description of which is here Specifically incorporated by reference.

  The present invention relates to a method for inactivating viruses belonging to the family Caliciviridae such as feline calicivirus and norovirus (hereinafter referred to as calicivirus).

[Background technology]
According to the Ministry of Health, Labor and Welfare, one-tenth of patients with acute gastroenteritis in winter are caused by norovirus infection. Most of the infection routes of norovirus are oral infections, and the method of inactivating or inactivating norovirus is important from the viewpoint of preventing secondary contamination from food handlers and cooking utensils.

  According to the Ministry of Health, Labor and Welfare, disinfecting ethanol and reverse soap are not effective in inactivating norovirus, and sodium hypochlorite aqueous solution is effective in inactivating norovirus (see Q16 for Q & A on Norovirus, Ministry of Health, Labor and Welfare) , Http://www.mhlw.go.jp/topics/syokuchu/03.html#3-3) (Non-Patent Document 1)

  However, the sodium hypochlorite aqueous solution has a strong odor, which causes problems such as adhesion to the skin and rough skin, and discoloration of clothes and carpets. In addition, when mixed with acid, it generates chlorine gas, which may cause poisoning and may be dangerous.

  On the other hand, since ethanol is a component contained in foods such as alcoholic beverages, it is highly safe, but it alone has no effect on norovirus inactivation as described above.

  On the other hand, a disinfectant solution for calicivirus using ethanol, natural extract (grapefruit seed extract) and phytic acid in combination has been reported (Patent Document 1).

  In addition, ethanol and organic acids are used in combination for the suppression and sterilization of general bacteria and are known as food preservatives (Patent Document 2), but the effect on norovirus is not known.

On the other hand, Patent Document 3, which has been recently released, includes the following (a), (b), (c) and (d), and does not include other disinfectants for the purpose of disinfecting. Disinfectants are disclosed. This disinfectant can be applied to fingers and skin and is also described as being for virucidal use.
(A) 40 to 90% (w / w) of ethanol, isopropyl alcohol, or a mixture thereof with respect to the entire disinfectant
(B) 0.1-2% (w / w) of lactic acid based on the whole disinfectant
(C) 0.01 to 2% (w / w) citric acid based on the entire disinfectant
(D) The zinc-containing compound that liberates zinc ions in the solution is 0.001 to 0.1% (w / w) with respect to the entire disinfectant.

[Patent Literature]
[Patent Document 1] Japanese Unexamined Patent Publication No. 2007-320924 [Patent Document 2] Japanese Patent Publication No. 60-35106 [Patent Document 3] Japanese Patent No. 4163249

[Non-patent literature]
[Non-Patent Document 1] http://www.mhlw.go.jp/topics/syokuchu/03.html#3-3
The entire description of the above patent documents and non-patent documents is specifically incorporated herein by reference.

[Summary of Invention]
[Problems to be solved by the invention]
As mentioned above, the sodium hypochlorite aqueous solution, which is said to be effective in inactivating norovirus by the Ministry of Health, Labor and Welfare, has several problems, and ethanol alone is effective in inactivating norovirus. However, it is only known as a sterilization solution for calicivirus by the combined use of a special natural extract (grapefruit seed extract) and phytic acid.

  According to the description of the specification, the disinfectant described in Patent Document 3 is highly effective against non-enveloped viruses such as Norovirus. However, the application target of this disinfectant was mainly limited to fingers and skin.

  On the other hand, disinfection of fingers and skin is also necessary, but it is equally necessary in the field of medical care and nursing care to prevent viruses against sputum and manure containing non-enveloped viruses such as norovirus. It is an activation method. At medical and nursing care sites, sputum and manure excreted by patients infected with or suspected of having non-enveloped viruses may sometimes be scattered on the floor, etc., and this must be handled safely. It is done. Until now, such excrement has been wiped with a disposable sheet or paper, and then treated with an aqueous sodium hypochlorite solution. However, in addition to the above-mentioned problems with sodium hypochlorite aqueous solution, sputum and feces and urine contain organic substances mainly composed of proteins in addition to viruses. There was a problem that hypochlorous acid was consumed before activation, and the inactivation effect was significantly reduced.

  Therefore, an object of the present invention is to have a calicivirus such as a norovirus contained in a disinfectant having no danger like an aqueous sodium hypochlorite solution in the presence of an organic substance mainly composed of a protein. Is to provide a method that can be effectively deactivated or deactivated.

By using an ethanol preparation (composition) in which an acid is present so as to exhibit a pH in a specific range with respect to ethanol water, the present inventors can eliminate calicivirus in which an organic substance mainly composed of protein is present. The present invention was completed by finding that it could be activated. In this specification, the inactivation and inactivation of calicivirus are used synonymously, and the inactivated or inactivated state of calicivirus means that the infectivity of the virus is substantially lost and detoxified. Specifically, it means that the infectious titer of the virus is 10 ^-4 or less compared to the control.

[Means for solving problems]
The present invention for solving the above problems is as follows.
[1]
The presence of calicivirus is suspected, and the substance containing protein is brought into contact with a composition comprising an aqueous solution containing ethanol and acid as active ingredients and having a pH in the range of 2.5 to 5.0. A method of inactivating calicivirus.
[2]
The presence of the calicivirus is suspected and the protein-containing material is present on the facility or article surface;
Coating at least a part or all of the facility or article surface with a water-permeable and / or retaining sheet;
The method according to [1], comprising the steps of supplying the composition onto the sheet, bringing the composition into contact with the surface under the sheet, and allowing the composition to stand for a predetermined time after the contact.
[3]
The method according to [2], wherein at least a part of the object existing on the surface is wiped off, and the sheet is coated on a facility or article surface after the object is wiped off.
[4]
The method according to [2] or [3], wherein the sheet contains the composition in advance.
[5]
The method according to any one of [1] to [4], wherein the presence of the calicivirus is suspected and the protein-containing product is vomit or manure.
[6]
The method according to any one of [1] to [4], wherein the presence of the calicivirus is suspected and the protein-containing product is a vomit or manure of a person infected with calicivirus.
[7]
The method according to any one of [1] to [6], wherein the ethanol concentration in the composition is in the range of 40 to 85% (w / w).
[8]
The method according to any one of [1] to [7], wherein the acid is an organic acid or an inorganic acid.
[9]
The method according to any one of [1] to [8], wherein the acid concentration is in the range of 1 to 10%.
[10]
The method according to any one of [1] to [9], wherein the acid is an acid having a pH of 2.5 to 5.0 in a concentration of 1 to 10%.
[11]
The method according to [8], wherein the acid is citric acid, lactic acid, or phosphoric acid.
[12]
The method according to any one of [1] to [11], wherein the composition further contains a polyglycerol fatty acid ester.
[13]
The method according to any one of [1] to [12], wherein the calicivirus is a norovirus.
[14]
[1] to [13] in which the presence of the calicivirus is suspected and the supply amount (volume ratio) of the composition is in the range of 0.5 to 100 with respect to the volume 1 of the protein-containing product. The method according to any one.

[Effect of the invention]
ADVANTAGE OF THE INVENTION According to this invention, the method which can inactivate the calicivirus which coexists with protein can be provided using an ethanol formulation (composition), and it is effective especially in the spread suppression of norovirus. Furthermore, according to the present invention, it is possible to provide an effective and safe method for inactivating viruses against vomit, feces and urine containing non-enveloped viruses such as norovirus, which are required in the field of medical care and nursing care.

[Mode for Carrying Out the Invention]
The present invention includes contacting a composition suspected of the presence of calicivirus and containing a protein with an aqueous solution containing ethanol and acid as active ingredients and having a pH in the range of 2.5 to 5.0. The present invention relates to a method for inactivating calicivirus in the presence of proteins.

  The composition used in the present invention contains ethanol and an acid as active ingredients. As described above, ethanol (ethyl alcohol) is said to have no effect on norovirus inactivation or inactivation when used alone. On the other hand, the inventors of the present invention have used a norovirus coexisting with proteins, in which an inactivation effect cannot be obtained with an aqueous sodium hypochlorite solution by using ethanol in combination with an acid and adjusting the pH to a range of 2.5 to 5.0. It has been found that an inactivation effect is exhibited.

  The ethanol concentration in the composition used in the present invention can be appropriately selected depending on the method of use of the composition (whether it is used as a stock solution or is appropriately diluted, etc.), for example, 40 to 85% ( The range of w / w) is appropriate from the viewpoint that inactivation of calicivirus is reliably obtained. If the ethanol concentration is in this range, an effect on bacteria is often expected at the same time, and it is generally said that this concentration is highly effective for sterilizing bacteria. In addition, if the ethanol concentration is low, drying after application such as spraying is delayed, and it takes time to move to the next operation. A more preferred concentration of ethanol is 40-59% (w / w). If it exceeds 60%, it becomes a dangerous item under the Fire Service Law, and handling is restricted. In addition, when the ethanol concentration is high, the solubility of citric acid, citrate, etc. as the acid and salt is low, which may be difficult to handle in terms of production and product stability.

  The acid used in combination with ethanol can be, for example, an organic acid or an inorganic acid. Examples of the organic acid include acetic acid, tartaric acid, succinic acid, malic acid, fumaric acid, phytic acid, and inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid, and phosphorus. An acid etc. can be mentioned. The acid concentration can be appropriately determined in consideration of the pH of the composition, and can be, for example, in the range of 1 to 10%. If the acid concentration is too low, the inactivation effect may be reduced due to the buffer capacity of the protein contained in the object to be used such as sputum. On the other hand, if the acid concentration is too high, water or ethanol will not be used after the ethanol preparation is used. The above-mentioned range is preferable because solid content remains after volatilization and needs to be removed by washing or the like. Further, depending on the type of acid, there is a possibility that the acid is concentrated to promote metal corrosion. A preferable concentration of the acid is about 1% to 5%. If the concentration is higher than 5%, the solid content tends to remain after the use of the composition and the water and ethanol volatilize, which may be visually noticeable. A more preferable concentration of the acid is about 1% to 3%.

  The composition sets the pH in the range of 2.5 to 5.0. If the pH is less than 2.5 and becomes too low, adverse effects such as metal corrosion become larger, so pH 2.5 or higher is preferable. A more preferable range of pH is in the range of 2.5 to 4.5, more preferably in the range of 2.8 to 4.0, and more preferably in the range of 3.0 to 3.5, from the viewpoint that the inactivation effect of calicivirus is reliably obtained. Within this range, the virus can be inactivated almost certainly, but if it exceeds 3.5, the inactivation effect may be reduced depending on the use conditions. For example, when the object of use such as vomit has a buffering capacity, the inactivation effect tends to decrease. However, if the pH is 4.0 or less, the virus inactivating effect is exhibited in many cases.

  The acid is suitably an acid having a pH of 2.5 to 5.0 in the aqueous solution as the composition in the concentration range of 1 to 10%. Specific examples of such acids include citric acid, lactic acid and phosphoric acid described above. The acid may be used alone or in combination with a plurality of acids as long as the pH range of the composition is within the above range. In addition, at least a part of the acid may be a salt, and when it is converted to a salt, the buffering action may also work to facilitate pH adjustment. Examples of the salt include alkali metal (lithium, sodium, potassium) salts of organic acids such as lactic acid and citric acid, or inorganic acids such as phosphoric acid.

  The above composition can be prepared by dissolving a water-soluble acid or acid metal salt in water and then adding ethanol.

  In addition to ethanol and acid, the composition enhances the bactericidal effect on bacteria and fungi by mixing antibacterial components such as glycerin fatty acid ester, polyglycerin fatty acid ester and glycine, plant extract and plant extract. Thus, it is possible to prepare an ethanol preparation having a comprehensively high function in terms of hygiene. Among them, polyglycerol fatty acid esters can increase the contact area by reducing the surface tension of ethanol preparations, and also easily penetrate into fine gaps. Can increase the sex. Preferable examples of the polyglycerin fatty acid ester include lauric acid tetraglyceride, lauric acid pentaglyceride, lauric acid hexaglyceride and the like.

  In addition, the said composition of this invention does not contain a zinc containing compound. This is because when a large amount of organic acid or inorganic acid is added, a salt with zinc ions is formed, and the solubility may deteriorate.

  The method of the present invention comprises contacting the composition with an object suspected of having a calicivirus and containing a protein. The thing in which the presence of calicivirus is suspected and the protein-containing material can be, for example, vomit or manure, and can be vomit or manure of a person infected with calicivirus or suspected of being infected. it can. When a large amount of protein is contained in vomit or feces and calicivirus is present therein, it is difficult to inactivate calicivirus with a sodium hypochlorite aqueous solution having a concentration usually used. On the other hand, in the composition used in the present invention, calicivirus can be inactivated even with such sputum or manure.

  When the presence of calicivirus is suspected as described above and the protein-containing material is vomit or manure, it may be excreted on the floor or bed, etc. It is desired at the site and nursing care site. In the method of the present invention, sputum or manure is excreted or present on the surface of a facility or article such as a bed or a bed, that is, the presence of calicivirus is suspected, and the protein-containing material is a floor or a bed. If present on the surface of the facility or article, at least part or all of the surface of the facility or article is covered with a water-permeable and / or retaining sheet. Preferably, at least a part of the object existing on the surface is wiped off, and the sheet is coated on the facility or article surface after the object is wiped off. Next, the composition is supplied onto the sheet, the composition is brought into contact with the surface under the sheet, and left for a predetermined time after the contact. The predetermined time can be, for example, 1 to 10 minutes, preferably 3 to 5 minutes. When the calicivirus is present in the object or the surface of the article, the calicivirus can be inactivated despite the presence of the protein. The amount of the composition to be supplied can be appropriately determined according to the amount of a protein containing protein that is suspected of the presence of calicivirus present on the surface of a facility or article such as a bed or bed. As shown in the examples described later, the composition has a protein amount of 40 mg / ml or less in a state where the composition is supplied to the protein-containing product (mixed state) according to the composition. Has an effect of inactivating calicivirus. Therefore, the supply amount of the composition can be appropriately determined in consideration of this point, and when the presence of calicivirus is suspected and the amount of the protein-containing material is large, the amount of the composition according to the amount Supply. Presence of calicivirus and the amount (volume ratio) of the composition to the amount 1 of the protein-containing product is, for example, in the range of 0.5 to 100, preferably in the range of 1 to 20. can do. Without covering the sheet, the composition may be supplied to and contacted with a suspected presence of calicivirus present on the facility or article surface and containing protein. However, since the presence of calicivirus is suspected, it is preferable to cover the sheet in order to prevent scattering. Furthermore, by contacting the sheet while covering the sheet, it is possible to suppress the evaporation of ethanol contained in the composition and to maintain the inactivation effect for a relatively long time. A coating sheet having a high liquid holding power is preferable from the viewpoint that a relatively large amount of the composition can be held and a high inactivation effect on calicivirus can be obtained. Examples of such a sheet having a high liquid holding ability include a water absorbent sheet (containing a water absorbent resin) and a nonwoven fabric.

  The said sheet | seat coat | covered on the plant | facility or article | item surface after wiping off the said thing can also contain the said composition previously. Moreover, the said composition can be previously contained also in the thing used for wiping off at least one part of the said thing which exists in the said surface, for example, a nonwoven fabric etc. Inclusion (supply) of the composition in the sheet or the like can be carried out immediately before use, but the composition in the sheet or the like is previously contained and sealed in a sealed container (for example, a bag). It can also be stored.

  Although there is no restriction | limiting in particular in the supply method to the said sheet | seat at the time of the contact of the said composition, For example, a composition can be poured or sprayed.

  In the method of the present invention, the surface of the facility or article treated by the above method can be further sprayed with the above composition to thoroughly inactivate calicivirus.

  The composition of the present invention is effective for inactivating norovirus among caliciviruses.

An example of a specific method of the present invention is as follows.
When handling vomit and feces from patients who are scattered on the floor, wear disposable gowns (aprons), masks and gloves, and gently dispose of feces and vomits with paper towels, etc. so that viruses in the filth do not scatter. It is appropriate to wipe off. After wiping, the floor is covered with a sheet, the composition is supplied to the coated sheet, and the floor under the sheet is disinfected. Furthermore, it is preferable to treat splashes that may have been scattered, for example, by spraying the composition on the floor not covered with a sheet. Alternatively, after wiping, the floor can be further wiped so as to be immersed in the composition without being covered with a sheet. A method that does not use a sheet may be appropriate when a small amount of sputum is scattered. After these treatments, if necessary, water can be wiped off.

  In addition, the above composition is not only for those suspected of having a calicivirus containing protein, but for example, tableware, food machinery, food equipment, gloves, fingers and other skin, table, cutting board, It can also be used to inactivate viruses attached to cloths and the like. It is effective to immerse the tableware and food machinery in order to inactivate viruses. When it cannot be immersed, it can also be used by spraying on these items. When immersion and spraying are not possible, the duster can be impregnated with the composition of the present invention, and the target surface can be wiped off. In this case, a higher concentration of acid or ethanol is preferable.

[Example]
Hereinafter, the present invention will be described in more detail based on examples.

Example 1 (Reference Example) Infectivity inactivation effect test against feline calicivirus (depending on ethanol concentration)
M / 100 phosphate buffer pH 7.2 (hereinafter referred to as “ethanol reagent special grade 99.5%” at 80%, 70%, 60%, 50%, 40%, 30%, 20% in W / W%) The sample was diluted with PBS (abbreviated as PBS), and the sample disinfection effect (virus inactivation effect) was measured by the test method shown below. The results are shown in Table 1.

Test Method 1) Feline calicivirus F9 strain was used as the virus used to measure the disinfection effect (virus inactivation effect) of the sample.
2) The stock solution of Sample 1 and Sample 2 and a 2-fold diluted solution (M / 100 phosphate buffered saline pH 7.2: diluted with PBS) were used. If the end point of the disinfection effect was not obtained, the sample was further diluted.
3) 100 μl of virus solution was added to 900 μl of each sample solution and allowed to act at room temperature for 1 minute and 30 minutes.
After the reaction, 100 μl was collected and added to 900 μl of broth liquid (5 mg of meat extract, 15 mg / ml of peptone) manufactured by Nissui Pharmaceutical Co., Ltd. containing a large amount of protein. The reason why the broth made by Nissui Pharmaceutical Co., Ltd. was used is to prevent the progress of the disinfection effect of the test sample after the action.
4) The virus control group of the virus used was diluted with a broth solution instead of the disinfectant sample, and the infectivity titer was measured.
5) The virus solution diluted 10 times with the bouillon solution was further diluted 10 ^-1 to 10 ^-4 with the bouillon solution (step dilution solution).
6) Remove the cell growth medium (MEM medium containing 10% fetal bovine serum) of CrFK cells cultured on a 6-well plate for 4 days using an aspirator, and use 3 well cells for each step diluted virus solution, Each was inoculated with 100 μl.
7) After adsorbing virus for 1 hour in the incubator, tissue culture solution without fetal bovine serum (MEM medium; MINIMUM ESSENTIAL MEDIUM EAGLE (MODIFIED) WITH EARLE`S SALTS (MIP Biomedicals, Inc) in sodium bicarbonate The cell was washed by adding and removing 3 ml per well. (To remove cell toxicity of test sample)
8) 3 ml of MEM agar medium (containing 0.03% L-glutamine and 0.22% sodium bicarbonate) containing 0.7% agar per plate (40 ° C.), and 6 holes when the agar medium solidifies The plate was inverted and cultured for 72 hours in a 5% CO ₂ incubator.
9) 1.5 ml of 10% formalin PBS solution per well was added to the 6-well plate after completion of the culture and left at room temperature for 1 hour. After fixing the cells, the agar medium was removed with running water and stained with methylene blue. This method allowed the virus count to be counted as plaques.
10) The disinfection effect was evaluated by comparing the infectivity value of the virus to which the disinfectant was added to the infectivity value of the virus to which no disinfectant sample was added.

Summary: The above results show that ethanol alone is not effective in inactivating feline calicivirus.

Example 2 (Reference Example) Infectivity inactivation effect test against feline calicivirus (pH dependence of ethanol-containing solution) 1
Samples having the compositions shown in Table 2 were prepared, and the disinfection effect (virus inactivation effect) of the samples was measured by the test method described in Example 1. The results are shown in Table 3.

* Glycerin fatty acid ester: (mono) capric acid monoglyceride

Summary: Sample 1 showing alkalinity did not show a disinfection effect against feline calicivirus. Sample 2 showing acidity has a disinfecting effect, and it has been found that the virus infectivity titer is reduced to 1 / 10,000 even when diluted 2 times below the detection limit in an action time of 1 minute.

Example 3 (Reference Example) Infectivity inactivation effect test against feline calicivirus (pH dependence of ethanol-containing solution) 2
An equal amount of feline calicivirus stock solution was added to the samples shown in Table 4, reacted for 1 minute at room temperature, and diluted 10-fold with a 5-fold bouillon solution (protein amount 80 mg / ml) to eliminate cytotoxicity. Further, MEM was used as a diluent in the dilution series of samples up to 10 ⁻⁶ by 10-fold serial dilution. 100 μl of each diluted solution was inoculated into 3 wells of a 3 day-cultured feline liver cell (CrFK) 6-well plate that had become confluent and 90 minutes after virus adsorption, MEM medium containing 0.7% agar was overlaid. The cells were cultured for 50 hours in a 34 ° C., 5% CO ₂ culture incubator. After fixing with 10% formalin for several hours, the agar medium was removed, methylene blue staining solution was added, and the number of plaques after staining was counted. The results are shown in Table 5.

*% Of composition indicates% (W / W).
* Sodium hypochlorite is manufactured by Junsei Chemical Co., Ltd., and the measured value of effective chlorine concentration is 5.6%.
* Feline calicivirus: F9 strain * The ethanol concentration of samples 1 to 4, the citric acid concentration of samples 1 to 8 and the sodium hypochlorite concentration of samples 9 to 13 are diluted twice after mixing with an equal amount of virus. As a result, the concentration is halved.

Summary 1. The mixed preparation of citric acid and ethanol showed an excellent virus inactivating effect from 5% to 1.25% citric acid against feline calicivirus, but the effect decreased at 0.625%.
2. On the other hand, the citric acid only group containing no ethanol had no effect even at 5%.
3. When the results of Example 1 were combined, it became clear that those that were not effective by themselves were effective when mixed.
4). A final concentration of 200 ppm was required for complete virus inactivation of sodium hypochlorite.

Example 4 (Example) Infectious inactivation effect on feline calicivirus in the presence of protein 1
Samples shown in Table 6 were prepared, and the disinfection effect (virus inactivation effect) was measured according to the method described in Example 3. However, the feline calicivirus liquid used was one containing normal bouillon having a different amount of protein and one containing only MEM medium not containing normal bouillon. As a comparison, the same test was conducted for sodium hypochlorite.

  As the bouillon, ordinary bouillon manufactured by Kyokuto Pharmaceutical Co., Ltd. was used at a 5-fold concentration (protein amount: 85 mg / ml). Since equal amounts of bouillon and virus were mixed, the amount was 42.5 mg / ml. Further, equal amounts of sample and virus solution were mixed, so the final protein concentration was 21.5 mg. The reaction time was 1 minute at room temperature.

Sample () shows final concentration when combined with virus solution

Summary 1. The addition of bouillon protein to feline calicivirus reduced the disinfection effect.
2. With 5% citric acid + ethanol, the disinfection effect did not decrease even in the presence of protein.
3. Lactic acid 5% alone did not show the virus inactivating effect, but the effect was recognized by adding ethanol.
4). Although sodium hypochlorite had a high concentration (final concentration 800 ppm), the disinfection effect disappeared in the presence of protein.
5. Since natural viruses are mixed with proteins in some form, the effect of inactivating viruses under mixed proteins such as 5% citric acid + ethanol is very useful.

Example 5 (Example) Infectious inactivation effect 2 on feline calicivirus in the presence of protein 2
The correlation between the amount of protein mixed in feline calicivirus and the inactivation effect was examined. Samples shown in Table 7 were prepared, and the disinfection effect (virus inactivation effect) was measured according to the method described in Example 3. However, the feline calicivirus liquid used was one containing normal bouillon having a different amount of protein and one containing only MEM medium not containing normal bouillon. As a comparison, the same test was conducted for sodium hypochlorite.

  However, the feline calicivirus solution was added with 5 times normal bouillon (85 mg / ml) and used as protein amounts of 40 mg / ml, 10 mg / ml and 8 mg / ml. For the protein amount of 0 mg / ml, MEM (tissue culture solution containing no protein) was substituted. The sample and virus solution were mixed in equal amounts and reacted at room temperature for 60 seconds.

* Numbers in parentheses indicate final drug concentration or protein concentration when mixed with virus solution.

Summary 1. Citric acid 5% (2.5%) + ethanol completely inactivated the virus containing 20 mg / ml protein.
2. Citric acid 2.5% (1.25%) + ethanol completely inactivated the virus containing 10 mg / ml protein.
3. Citric acid 1.25% (0.625%) + ethanol inactivated virus containing 4 mg / ml protein up to 10 ³ but not completely inactivated.
4). Sodium hypochlorite 1600 ppm (800 ppm) completely inactivated a virus containing 4 mg / ml protein, but could not inactivate viruses containing 10 mg / ml and 20 mg / ml protein.
5. Sodium hypochlorite 800 ppm (400 ppm) and sodium hypochlorite 400 ppm (200 ppm) could not be inactivated against viruses containing 4 mg / ml protein.

Example 6 (Example) Infectious inactivation effect on feline calicivirus in the presence of protein 3
Samples shown in Table 8 were prepared, and the disinfection effect (virus inactivation effect) was measured according to the method described in Example 3. However, the feline calicivirus liquid used was one containing normal bouillon having a different amount of protein and one containing only MEM medium not containing normal bouillon. As a comparison, the same test was conducted for sodium hypochlorite. However, a normal bouillon (170 mg / ml) having a 10-fold concentration was added to feline calicivirus to prepare a virus solution containing a protein amount of 80 mg / ml, 40 mg / ml, or 20 mg / ml, and this was used. For the protein amount of 0 mg / ml, MEM (tissue culture solution containing no protein) was substituted. Equal amounts of feline calicivirus solutions having different protein amounts were added to citric acid, ethanol, and sodium hypochlorite solutions having different concentrations, and reacted at room temperature for 60 seconds.

NT is Non Test
* The numbers in parentheses are the final formulation concentration or protein concentration when mixed with the virus solution.

Summary 1. Citric acid 5% (2.5%) + ethanol completely inactivated the virus in the presence of protein at a final concentration of 40 mg / ml.
2. Citric acid 2.5% (1.25%) + ethanol showed a reduction in infectivity of about 10 ^{6 in} the presence of protein at a final concentration of 10 mg / ml.
3. Sodium hypochlorite 3200 ppm (1600 ppm) completely inactivated the virus in the presence of 10 mg / ml protein, but the virus inactivation effect decreased rapidly as the protein concentration increased.
4). Sodium hypochlorite was found to lose its virus inactivating action in the presence of protein at a concentration of 1600 ppm (800 ppm) or less.

Example 7 (Reference Example) Effect on Norovirus
The inactivation effect on anti-norovirus was evaluated by RT-PCR. The composition of the test sample was ethanol 68%, lactic acid 10%, and water 22%, and stool-derived norovirus (belonging to NV gene group 2) was used as the test material.

  400 μL of a test sample was added to 100 μL of Norovirus suspension, and the mixture was stirred with a vortex mixer, and RNA extraction was performed 30 minutes later. The RNA extract was treated with DNase, followed by RT-PCR (reverse transcription + polymerase chain reaction), and further amplified by the PCR method. The amplified product was subjected to 2.5% agarose gel electrophoresis, and an electrophoresis photograph was taken. The same operation was performed twice for the test sample.

  For comparison, (1) Norovirus suspension 500 μL, (2) Norovirus suspension 100 μL with disinfecting ethanol 400 μL, (3) Norovirus suspension 100 μL with sodium hypochlorite solution (200 ppm) 400 μL. The same operation was performed for the added one.

  As a result, norovirus band (344 bp) was not observed for the test sample and the system using sodium hypochlorite solution, and norovirus band (344 bp) was used for the system using only norovirus suspension and disinfecting ethanol. Was observed.

Example 8 (Example)
Material 1. Virus: Feline calicivirus F9 strain2. 2. Cell: CrFK cell Bouillon liquid: 10 times concentration 160mg / ml
4). Virus dilution: MEM medium (FCS-)

Sample 5: Sodium hypochlorite 1600ppm aqueous solution

Method:
1. Samples shown in Table 9 and feline calicivirus stock solutions containing protein amounts of 80 mg / ml, 20 mg / ml, 10 mg / ml, and 0 mg / ml were mixed in an equal volume of 100 μl and reacted at room temperature for 60 seconds.
2. Immediately after the reaction, 100 μl of the sample / virus mixture was added to 900 μl of a bouillon solution with a concentration of 80 mg / ml to stop the reaction. This was serially diluted 10-fold from 10 ^-2 to 10 ^-5 with MEM medium.
3. Inoculate 100 μl of virus sample solution diluted 10-fold in a 6-well plate on the third day of culture, and after 90 minutes of virus adsorption, overlay with 0.7% agar medium in a 5% CO ₂ incubator. The cells were cultured at 34 ° C for 60 hours.
4). After completion of the culture, the number of plaques formed by 10% formalin fixation and methylene blue staining was counted to determine the infectious titer.
5. For virus control, MEM was used instead of the sample, and the method was based on the sample.
The results are shown in Table 10.

Summary 1. Sample 1 showed a virus inactivating effect, and virus infectivity was completely inactivated even in the presence of a protein amount of 10 mg / ml.
2. For sample 2, no virus inactivation was observed. However, as shown in Example 11 to be described later, even with the composition of sample 2, virus inactivation can be recognized when the amount used is increased with respect to the amount of virus solution.
3. Samples 3 and 4 showed a virus inactivating effect, but did not reach sample 1.

Example 9 (Example)
Material 1. Virus: Feline calicivirus F9 strain2. 2. Cell: CrFK cell Bouillon liquid: 10 times concentration 160mg / ml
4). Virus dilution: MEM medium (FCS-)

Method:
1. The sample shown in Table 11 and feline calicivirus stock solution containing 20 mg / ml, 10 mg / ml, and 0 mg / ml of protein were mixed in an equal volume of 100 μl and reacted at room temperature for 60 seconds.
2. Immediately after the reaction, 100 μl of the sample / virus mixture was added to 900 μl of a bouillon solution with a concentration of 80 mg / ml to stop the reaction. This was serially diluted 10-fold from 10 ^-2 to 10 ^-5 with MEM medium.
3. Inoculate 100 μl of virus sample solution diluted 10-fold in a 6-well plate on the third day of culture, and after 90 minutes of virus adsorption, overlay with 0.7% agar medium in a 5% CO ₂ incubator. The cells were cultured at 34 ° C for 60 hours.
4). After completion of the culture, the number of plaques formed by 10% formalin fixation and methylene blue staining was counted to determine the infectious titer.
5. For virus control, MEM was used instead of the sample, and the method was based on the sample.

Summary 1. For samples 1 to 6, the virus inactivating effect was observed, and the virus infectivity was completely inactivated even in the presence of a protein amount of 20 mg / ml.
2. For sample 7, the inactivation effect was low for viruses in virus solution containing 20 mg / ml of protein, but for viruses in virus solution containing protein at a concentration lower than 10 mg / ml. An inactivation effect was observed.

Example 10 (Example)
Material virus: Feline calicivirus F9 strain normal bouillon 10 times concentration: 160mg / ml
Sodium hypochlorite: effective chlorine concentration 4%

Method 1. Four types of virus solutions with different protein concentrations, adjusted to the final protein concentrations of 80 mg / ml, 40 mg / ml, 20 mg / ml, and 0 mg / ml by adding normal broth to the feline calicivirus added to the samples shown in Table 13. Prepared.
2. Viruses used for each sample were viruses with different protein concentrations, and the virus inactivating activity was measured.
3. 100 μl of the sample was mixed with 100 μl of the virus in an equal amount, and vortexed at room temperature at 25 ° C. for 60 seconds, then 100 μl was taken and added to 900 μl of 80 mg / ml protein broth and diluted 10 times. The reason why such a high-concentration bouillon solution is used is to detoxify the cytotoxicity of the disinfectant against CrFK cells used to stop the action of the sample and to form plaques.
4. MEM was used for the dilutions from 10 ⁻² to 10 ^−5, and 100 μl of each dilution was inoculated into a 6-well plate in duplicate for each dilution.
5. In the control virus group, MEM was added in an equal amount to the virus containing each concentration of protein.
6. Adsorption of virus to cells for 90 minutes, overlay of agar medium, culture at 34 ° C. for 60 hours.
7). Plaques formed on the plate after culture were fixed with formalin, stained with methylene blue, and the infectious titer was calculated from the number of plaques.

* NT: Not tested

Summary Sample 3 appeared to be superior in inactivating viruses in the presence of proteins.

Example 11 (Example)
Material 1. Virus: Feline calicivirus F9 strain2. 2. Cell: CrFK cell Bouillon liquid: 10 times concentration 160mg / ml
4). Virus dilution: MEM medium (FCS-)

Method 1. Samples shown in Table 15 and feline calicivirus stock solutions containing protein amounts of 80 mg / ml, 20 mg / ml, 10 mg / ml, and 0 mg / ml were mixed in equal amounts of 100 μl and reacted at room temperature for 60 seconds.
6.2. Immediately after the reaction, 100 μl of the sample / virus mixture was added to 900 μl of a bouillon solution with a concentration of 80 mg / ml to stop the reaction. This was serially diluted 10-fold from 10 ^-2 to 10 ^-5 with MEM medium.
3. Inoculate 100 μl of virus sample solution diluted 10-fold in a 6-well plate on the third day of culture, and after 90 minutes of virus adsorption, overlay with 0.7% agar medium in a 5% CO ₂ incubator. The cells were cultured at 34 ° C for 60 hours.
4). After completion of the culture, the number of plaques formed by 10% formalin fixation and methylene blue staining was counted to determine the infectious titer.
5. For virus control, MEM was used instead of the sample, and the method was based on the sample.

Summary 1. In the system of virus: drug = 1: 1, the samples B, F, and G showed a virus inactivating effect even in the presence of 10 mg / ml of the final protein.
2. Samples A and C also showed a virus inactivating effect, but did not reach B, F, and G.
3. In the system of virus: drug = 1: 9, virus inactivation effect was observed for samples A to D even in the presence of a final protein concentration of 16 mg / ml.
4). Sample E also showed a virus inactivating effect, but did not reach AD.

  INDUSTRIAL APPLICABILITY The present invention is useful in drug production and public health related to the inactivation of caliciviruses, particularly noroviruses.

Claims (10)

A method of inactivating calicivirus present in vomit or feces,
A method comprising contacting a composition comprising an aqueous solution containing ethanol, phosphoric acid , glycerin fatty acid ester and polyglycerin fatty acid ester and having a pH in the range of 2.5 to 5.0 with the sputum or manure. The vomit or manure is present on a facility or article surface;
Coating at least a part or all of the facility or article surface with a water-permeable and / or retaining sheet;
The method according to claim 1, comprising: supplying the composition onto the sheet, bringing the composition into contact with the surface under the sheet; and allowing the composition to stand for a predetermined time after the contact.   The method according to claim 2, wherein at least a part of the object existing on the surface is wiped off, and the sheet is coated on a facility or article surface after the object is wiped off.   The method according to claim 2 or 3, wherein the sheet contains the composition in advance.   The method according to any one of claims 1 to 4, wherein the vomit or manure is vomit or manure of a person infected with calicivirus.   The method according to claim 1, wherein the ethanol concentration in the composition is in the range of 40 to 85% (w / w).   The method according to any one of claims 1 to 6, wherein the concentration of the phosphoric acid is in the range of 1 to 10% (w / w).   The method according to any one of claims 1 to 7, wherein the calicivirus is a norovirus.   The method according to claim 1, wherein a supply amount (volume ratio) of the composition is in a range of 0.5 to 100 with respect to a volume 1 of the vomit or manure.   The method according to claim 1, wherein the polyglycerol fatty acid ester is lauric acid pentaglyceride.