WO2015129278A1 - Virus inactivation effect test - Google Patents

Abstract

The present invention addresses the problem of providing a technique by which the effect of a germicide against norovirus can be easily tested. The present invention provides a method of measuring the inactivation effect of chlorous acid water or a chlorous acid water preparation against norovirus, the method comprising: (A) a step for inoculating host cells with a feline calicivirus (FCV) strain or other norovirus-equivalent strain; (B) a step for culturing the inoculated host cells in a serum-containing medium; (C) a step for recovering the culture solution and cells; (D) a step for centrifuging the recovered culture solution and cells, and obtaining a supernatant; (E) a step for using the supernatant as a test virus solution, and performing an inactivation treatment on the test virus solution using chlorous acid water or a chlorous acid water preparation; (F) a step for neutralizing by diluting with BSA-added PBS after the inactivation treatment; and (G) a step for culturing host cells inoculated with the neutralized virus solution, and measuring the TCID₅₀.

Description

Virus inactivation test

The present invention relates to a technique for simply testing a virus inactivation effect.

Recently, food poisoning caused by norovirus and infectious diseases have become very serious problems. In particular, norovirus infection in infants under 5 years old accounts for about 60% of the total, and death cases occur. It is developing into a problem.

The government authorities, including the Japanese Ministry of Health, Labor and Welfare, are now considering a new anti-norovirus measure for infants under 5 years old. Currently, the only effective disinfectant and disinfectant is sodium hypochlorite, and sodium hypochlorite is a drug that can be used by diluting it as much as necessary. Can't prepare for. Alcohols are the only bactericidal / disinfecting agents that can be used in sudden situations, but these alcohols are generally expected to have little effect on viruses. Is desired.

The present inventors have developed chlorous acid water or a preparation thereof as a novel disinfectant / disinfectant and confirmed the inactivation effect of norovirus using the plaque method. As a result, it was found that it has an inactivating effect on Norovirus (International Publication No. 2014/188311 (Patent Document 1)), and this aqueous chlorite (International Publication No. 2008/026607 (Patent Document 2)) We tried to apply these preparations as disinfectants and disinfectants for infants aged 5 years and under, vomit, and filth.

The method currently employed by the Ministry of Health, Labor and Welfare is the TCID ₅₀ method, which uses each disinfectant and disinfectant and has been evaluated for its inactivation effect on feline calicivirus, a substitute for norovirus.

International Publication No. 2014/188311 International Publication No. 2008/026607

The present inventors have developed a new method that can efficiently test the killing effect against feline calicivirus for the test of norovirus. The present invention also provides the following.

In one aspect, the present invention provides (A) inoculating a host cell with a feline calicivirus (FCV) strain or other Norovirus equivalent strain; (B) culturing the inoculated host cell in a serum-containing medium; (C) a step of collecting the culture medium and cells; (D) a step of centrifuging the collected culture medium and cells to obtain a supernatant; and (E) a sample using the supernatant as a test virus solution. A method for measuring the inactivation effect of a sample against norovirus without freezing and thawing, comprising the step of performing an inactivation test.

In one embodiment, the serum-containing medium used in the present invention is fetal bovine serum-containing Eagle MEM.

In one embodiment, centrifugation is performed at about 1,500 to 2,000 × g.

In another embodiment, the feline calicivirus (FCV) strain or other Norovirus equivalent strain uses a plurality of strains.

In yet another embodiment, the feline calicivirus (FCV) strain or other norovirus equivalent strain comprises feline calicivirus (FCV) F4 strain and F9 strain.

In yet another embodiment, the step (C) is performed when all cytopathic effects (CPE) have occurred.

The present invention further provides the following items.
(Item 1)
(A) inoculating a host cell with a feline calicivirus (FCV) strain or other Norovirus equivalent strain;
(B) culturing the inoculated host cells in a serum-containing medium;
(C) recovering the culture solution and cells;
(D) centrifuging the collected culture medium and cells to obtain a supernatant;
(E) A step of inactivating the test virus solution with a chlorite aqueous solution or a chlorite water preparation using the supernatant as the test virus solution;
(F) The step of neutralizing by diluting with albumin phosphate buffered saline (PBS) after the inactivation treatment;
(G) culturing host cells inoculated with the neutralized virus solution and measuring TCID ₅₀ ,
A method for measuring the inactivation effect of chlorite water or a chlorite water preparation against norovirus.
(Item 2)
Item 2. The method according to Item 1, wherein the albumin is bovine serum albumin (BSA).
(Item 3)
Item 4. The method according to Item 1, wherein the step (F) is performed by diluting with PBS containing 0.1% BSA.
(Item 4)
Item 4. The method according to Item 3, wherein the step (F) is performed by diluting 10 times with PBS containing 0.1% BSA.
(Item 5)
Item 5. The method according to any one of Items 1 to 4, wherein the serum-containing medium is fetal bovine serum-containing Eagle MEM.
(Item 6)
Item 6. The method according to any one of Items 1 to 5, wherein the centrifugation is performed at about 1,500 to 2,000 × g.
(Item 7)
Item 7. The method according to any one of Items 1 to 6, wherein a plurality of strains are used as the feline calicivirus (FCV) strain or other Norovirus equivalent strain.
(Item 8)
Item 8. The method according to any one of Items 1 to 7, wherein the feline calicivirus (FCV) strain or other Norovirus equivalent strain comprises feline calicivirus (FCV) F4 strain and F9 strain.
(Item 9)
Item 9. The method according to Item 8, wherein the feline calicivirus (FCV) strain or other Norovirus equivalent strain is feline calicivirus (FCV) F4 strain.
(Item 10)
Item 10. The method according to any one of Items 1 to 9, wherein the step (C) is performed when all cytopathic effects (CPE) are generated.
(Item 11)
Item 11. The method according to Item 10, wherein the step (C) is performed when the cells are cultured for about 2 days.
(Item 12)
The method according to any one of items 1 to 11, wherein the culture in the step (G) is performed for 7 to 8 days.
(Item 13)
Item 13. The method according to Item 12, wherein the culture in the step (G) is performed for 8 days.

It will be understood that the features described above can be implemented in combination, and that such embodiments are within the scope of the present invention.

Still further embodiments and advantages of the invention will be recognized by those of ordinary skill in the art upon reading and understanding the following detailed description as needed.
(The invention's effect)
According to the present invention, a technique capable of simply testing the effect of a bactericide on a norovirus is provided, and the possibility of being widely used in the food industry, the medical field and the like is further increased.

Hereinafter, the present invention will be described. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

In the present specification, “chlorous acid water” refers to an aqueous solution containing chlorous acid (HClO ₂ ) used as a disinfectant. Chlorous acid water can be produced, for example, by the production method described in International Publication No. 2014/1888311 and PCT / JP2014 / 006379. Chlorous acid water produced by the production method described in PCT / JP2014 / 006379 can stably maintain chlorous acid (HClO ₂ ) over a long period of time by creating a transition state and delaying the decomposition reaction. When a specimen of chlorite water was measured with a spectrophotometer, an absorption part containing acidic chlorite ions (H ⁺ + ClO ₂ ⁻ ) that show a peak in the vicinity of 260 nm between wavelengths of 240 to 420 nm in the UV spectrum and around 350 nm When _two absorption parts containing chlorine dioxide (ClO ₂ ) representing a peak can be confirmed at the same time, that is, in the case of showing a twine, the presence of chlorous acid water can be recognized. At this time, it is considered that the cycle reaction of chlorine dioxide (ClO ₂ ) and acidic chlorite ion (H ⁺ + ClO ₂ ⁻ ) is proceeding mainly with chlorous acid (HClO ₂ ) as a main component.

In the present specification, “chlorite water” and “CAW (chloro acid water)” can be used interchangeably.

“Chlorous acid aqueous preparation” can be manufactured using chlorous acid water. Although it is not limited to this as a typical composition of a chlorite aqueous formulation, chlorite water (5% product) 60.00% (w / v) (the concentration of chlorous acid is 50,000 ppm) ), Potassium dihydrogen phosphate 1.70% (w / v), potassium hydroxide 0.50% (w / v) and purified water 37.8% (w / v) (Sold under the name "Outturlock Super" by the applicant), but in the case of this composition, chlorous acid water is 0.25% (w / v) to 75% (W / v), potassium dihydrogen phosphate is 0.70% (w / v) to 13.90% (w / v), potassium hydroxide is 0.10% (w / v) to 5. It may be 60% (w / v). Sodium dihydrogen phosphate may be used instead of potassium dihydrogen phosphate, and sodium hydroxide may be used instead of potassium hydroxide.

In the invention described in PCT / JP2014 / 006379, a conventional sodium chlorate aqueous solution is added with sulfuric acid or an aqueous solution thereof in an amount and concentration capable of maintaining the pH value of the aqueous solution within 2.3 to 3.4. Chloric acid was generated, and hydrogen peroxide was added in an amount equal to or greater than that required for the reduction reaction of the chloric acid. Rather, chlorine dioxide gas (ClO ₂ ) is added to any one of inorganic acids, inorganic acid salts, organic acids or organic acid salts, or two or more of them, or a combination of these (aqueous solution A). This is achieved by providing a method for producing chlorous acid including a process. By using chlorine dioxide gas (gas) as a raw material, chlorite ions are generated with high alkalinity, and at that time, the pH drops to below neutral. By transitioning to the state of chlorous acid, a transition state is created, and as a result, the advantage of being able to stably maintain chlorous acid (HClO ₂ ) over a long period of time by delaying the decomposition reaction. Can do. Chlorine dioxide (ClO ₂ ) is trapped in an aqueous solution A containing any one or more of inorganic acids, inorganic acid salts, organic acids or organic acid salts, or a combination of these. This effect is achieved. The expression “trap” means adsorption or trapping, etc., preferably gaseous chlorine dioxide is any one of inorganic acid, inorganic acid salt, organic acid or organic acid salt, or a combination of two or more. Any operation may be performed as long as the state coexists with the system. As such an operation, in general, a method of directly blowing into the aqueous solution A, or a method of adsorbing by spraying the aqueous solution A in a mist form from the upper part and releasing chlorine dioxide gas from the lower part, spraying, and the like can be mentioned. However, it is not limited to them. Although not wishing to be bound by theory, the chlorite water of the present invention (described in PCT / JP2014 / 006379) manufactured using a manufacturing plant as shown in FIG. 1 described in PCT / JP2014 / 006379. Examples 1 to 6) have been proven to exhibit a stable bactericidal effect at least for 10 days at refrigeration (4 ° C.) as shown in Example 7 described in PCT / JP2014 / 006379. It is understood that the invention described in PCT / JP2014 / 006379 provides a method for producing so-called chlorous acid water in which chlorous acid is stable in an aqueous solution.

In this specification, “antibacterial (action)” refers to inhibiting the growth of microorganisms such as filamentous fungi, bacteria and viruses having pathogenicity and harmfulness. Those having antibacterial action are called antibacterial agents.

In this specification, “sterilization (action)” in a narrow sense means to kill microorganisms such as filamentous fungi, bacteria and viruses having pathogenicity and harmfulness. What has a bactericidal action is called a narrowly defined bactericidal agent.

The antibacterial action and bactericidal action are collectively referred to as killing (action), but unless otherwise specified in this specification, bactericidal (action) is used in a broad concept including antibacterial (action). Accordingly, those having antibacterial action and bactericidal action are generally referred to as “bactericidal agents” in the present specification, and are generally understood as drugs having both an antibacterial action and a narrowly defined bactericidal action. The

In this specification, the “inactivation effect” means that a certain microorganism (eg, norovirus) is reduced or eliminated to the extent that there is no pathogenic or harmful effect.

In this specification, “neutralization” means stopping virus inactivation by chlorite water or a chlorite water preparation. Neutralizing the inactivated virus solution is called “neutralization treatment”. The neutralization treatment is performed by diluting with a buffer solution containing albumin immediately after the inactivation treatment. Usually, the neutralization treatment is performed by diluting with 0.1% BSA-added PBS for 10 times, but is not limited thereto, and may be performed with PBS containing 0% to 5% BSA, for example, 0%, 0.05% , 0.1%, 0.5%, 1%, 2%, 3%, 4% or 5% BSA in PBS.

In this specification, “PBS with BSA” means PBS containing BSA. “BSA” refers to bovine serum albumin and “PBS” refers to phosphate buffered saline.

Performing the neutralization treatment after the inactivation treatment is important in measuring the inactivation effect of the chlorite aqueous solution or the chlorite aqueous preparation. This is because if neutralization is not performed, virus inactivation proceeds in the subsequent cell culture step, and an appropriate virus inactivation effect cannot be measured. In the virus inactivation effect test using the conventional TCID ₅₀ method, there was no concept of neutralization after virus inactivation treatment. However, in the present invention, a neutralization treatment step was newly introduced. For neutralization, BSA-added PBS was used. BSA-added PBS shows an excellent neutralizing effect on chlorite water or chlorite aqueous preparations. As a result of using BSA-added PBS for neutralization treatment, chlorite water or chlorous acid was used in the same manner as the plaque method. It became possible to appropriately evaluate the virus inactivation effect of water preparations.

As used herein, “antiviral (action)” refers to inhibiting virus growth. Those having an antiviral action are called antiviral agents.

In this specification, “virus killing (action)” refers to inactivating the infectivity of virus particles. Virus inactivation is considered to be due to a three-dimensional structural change of viral particle components such as nucleic acid proteins and lipids, or modulation of the interaction between them. What has a virucidal action is called a virucidal agent.

In this specification, “virus killing (action)” refers to a broad concept that summarizes antiviral action and virucidal action. “Viral killing agent” refers to any agent having antiviral and virucidal action. Viral killing agents can be used as pharmaceuticals, quasi drugs, food additives, disinfectants and the like.

Antiviral agents act against specific viruses in principle, whereas virucidal agents are effective against a wide variety of viruses. The use of a virucidal agent always gives rise to drug-resistant virus variants, but virucidal agents do not in principle give rise to drug-resistant virus strains. This is because there are multiple target molecules for the virucidal agent. Therefore, it is preferable in that resistance does not occur.

The following tests are typically used as conventional methods for measuring the action of a virucidal agent.
1) Add 180 μl of buffer solution at the specified pH to a 2 ml plastic tube (assist tube). 2) Add 10 μl of the specified concentration of chlorous acid aqueous solution. 3) Add 10 μl of virus solution, stir well, and keep warm in a constant temperature water bath at the specified temperature. 4) Immediately after the incubation, cool with ice water and dilute 100 times with the virus diluted solution containing protein. 5) Measure residual viral load by plaque method.

The virus targeted by the present invention can include any virus that can evaluate Norovirus. For example, such viruses include noroviruses and norovirus equivalent strains such as feline calicivirus (FCV) strains.

Norovirus is a genus of viruses that cause non-bacterial acute gastroenteritis. In addition to causing food poisoning due to the consumption of shellfish such as oysters, it is also orally transmitted through the dust of the infected human feces and vomit, or the dried ones. When testing for norovirus, feline calicivirus, a related species, is used. This related species test has been recognized in the field. For norovirus, test method using alternative virus and feline calicivirus in Norovirus inactivation efficacy evaluation test, EPA, 2007 Survey report on inactivation conditions of norovirus, National Institute of Health Sciences, Food Sanitation Management Department See Shigeki Yamamoto and Mamoru Noda, Ministry of Health, Labor and Welfare. The virus-killing effect of norovirus can be replaced by a survey by feline calicivirus (FCV), a related bacterium (in addition to this literature, Gehrke, C et al: Inactivation of feline calicivirus, a surrogate of norovirus (formerly Norwalk-like viruses), by different types of alcohol in virtual and in vivo, J Hosp Infect (2004) 46: 49-55; Doultree, JC et al: Inactivation of feline calicivirus, a norwalk virussurrofect J (1999) 41: 51-57); Jennifer, L et al: Surrogates for the study of norovirus stability and inactivation in the environment: A comparison of murine norovirus and feline calicivirus, J Food Protect 1 (765) 11: 765 Hirotaka Takagi et al .: Examination of norovirus (NV) inactivation effect by replacing feline calicivirus (FCV)-Alikari agent, hydrogen peroxide And the inactivation effect by sodium percarbonate, Medicine and pharmacy (2007) 57: 311-312), which is also incorporated herein.

For example, the measurement of the inactivation rate of virus by chlorite water can be determined by conducting a normal experiment (mixing, etc.) and measuring the amount of remaining infectious virus, and the concentration of chlorite is 5 ppm. The virus (for example, influenza virus etc.) can be completely inactivated by contact within 1 minute under the condition of pH 6.5.

Therefore, in the present specification, the “norovirus equivalent strain” is not limited to the feline calicivirus (FCV) strain, and any virus strain can be used as long as the inactive effect can be exerted as described in the above research. It is understood that

In order to investigate heat resistance against viruses, resistance to disinfectants, viability in the environment, etc., it is necessary to quantitatively measure live (infectious) viruses. Methods for quantifying infectious viruses include methods that use animals that are the original host or experimental animals that are sensitive to the virus, and methods that use cultured cells. Generally, cultured cells that are simple and highly quantitative are used. The method is used. However, since human norovirus has not been successfully cultured in cultured cells, the method using cultured cells cannot be performed. Therefore, historically, research on the inactivation of norovirus, etc. has been carried out by (1) infection experiments with human fecal norovirus by volunteers, (2) experiments with cultured cells using substitute viruses closely related to norovirus. It has been done by the estimation method. Volunteer infection experiments have been carried out mainly in the United States to examine the presence or absence of gastrointestinal inflammation symptoms such as vomiting and diarrhea by directly drinking the juice containing fecal human norovirus. Although the inactivation conditions of norovirus can be known directly, there are problems such as laborious experiments and lack of quantitativeness. In recent years, it has been clarified that there is a difference in susceptibility to norovirus depending on the individual, and there is a possibility that the experimental results of the research before the discovery may remain doubtful.

Under such circumstances, viruses that are taxonomically related are attracting attention as Norovirus equivalent strains. Such closely related viruses often show similar heat resistance and sensitivity to disinfectants, so instead of norovirus that cannot be cultured, the resistance of norovirus is inferred from the results of inactivation experiments with various viruses, Various viruses are used for inactivation experiments and the like for the purpose of knowing the resistance of norovirus. Initially, enteroviruses such as poliovirus, coxakey virus and echovirus, which are the same human intestinal viruses and are non-enveloped 1 RNA viruses, are used for the purposes of the present invention. Viruses can also be targeted.

In addition, in the late 1990s, feline calicivirus, which belongs to the same Caliciviridae family as Norovirus and has been successfully cultured in cell culture, came to be mainly used. The current resistance to norovirus, such as heating and disinfectant, is mainly based on this feline calicivirus data, and since it is substantially equivalent, feline calicivirus is a preferred norovirus equivalent strain. It is not limited to. As a test method for the antiviral effect of US Anti-microbials Division of US EPA, a protocol using feline calicivirus is described as an inactivation test for norovirus (Antimicrobials Division USPA, Initial-virucidal-effectiveness test, using feline calicivirus as surrogate for norovirus, http: //epa.gov/oppad001/pdf_files/initial_virucidal_test.pdf). In addition, data concerning hepatitis A virus, which is suggested to be transmitted through bivalves as well as norovirus, may be used. For the purposes of this specification, it is understood that it falls within the category of norovirus equivalent strains. The More recently, mouse norovirus has been reported to be isolated and propagated in cultured cells for the first time as a virus belonging to the same norovirus genus as human norovirus (Wobus, CE et al: Replication of norovirus in cell culture reveals a tropism for dendritic cell sand macrophages, Plos Biol (2004) 2: 2076-2084). Since then, inactivation experiments using murine norovirus have begun. In addition, human noroviruses were reported to be susceptible only to chimpanzees except for humans, but human norovirus infection was reported in pigs in 2006 (Cheetham, S et al, Pathogenesis of a genogroup II human norovirus in gnotobiotic pigs, J Virol (2006) 80: 10372-10381)), it can be applied to experiments using the infectivity of human norovirus to pigs as an index. In addition, it was reported that human norovirus growth was confirmed by Ca3 three-dimensional culture method of CaCo2 cells derived from human colon cancer (Straub, TM et al: In vitro cell culture infectivity assay for human noroviruses, Emerg Infect Dis (2007) 13: 396-403), it is understood that such Caco2 cells fall within the category of norovirus equivalent strains for the purposes of this specification.

(Preferred embodiment)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification. It will also be appreciated that the following embodiments of the invention may be used alone or in combination.

In one aspect, the present invention comprises (A) inoculating a host cell with a feline calicivirus (FCV) strain or other Norovirus equivalent strain; (B) culturing the inoculated host cell in a serum-containing medium; (C) a step of collecting the culture medium and cells; (D) a step of centrifuging the collected culture medium and cells to obtain a supernatant; and (E) a sample using the supernatant as a test virus solution. A method for measuring the inactivation effect of a sample against norovirus without freezing and thawing, comprising the step of performing an inactivation test.

Any method can be used in carrying out the culture of the present invention. For example, the method and virus for culturing primary cells under serum-free conditions described in JP2010-148522A (P2010-148522A) Can be referred to and is incorporated herein by reference.

In one embodiment, the serum-containing medium is fetal bovine serum-containing Eagle MEM, but is not limited thereto. For example, DMEM (Dulbecco's modified Eagle medium) can be used.

In one embodiment, the centrifugation is advantageously performed at about 1,500 to 2,000 × g, but is not limited thereto. For example, in addition to this, centrifugation conditions of usually about 3,000 × g to 5000 × g are used, but are not limited thereto.

In one embodiment, the feline calicivirus (FCV) strain or other Norovirus equivalent strain uses a plurality of strains. This is because the inactivation effect can be more reliably verified by two or more strains. Examples of such strains include, but are not limited to, feline calicivirus (FCV) F4 strain and F9 strain (for example, ATCC VR-782 strain). For example, in addition to this, caliciviridae viruses other than feline calicivirus, FCV2280 strain, poliovirus, enteroviruses such as coxakey virus and echovirus, mouse norovirus CaCo-2 cells, and the like can be used. Although experimental data is not shown, data that actually functions is obtained even with FCV2280 strain, poliovirus and the like.

In another embodiment, the feline calicivirus (FCV) strain or other norovirus equivalent strain includes feline calicivirus (FCV) F4 strain and F9 strain. This is because it is recommended as a stock at the National Pharmaceutical Food Sanitation Laboratory.

In another embodiment, the step (C) is performed when all cytopathic effects (CPE) have occurred. This is because the virus recovery rate increases. However, the present invention is not limited to this, and the step (C) may also be performed when CRP occurs in cells of 50% or more. For example, 60% or more, 70% or more, 80% or more, 90% or more Any suitable numerical value such as 95% or more, 98% or more can be mentioned, and it is usually carried out at 70% or more.

In another aspect, the present invention provides (A) inoculating a host cell with a feline calicivirus (FCV) strain or other Norovirus equivalent strain; (B) culturing the inoculated host cell in a serum-containing medium; (C) a step of collecting the culture solution and cells; (D) a step of centrifuging the collected culture solution and cells to obtain a supernatant; (E) sublimation using the supernatant as a test virus solution. A step of inactivating the test virus solution with acid water or a preparation thereof; (F) a step of neutralizing by dilution with BSA-added PBS after the inactivation treatment; (G) a virus solution after the neutralization; A method for measuring the inactivation effect of chlorite water or a preparation thereof against Norovirus, comprising culturing host cells inoculated with, and measuring TCID ₅₀ is provided.

In one embodiment, the step (F) is advantageously performed by diluting with PBS containing 0.1% BSA, but is not limited thereto. For example, 0%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4% or 5% BSA-added PBS may be used.

In another embodiment, the step (F) is performed by diluting 10 times with PBS containing 0.1% BSA. The dilution factor may be, for example, 100 times or 1000 times. After 10-fold dilution with PBS containing 0.1% BSA, a further 10-fold serial dilution may be performed.

In another embodiment, the feline calicivirus (FCV) strain or other norovirus equivalent strain is feline calicivirus (FCV) F4 strain. It is preferable to verify the inactivation effect with a plurality of strains, but it is also possible to verify with the feline calicivirus (FCV) F4 strain alone. This is because if the method of the present invention is used, the feline calicivirus (FCV) F4 strain alone can sufficiently evaluate the virus inactivating effect of chlorite aqueous solution or chlorite aqueous preparation.

In yet another embodiment, the step (C) is performed when cultured for about 2 days. This is because empirically, all cytopathic effects (CPE) occur approximately two days after the start of culture. Thus, it is possible to incubate for longer than 2 days until all cytopathic effects (CPE) have occurred.

In still another embodiment, the culture in the step (G) is advantageously performed for 7 to 8 days, but is not limited thereto, for example, it may be longer than 7 days and 8 days, It may be 8 days.

The inactivation effect of feline calicivirus, which is a substitute virus for Norovirus in the plaque method that has been obtained so far, was also evaluated with the TCID ₅₀ method. It can be verified that the preparation can be an effective disinfectant / disinfectant for norovirus.

It should be noted that the references cited in this specification, such as scientific literature, patents, and patent applications, are incorporated herein by reference to the same extent as if they were specifically described.

As described above, the present invention has been described by showing preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

When necessary, the animals used in the following examples were handled based on the Declaration of Helsinki. Specifically, the reagents described in the examples were used as reagents, but equivalent products from other manufacturers (Sigma, Wako Pure Chemical, Nacalai Tesque, etc.) can be substituted.

(Example 1: Comparative test between Plack method and TCID ₅₀ method)
In this example, the test method of the present invention (modified TCID ₅₀ method) was compared with the conventional test method (Plac method).

(Method)
The test was conducted by the following method.

(Preparation of test virus solution)
The feline calicivirus (FCV) F4 strain was inoculated into CRFK cells, and then statically cultured in an Eagle's MEM medium containing 2% fetal calf serum in a CO ₂ furan vessel at 37 ° C. When all the cytopathic effects (CPE) occurred (cultured for about 2 days), the culture medium and cells were collected and centrifuged at 3,000 rpm (about 1,500 to 2,000 × g) for 30 minutes. The supernatant was used as a test virus solution.

(Confirmation test (1) Comparison test between Plack method and TCID ₅₀ method)
Immediately before use, after confirming the concentration of each disinfectant based on the iodokari titration method, use a diluted solution prepared with sterile ion-exchanged water so that the effective chlorine concentration is 1,000 ppm. The final concentration is 400 ppm, 200 ppm. The diluted solution of 1,000 ppm and sterilized ion-exchanged water were mixed so that the final concentration was 400 ppm for the final concentration of 400 ppm and diluted to 500 μl of ion-exchanged water for the final concentration of 400 ppm. The solution was diluted to 200 μl and ion-exchanged water 700 μl). 100 μl of the virus solution was added, and after 5 and 10 minutes, 100 μl of the treatment solution was sampled, diluted with cold 0.1% BSA-added PBS, and the residual virus infection titer was measured based on the plaque method and the TCID ₅₀ method.

<Pluck method>
The solution was diluted 10-fold with PBS containing 0.1% BSA, 0.5 ml was inoculated into a monolayer culture of CRFK cells, and virus adsorption was performed on a rocker platform for 60 minutes at room temperature with mechanical locking. For the formation of plaques, CRFK cells after virus adsorption were cultured in MEM containing 0.3 to 0.5% methylcellulose and 0.5% FBS at 37 ° C. in a CO 2 furan vessel. After confirming the generated plaques, the cells of the petri dish were single-stained with 0.5% (w / v) crystal murasaki staining solution containing 10% formalin, and the number of plaques was counted visually.
<TCID ₅₀ method>
The reaction solution diluted in serum-free Eagle MEM medium is further diluted 10-fold in serum-free MEM medium, and 25 μl of each dilution is inoculated into CRFK cells cultured in a 96-well plate (4 holes for each dilution). Used), and cultured in an MEM medium supplemented with 2% fetal calf serum at 37 ° C. in a CO ₂ -furan chamber. After inoculation, the presence or absence of CPE was observed on days 7 to 8, and when CPE was observed in 50% or more of the cells, it was regarded as CPE positive (not inactivated). The amount of surviving virus was shown as the amount of surviving virus (TCID ₅₀ ) in 25 μl of the virus solution subjected to the inactivation test. Results of quantitative test, if the surviving virus can not be confirmed at all, the following survival viral load 32TCID ₅₀ / 25μl.

(Inactivation process)

(Neutralization step dilution)
100 μl of the treatment solution was immediately diluted 10-fold with 900 μl of 0.1% BSA-added PBS, and appropriate serial dilution was performed.

(infection)
CRFK cells were cultured in 6-well dishes to form a single-layer sheet, and 500 μl of each treatment solution was inoculated, followed by infection treatment for 1 hour. Thereafter, the cells were cultured in a MEM containing 0.3 to 0.5% methylcellulose or 0.75% agar and FBS at 37 ° C. in a CO 2 furan vessel.

(Evaluation)
The number of plaques was measured visually, and the amount of surviving virus was calculated.

(result)
IBSA additive-free version

(Plac method)
CRFK cells monolayer-cultured on 6-well plates were infected with 500 μl of a diluted solution obtained by diluting the inactivated stock solution 10-fold with 900 μl of 0.1% BSA-added PBS.

(TCID ₅₀ method)
CRFK cells cultured in a monolayer on a 96-well plate were infected with 25 μl of a diluted solution obtained by diluting the inactivated stock solution 10-fold with 900 μl of 0.1% BSA-added PBS. The dilution series was evaluated using 4 Well.

(II. Version with 0.05% final BSA concentration)

(Summary)
The inactivation effect of each disinfectant on feline calicivirus when inactivated for 5 minutes in the absence of organic matter was summarized.

Next, the inactivation effect of each disinfectant on feline calicivirus when inactivated for 10 minutes in the absence of organic matter was summarized.

Next, the inactivation effect of each disinfectant against feline calicivirus when inactivated for 5 minutes in the presence of organic substances (final concentration of BSA 0.05%) was summarized.

Next, the inactivation effect of each disinfectant against feline calicivirus when inactivated for 10 minutes in the presence of organic substances (final concentration of BSA 0.05%) was confirmed.

(Discussion)
In order to compare the evaluation between the plaque method and the TCID ₅₀ method, first, the virus titer was confirmed using the feline calicivirus F4 strain, which is an alternative virus to Norovirus. As a result, it was found that the virus titer was 10 6 to the 6th level of virus even with both evaluation methods (Table I-1).

Next, as each disinfectant, the inactivation effect against viruses of “chlorite aqueous solution”, “chlorite aqueous preparation” and “sodium hypochlorite solution” was compared with both the plaque method and the TCID ₅₀ method. It confirmed with the evaluation method. As a result, regardless of the plaque method, TCID ₅₀ method, or any of the evaluation methods, there is no anti-virus against the viruses of “chlorite aqueous solution”, “chlorite aqueous preparation”, and “sodium hypochlorite solution”. It was found that the same effect was obtained for the activation effect. (Table I-2).

In addition, regarding the inactivation effect against viruses, “chlorite solution” and “chlorite solution formulation” in which “chlorite solution” is stabilized with a buffer solution are 400 ppm as chlorite concentration, It can be seen that if the inactivation treatment is performed for 5 minutes, the amount of virus in the 10th to the fifth power can be inactivated to below the detection limit. Even if the concentration of chlorous acid is 200 ppm, inactivation for 5 minutes If the treatment is performed, the amount of virus in the 10 3rd range can be inactivated, and if the inactivation process is performed for 10 minutes, the amount of virus in the 10th 5th range is inactivated to the detection limit or less. I knew that I could do it.

Next, the sodium hypochlorite solution inactivates the amount of virus in the 10th power of 5 to below the detection limit if it is inactivated for 5 minutes using an effective chlorine concentration of 200 ppm. I knew that I could do it. From this, it can be judged that the same evaluation can be obtained by using either the plaque method or the TCID ₅₀ method for the evaluation of the inactivation effect on the virus (Tables III-1 and III- 2). However, with the use of a chlorine-based disinfectant, the main active ingredient disappears under the conditions in which organic substances are present, and the chlorine concentration is higher than in the absence conditions in which organic substances are not present ( High) may be necessary. From this, we decided to confirm the appropriate concentration of “chlorite water” that can inactivate the virus in the presence of organic matter. As organic substances, bovine serum albumin (hereinafter referred to as BSA) adjusted to a final concentration of 0.05% based on the “Survey Report on Inactivation Conditions of Norovirus” conducted by the National Institute of Health Sciences. In order to confirm whether there is a difference between the plaque method and the TCID ₅₀ method due to the influence of organic matter, we will try to confirm using liquid. It confirmed with the evaluation method. First, the virus titer was confirmed using the feline calicivirus F4 strain, which is a norovirus substitute virus.

As a result, it was found that the virus titer was 10 6 to the 6th level of virus even in both evaluation methods, and both evaluation methods were not affected by organic matter and in the absence of organic matter. (Table II-1).

Next, the inactivation effect against viruses of “chlorite aqueous solution”, “chlorite aqueous preparation” and “sodium hypochlorite solution” was evaluated by the plaque method and the TCID ₅₀ method. As a result, regardless of the plaque method, TCID ₅₀ method, or any of the evaluation methods, there is no anti-virus against the viruses of “chlorite aqueous solution”, “chlorite aqueous preparation”, and “sodium hypochlorite solution”. As for the activation effect, it turned out that an equivalent result is obtained similarly to the organic substance absence condition. (Table II-2).

In addition, regarding the inactivation effect against viruses, “chlorite solution” and “chlorite solution preparation” in which “chlorite solution” is stabilized with a buffer solution are 1000 ppm as chlorite concentration, It turns out that the inactivation for 5 minutes can inactivate the amount of virus in the 5th to the 5th power level to below the detection limit. Even if the concentration of chlorous acid is 400 ppm, inactivation for 10 minutes If the treatment is performed, the amount of virus in the 10 3rd range can be inactivated, and if the inactivation process is performed for 10 minutes, the amount of virus in the 10th 5th range is inactivated to the detection limit or less. I knew that I could do it. However, the sodium hypochlorite solution cannot inactivate the amount of virus in the 10th power to the detection limit or less unless it is inactivated for 5 minutes using an effective chlorine concentration of 1000 ppm. I understood that. (Table III-1 and Table III-2)
From these results, the virus titer and the virus inactivation effect using disinfectant are the same as the evaluation results of the plaque method and the modified TCID ₅₀ method in the absence of organic matter or in the presence of organic matter. It was found that the inactivation effect confirmation test against viruses was evaluated using the plaque method, but this data shows that the same result can be obtained even if the modified TCID ₅₀ method is used. I understood.

If there is no organic matter, the sodium hypochlorite solution will inactivate feline calicivirus sufficiently if it is inactivated at 200 分 間 ppm for 5 minutes as the effective chlorine concentration. In contrast, “chlorite water” and “chlorite aqueous preparations” are 10 to the third power of the virus when inactivated for 5 minutes at 200 ppm chlorite concentration. The amount can be inactivated, and further, if it is inactivated for 10 minutes, it can be said that it is a disinfectant having an inactivating effect equivalent to that of sodium hypochlorite solution.

In addition, under conditions where organic substances are present, sodium hypochlorite solution can inactivate viral loads on the 10 3rd power range by inactivating the effective chlorine concentration at 400 ppm for 5 minutes. However, if the effective chlorine concentration is not 1000 ppm, it cannot be inactivated to below the detection limit, whereas “chlorite aqueous solution” and “chlorite aqueous preparation” are the same sub-phase as sodium hypochlorite solution. It can be seen that a virus with a concentration of 400 3 (800 ppm as an effective chlorine concentration) and inactivation treatment for 5 minutes can inactivate the amount of virus in the 10th power range, and 1000 ppm as the concentration of chlorous acid. It was found that a liquid with an effective chlorine concentration of 2000 ppm can be inactivated to below the detection limit. In addition, an inactivation effect confirmation test for feline calicivirus using the plaque method was carried out by performing a reproducibility confirmation test (cross check test) using the TCID ₅₀ method. "Water" and its preparations have an inactivation effect against feline calicivirus, which is a substitute for norovirus, and the inactivation effect lasts longer than sodium hypochlorite. (Stock solution, 6-fold dilution, 30-fold dilution) (storage period; up to D + 360).

(Example 2: Comparison between modified methods)
About this test example, about the feline calicivirus inactivation effect of the chlorite aqueous preparation carried out at the university, a reproducibility confirmation test (cross check test) was conducted at another private facility, and the results were summarized.

(Comparison study of test methods by inspection organizations)
The following table summarizes the different test materials and methods for the test contents of conventional facilities (university medical school) and private facilities.

(Test method)
(material)
(Drug)
・ Sodium hypochlorite (200ppm and 400ppm)
"Purelux;Oyalux" (sodium hypochlorite 6%)
・ Chlorous acid water (200ppm and 400ppm)
“Chlorous acid aqueous formulation: Sankei Co., Ltd.” (Chlorous acid 4%)
・ Control citrate-phosphate buffer (use adjusted to each pH)
About sodium hypochlorite and chlorous acid water, those diluted with distilled water to a concentration of 400 ppm and 800 ppm are used. Since the concentration changes with time, adjustment is performed immediately before the start of the test.
Dilute to final concentrations of 200 and 400 ppm by addition of virus and buffer.
-Virus used: "Feline calicivirus (F9 strain)"; as a substitute virus for Norovirus-Cells used: CrFK cells (cat kidney-derived cells)-Addition protein: To examine whether the presence or absence of organic substances affects the effect of the drug Add "7.5% bovine serum albumin (BSA)" ... mix with virus solution to 1% BSA. (The final concentration is 0.05% BSA by mixing the drug and buffer.)
-Drug reaction stop solution: added to stop the effect of the drug on the virus solution at the set time 1N sodium thiosulfate (pentahydrate, molecular weight 248.19)
Polypeptone (PP) 80mg / ml
-Buffer solution: In order to investigate the effect of the virus inactivation effect of each drug due to the difference in pH range, three points of acid, neutral, and basic regions are provided.
(1) pH 5.5 (acidic range)
(2) pH6.5 (neutral range)
(3) pH7.5 (alkali (basic) range)
<Adjustment of citrate-phosphate buffer is as follows>
・ 0.1M citric acid: 19.21g / L (molecular weight 192.1)
・ 0.2M disodium hydrogen phosphate: 35.6g / L (dodecahydrate, molecular weight 358.14)
Add 0.2M disodium hydrogenphosphate solution to the adjusted 0.1M citric acid solution, and adjust the pH using a pH meter.
(See the table below for the mixing ratio of each solution)

(Method)
The mixing ratio of the used virus solution, drug, and citrate-phosphate buffer is shown in the table below.

(Reaction between test solution and virus)
(1) Sodium hypochlorite group or chlorite water group added 1.8 ml of buffer solution of each pH to a small tube, and control group added 1.9 ml of buffer solution to a small tube, sodium hypochlorite 0.1 ml of sodium hypochlorite or chlorous acid water adjusted to 200 ppm was added to the section or chlorite water section.
(2) 0.1 ml of virus solution (adjusted with BSA) was added thereto and reacted for 10 minutes.
(3) PP 0.9 ml was dispensed into a small tube, and 0.1 ml of the sample solution of (2) was added after the designated reaction time had elapsed to stop the reaction. At this time, sodium hypochlorite and aqueous chlorite were added immediately after the reaction, sodium thiosulfate (1N 0.1 ml) was added to stop the reaction, and then added to PP.
(4) Separately, 0.9 ml of Eagle's minimum essential medium (MEM) with no fetal bovine serum added to a small tube, add 0.1 ml of the sample solution of (3), and dilute 10 times. Subsequently, a 10-fold serial dilution was performed immediately.
(5) The cell fluid was drained and immediately 100 μl was inoculated.

(Infectious titer measurement)
Infectious titer was measured by a standard plaque assay method. (BMSA regular method)
-Cells were seeded in 6-well plates and used after 3-5 days in culture, when the cells became confluent.
(1) 0.1 ml of the test virus solution was added to 0.9 ml of PP, and the 0.1 ml was diluted 10-fold with Eagle's minimum essential medium (MEM) without fetal bovine serum. (Same as above (reaction between test solution and virus) (4))
(2) The plate solution was removed and the diluted virus solution was inoculated at 100 μl / well.
(3) Placed in a 34 ° C. CO ₂ incubator for 1 hour to adsorb the virus.
(4) After 1 hour, 3 ml / well of agar medium was added to each well (see Table 9 below).
(5) The plate was turned over and cultured in a 34 ° C. CO ₂ incubator for 2 days.
(6) Two days later, the plate was removed from the 34 ° C. CO ₂ incubator, about 3% formalin solution was added at 2 ml / well, and allowed to stand at room temperature for 1 hour or more to fix the cells.
(7) After fixation, the agar medium is washed with running water, 2 ml of methylene blue staining solution is added, and left at room temperature for 30 minutes or longer. Thereafter, the plaques were counted after washing with running water and drying.

(Evaluation)
When the difference in virus infectivity between the test solution and the control is 1/1000 or less, the test solution is determined to be significant.

(Result)

(Discussion)
In the absence of protein, the infectious titer in a diluted solution of 200 ppm chlorite water as the concentration of chlorite was below the detection limit regardless of the pH range, and feline calicivirus could be inactivated. However, with sodium hypochlorite, it was found that the infectious titer was less than the detection limit only in the alkaline region, even with 400 ppm and 200 ppm effective chlorine solutions. In addition, in the presence of protein, the closer to the acidic range, the higher the virus inactivation effect of chlorite water, especially in the case of 400 ppm dilute chlorite water as a chlorite concentration. For example, the infectious titer was below the detection limit even in the acidic range and neutral range, indicating that feline calicivirus can be inactivated.

Furthermore, comparing the results obtained so far, the concentration that could be inactivated in 10 minutes and the results in the presence of protein in this test, the results obtained so far are also In any of the examples, it was confirmed that “chlorite aqueous solution” had an inactivation effect equivalent to or higher than that of sodium hypochlorite.

From the above, the results of the present example were the same as or better than the conventional results obtained by the university. Therefore, it can be judged that the evaluation that “chlorite aqueous solution” and its preparations have a sufficient inactivation effect against feline calicivirus is an appropriate evaluation. Therefore, it can be evaluated that the inspection technique employed in this embodiment is also appropriate.

For the Ministry of Health, Labor and Welfare, the evaluation of the inactivation effect of each disinfectant / disinfectant against viruses was conducted by the National Institute of Health Sciences in 2009, “Survey on Norovirus Inactivation Conditions”. This report is based on the TCID ₅₀ method for evaluating the inactivation effect of feline calicivirus, which is a substitute for norovirus, using each disinfectant and disinfectant. It is said that the sterilizing / disinfecting agent for which inactivation effect was recognized by this evaluation method is also recognized as a sterilizing / disinfecting / disinfecting agent for norovirus measures. The method of the present invention is a simplified method unlike these methods, but it was found that the method can be similarly evaluated.

(Summary)
Food poisoning caused by norovirus and infectious diseases are very serious problems. In particular, norovirus infection in infants under 5 years old accounts for about 60% of the total, and death cases have occurred, and development of a disinfectant for the prevention of infectious diseases is desired, but chlorous acid (HClO) _{As a result} of examining the inactivation effect of “chlorite aqueous solution” containing ₂ ) as a main component against norovirus, it was found that the method of the present invention can be successfully measured.

To summarize the method, feline calicivirus F4, a norovirus substitute virus, was inactivated with a chemical solution with a chlorine concentration of 100 ppm to 1,000 ppm for 10 minutes, and then the TCID ₅₀ method was used for CAW (sublimation). The inactivation effect of chloric acid water) was confirmed, and the inactivation effect in the presence of organic matter was confirmed using bovine serum albumin (BSA). As a result, under conditions where organic matter (BSA) was not added, Sodium hypochlorite can be used under the condition that 5logCFU / ml virus at 400ppm chloric acid concentration can be inactivated below the detection limit (1.3LogCFU / ml or less) and BSA is added to a final concentration of 0.05%. Both CAW and CAW were able to inactivate 5Log CFU / ml virus below the detection limit (1.3LogCFU / ml or less) at 1,000 ppm chlorite concentration. Moreover, the inactivation effect with respect to a feline calicivirus can also be confirmed using the chlorous acid liquid contained in the wet sheet of the cotton material nonwoven fabric impregnated with the diluted CAW solution.

As described above, the present invention has been exemplified by using the preferred embodiments of the present invention, but it is understood that the scope of the present invention should be interpreted only by the scope of the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood. This application claims the benefit of priority of Japanese Patent Application No. 2014-39373 filed on February 28, 2014, the contents of which are hereby incorporated by reference in their entirety.

According to the present invention, a technique capable of easily testing the effect of a bactericide on a norovirus is provided, and the possibility of being widely used in the food industry, the medical field, etc. is further increased.

Claims (13)

1. (A) inoculating a host cell with a feline calicivirus (FCV) strain or other Norovirus equivalent strain;
   (B) culturing the inoculated host cells in a serum-containing medium;
   (C) recovering the culture solution and cells;
   (D) centrifuging the collected culture medium and cells to obtain a supernatant;
   (E) A step of inactivating the test virus solution with a chlorite aqueous solution or a chlorite water preparation using the supernatant as the test virus solution;
   (F) The step of neutralizing by diluting with albumin phosphate buffered saline (PBS) after the inactivation treatment;
   (G) culturing host cells inoculated with the neutralized virus solution and measuring TCID ₅₀ ,
   A method for measuring the inactivation effect of chlorite water or a chlorite water preparation against norovirus.
2. The method of claim 1, wherein the albumin is bovine serum albumin (BSA).
3. The method according to claim 1, wherein the step (F) is performed by diluting with PBS containing 0.1% BSA.
4. The method according to claim 3, wherein the step (F) is performed by diluting 10 times with PBS containing 0.1% BSA.
5. The method according to any one of claims 1 to 4, wherein the serum-containing medium is fetal calf serum-containing Eagle MEM.
6. The method according to any one of claims 1 to 5, wherein the centrifugation is performed at about 1,500 to 2,000 x g.
7. The method according to any one of claims 1 to 6, wherein a plurality of strains are used as the feline calicivirus (FCV) strain or other Norovirus equivalent strain.
8. The method according to any one of claims 1 to 7, characterized in that the feline calicivirus (FCV) strain or other norovirus equivalent strain comprises feline calicivirus (FCV) F4 and F9 strains.
9. 9. The method of claim 8, wherein the feline calicivirus (FCV) strain or other norovirus equivalent strain is feline calicivirus (FCV) F4 strain.
10. The method according to any one of claims 1 to 9, wherein the step (C) is performed when all cytopathic effects (CPE) have occurred.
11. The method according to claim 10, wherein the step (C) is performed when the culture is performed for about 2 days.
12. The method according to any one of claims 1 to 11, wherein the culture in the step (G) is performed for 7 to 8 days.
13. The method according to claim 12, wherein the culture in the step (G) is performed for 8 days.