Virus infection inhibitors

Abstract

The present invention addresses the problem of providing a novel environmental symbiotic infection inhibitor against infectious disease viruses (RNA virus and DNA virus). The present invention provides a virus infection inhibitor comprising a microorganism of the genus Caldibacillus and a microorganism of the genus Paenibacillus.

Description

Technical Field

[0001] The present invention relates to an inhibitor of virus infection. Background Art

[0002] Viruses are protein structures containing nucleic acids and can self-replicate using host cells. Due to these characteristics, there has long been a debate about whether they are living organisms or non-living organisms. If viruses are regarded as living organisms, then viruses are the most numerous on Earth, and the predicted number of existing viruses is 10 31 individuals (Non-Patent Document 1). Historically, viruses have played a wide range of roles in ecosystems and have played an important role in the evolution of animals. An obvious example is that a gene necessary for placenta formation, which is important during pregnancy, has been integrated into the human genome (an example of exaptation, Non-Patent Document 2). In addition, from a macroscopic perspective, it has been reported that they affect the balance of ecosystems and are involved in suppressing the occurrence of marine red tides (Non-Patent Document 3).

[0003] As such, viruses have a wide range of effects on the physiological functions of animals and plants and ecosystems, but their most common function is as one of the pathogens. The spread of infections by increasingly virulent pathogenic viruses causes huge damage and leads to serious social problems. As such pathogenic viruses, coronaviruses, influenza viruses, classical swine fever viruses, etc. can be cited. These above viruses are all classified as enveloped RNA viruses and are known as viruses that cause infection and disease in humans or animals.

[0004] There are various known types of coronaviruses, including: Porcine epidemic diarrhea (PED) virus, which belongs to alpha coronavirus and can cause acute diarrhea in pigs; Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which belongs to beta coronavirus and has caused the recent global pandemic of coronavirus disease 2019 (COVID-19); Infectious bronchitis virus (IB) of poultry, which belongs to gamma coronavirus; and seasonal influenza viruses. In addition, avian influenza (H5N1) virus and classical swine fever virus (Flaviviridae, Classical swine fever virus genus) are not only worrying due to their danger but may also cause serious economic losses due to culling. Due to the characteristics of RNA viruses, these viruses mutate quickly, and various subspecies can appear in a relatively short period of time, which means that the emergence of unknown mutant strains has always been a problem. Inevitably, different mutant strains may exist in different regions, and there is a problem that vaccines effective against the mutant strains cannot be developed in time.

[0005] Due to these problems, there is an urgent need to develop technologies for inhibiting virus infection. As an example, various technologies for preventing and inhibiting coronavirus infection have been proposed (for example, Patent Documents 1 and 2).

[0006] However, key infection control technologies mainly use synthetic pharmaceutical ingredients such as ethanol and benzalkonium chloride, without considering the protection of the ecosystem or the interaction with symbiotic bacteria that coexist with humans and animals. Therefore, even if it has an effect of inhibiting infection against viruses, it may cause other adverse effects. That is, as proposed by the "hygiene hypothesis" in 1989, in modern society, excessive hygiene management is a trigger for various diseases (Non-Patent Document 4). Therefore, it is necessary to achieve the inhibition of infection by pathogenic viruses while maintaining a healthy symbiotic relationship among humans, animals, and microorganisms. Prior Art Documents Patent Documents

[0007] Patent Document 1: Japanese Unexamined Patent Application Publication No. 2000-044473 Patent Document 2: Japanese Unexamined Patent Application Publication No. 2022-067656 Non-Patent Documents

[0008] Non-Patent Document 1: Supervised translation: Kunitada Shimono / Tsukasa Sega, Mechanisms of Viral Infection and Host Response in Life Sciences and Their Applications in Medicine, Nankodo Non-Patent Document 2: Hirokuni Miyamoto, "Chapter 22 Environmental Microorganisms and Animals," edited by Hiroshi Ohno, Symbiotic Microorganisms, Kagaku Dojin (Dojin Bioscience series No. 27), pp. 247-256 (2016) Non-Patent Document 3: Yuji Tamaru, Yoko Shirai, Shojo Takao, Keizo Nagasaki, Special Issue "Aquatic Biomass and Environmental Remediation," "The Smallest Biological Factor in Seawater - Aquatic Virus Ecology -," Bull. Soc. SeaWater Sci., Jpn., 61, 307-315 (2007) Non-Patent Document 4: Strachan DP (1989) Hay fever, hygiene, and household size. BMJ 299(6710): 1259-1260 Summary of the Invention Problems to be Solved by the Invention

[0009] There is a need for a technology that does not have an adverse effect on the ecosystem or symbiotic microorganisms of humans and animals like synthetic pharmaceuticals, and that has an effect of inhibiting the infection ability of viruses with a high mutation rate for which the development of vaccines takes a long time. Through such technologies, from the perspective of further strengthening infection prevention and control, not only can the infection source be cut off, but also the application in a sustainable society can be expected.

[0010] The present invention has been completed in view of the above circumstances, and an object thereof is to provide a novel environmentally symbiotic infection inhibitor against infectious disease viruses (RNA viruses and DNA viruses). Means for solving the problem

[0011] The inventors of the present invention found that the above problems can be solved by specific mixed microorganisms, and thus completed the present invention. Specifically, the present invention provides the following solutions.

[0012] (1) A virus infection inhibitor comprising a microorganism belonging to the genus Caldibacillus or a culture thereof.

[0013] (2) The virus infection inhibitor according to (1), wherein the microorganism belonging to the genus Caldibacillus includes the NITE accession number "BP-863".

[0014] (3) The virus infection inhibitor according to (1), which is an environmentally symbiotic virus infection inhibitor comprising a mixed microorganism having a symbiotic function for humans, animals, plants, and ecosystems, wherein the mixed microorganism is the genus Caldibacillus (Thermobacillus) and the genus Paenibacillus (Paenibacillus).

[0015] (4) The environmentally symbiotic virus infection inhibitor according to (3), wherein the mixed microorganism includes the NITE accession number "BP-03693".

[0016] (5) The environmentally symbiotic virus infection inhibitor according to (4), wherein the mixed microorganism including the NITE accession number "BP-03693" includes the ATCC accession number "PTA-1773".

[0017] (6) The virus infection inhibitor according to (5), wherein the ATCC accession number "PTA-1773" includes the NITE accession number "BP-863" and the NITE accession number "BP-1051".

[0018] (7) The virus infection inhibitor according to any one of (1) to (6), wherein the virus is Porcine Epidemic Diarrhea Virus belonging to α-coronavirus, SARS-CoV-2 belonging to β-coronavirus, or Infectious Bronchitis Virus of poultry (IB) belonging to γ-coronavirus.

[0019] (8) The virus infection inhibitor according to any one of (1) to (6), wherein the virus is a seasonal influenza virus, an avian influenza (H5N1 type) virus, or a classical swine fever virus (Pestivirus in the family Flaviviridae).

[0020] (9) The virus infection inhibitor according to any one of (1) to (6), which has no adverse effects on symbiotic organisms, wherein the virus is a norovirus of the family Caliciviridae, feline calicivirus, norovirus, Norwalk Virus, rabbit hemorrhagic disease virus, a plant infectious virus (Begomovirus, Tobamovirus, Nepovirus, tobravirus), a bacteriophage (Cystoviridae (family Cystoviridae)), or a hepatitis virus. Advantages of the Invention

[0021] According to the present invention, there is provided a novel infection inhibitor against environmentally symbiotic viruses (RNA viruses, DNA viruses). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a diagram showing the effect of the infection inhibitor of the present invention against porcine epidemic diarrhea virus (PED virus P-5V strain). Figure 2 It is a diagram showing the effect of the infection inhibitor of the present invention against SARS-CoV-2 coronavirus (Delta strain). Figure 3 It is a diagram showing the effect of the infection inhibitor of the present invention against influenza virus (swine influenza virus H1N1 IOWA strain). Figure 4 It is a diagram showing a use example of the infection inhibitor of the present invention. Figure 5 It is a diagram showing a use example of the infection inhibitor of the present invention. Figure 6 It is a diagram showing a social implementation example of the present invention. Figure 7 It is a diagram showing a social implementation example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0023] Hereinafter, embodiments of the present invention will be described in detail. It should be noted that the present invention is not limited to the following embodiments.

[0024] <Virus Infection Inhibitor> The virus infection inhibitor of the present invention (hereinafter, also referred to as "the infection inhibitor of the present invention") contains a microorganism of the genus Caldibacillus or its culture.

[0025] The infection inhibitor of the present invention preferably may contain one or more bacteria selected from the group consisting of the ATCC deposit number "PTA-1773", the NITE deposit number "BP-03693", and the NITE deposit number "BP-863". All of the above contain microorganisms of the genus Caldibacillus.

[0026] In the present invention, "the culture of a microorganism" includes any culture obtained by culturing under conditions (culture medium, temperature, etc.) capable of culturing the microorganism. The culture solution can be, for example, a culture solution containing the microorganism (sterilized bacterial solution, non-sterilized bacterial solution, etc.), a culture supernatant not containing the microorganism, and the like. Examples of the culture medium include animal or plant proteins.

[0027] (1) ATCC deposit number "PTA-1773" The mixed microorganism "PTA-1773" is a thermophilic strain, deposited on May 10, 2000, by the American Type Culture Collection (ATCC), which is an international depository institution based on the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (address: 10801 University Boulevard Manassas, Virginia 20110-2209 U.S.A.). The ATCC deposit number "PTA-1773" includes the NITE deposit number "BP-863", the NITE deposit number "BP-1051", and the NITE deposit number "BP-03693".

[0028] "PTA-1773" includes microorganisms of the genus Caldibacillus and the genus Paenibacillus.

[0029] (2) NITE deposit number "BP-03693" The mixed microorganism "BP-03693" is a thermophilic strain, which was deposited on July 27, 2022, by the "National Institute of Technology and Evaluation Patent Microorganisms Depositary Center" (address: 2-5-8 Kamigoura, Kisarazu City, Chiba Prefecture), an international depositary authority based on the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The mixed microorganism "BP-03693" was isolated from the mixed microorganism "PTA-1773" and consists of three strains: Caldibacillus hisashii (formerly known as Bacillus hisashii, NITE deposit number "BP-863", internationally deposited on February 8, 2010), Paenibacillus No. 39 strain, and Paenibacillus No. 139 strain. In addition, the "National Institute of Technology and Evaluation Patent Microorganisms Depositary Center" (address: 2-5-8 Kamigoura, Kisarazu City, Chiba Prefecture) confirmed on October 31, 2022, that "BP-03693" consists of three strains: Caldibacillus hisashii, Paenibacillus No. 39 strain, and Paenibacillus No. 139 strain.

[0030] It should be noted that "BP-863" was internationally deposited as a related species of Bacillus thermoamylovans on January 15, 2010, but was subsequently registered as a new species of Bacillus hisashii (Nishida, A. et al. Bacillus hisashii sp. nov., isolated from the caeca of gnotobiotic mice fed with thermophile-fermented compost. Int J Syst Evol Microbiol 65, 3944-3949, doi: 10.1099 / ijsem.0.000516 (2015)). In addition, due to changes in international microbial classification, it has been changed to Caldibacillus hisashii (Gupta, R.S., Patel, S., Saini, N. & Chen, S. Robust demarcation of 17 distinct Bacillus species clades, proposed as novel Bacillaceae genera, by phylogenomics and comparative genomic analyses: description of Robertmurraya kyonggiensis sp. nov. and proposal for an emended genus Bacillus limiting it only to the members of the Subtilis and Cereus clades of species.) Int J Syst Evol Microbiol 70, 5753-5798, doi:10.1099 / ijsem.0.004475 (2020)). As recorded by Gupta et al., with the above classification changes, it was determined that the previous Bacillus thermoamylovorans and Bacillus hisashii bacteria are closely related and are both classified in the genus Caldibacillus. In addition, the "National Institute of Technology and Evaluation Patent Microorganism Depositary" (address: 2-5-8 Kamigyo Renjaku, Kisarazu City, Chiba Prefecture) confirmed on October 31, 2022 that "BP-863" was classified as Caldibacillus hisashii.

[0031] "BP-03693" includes microorganisms of the genera Caldibacillus and Paenibacillus.

[0032] (3) NITE Deposit Number "BP-1051" The mixed microorganism "BP-1051" is a thermophilic strain, which was deposited on January 18, 2011, by the "National Institute of Technology and Evaluation Patent Microorganisms Depositary" (address: 2-5-8 Kamigeneral Kamenosuke, Kisarazu City, Chiba Prefecture), an international depositary authority based on the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0033] (4) Actions, etc. of the infection inhibitor of the present invention In past research, the present inventors found that "PTA-1773" and "BP-863" can activate the innate immune system of animals; and reduce fat accumulation, etc. by controlling the intestinal flora (WO2011 / 099514). In addition, "PTA-1773" and "BP-1051" are effective for plant growth and have the function of suppressing the emission of greenhouse gases from the soil. Furthermore, through further investigation, it was found that the fermentation solution produced by the mixed microorganism containing "BP-03693" contained in "PTA-1773" has a significant infection inhibitory effect at least on specific viruses (for example, RNA viruses (coronaviruses, influenza viruses, etc.) and DNA viruses (hepatitis B virus, etc.)).

[0034] In the present invention, the "mixed microorganism" refers to an aggregate of multiple types of microorganisms. It should be noted that as a result of genomic analysis of the above-deposited microorganisms, it has been confirmed that there may be phage-related proteins and lantibiotic peptides. Therefore, they may be involved in inhibiting virus infection.

[0035] In the present invention, the "virus infection inhibitory effect" includes inhibiting the proliferation of viruses in the presence of the infection inhibitor of the present invention. The virus infection inhibitory effect can be evaluated by the method shown in the examples.

[0036] Moreover, the above-deposited microorganisms (especially microorganisms belonging to the genus Caldibacillus and the genus Paenibacillus) are expected to have a symbiotic function for humans, animals and plants, and the ecosystem, and thus are environmentally symbiotic.

[0037] In the present invention, "environmentally symbiotic" includes not damaging the balance of flora such as resident bacteria on the skin and resident bacteria in the intestine. For example, existing disinfectants (such as ethanol) can easily disrupt this balance of flora and thus cannot be considered environmentally symbiotic.

[0038] The infection inhibitor of the present invention is expected to be environmentally symbiotic. Therefore, in addition to its function of inhibiting virus infection, it can also reduce adverse effects on ecosystems and various environments (such as soil, wastewater, the body, rivers, seawater, sludge, etc.). Figure 4 )

[0039] The infection inhibitor of the present invention is expected to be environmentally symbiotic. Therefore, it can achieve low-load skin disinfection, nasal cavity disinfection, etc., as well as disinfection of livestock houses, etc. Figure 5 )

[0040] (5) Composition, etc. of the infection inhibitor of the present invention The infection inhibitor of the present invention may contain microorganisms of the genus Caldibacillus (e.g., "PTA-1773", "BP-03693", "BP-1051", or "BP-863", etc.) or their cultures, and there are no particular limitations otherwise. It should be noted that "BP-03693", "BP-1051", and "BP-863" are included in "PTA-1773".

[0041] The content of each of "PTA-1773", "BP-03693", "BP-1051", and "BP-863" can be appropriately set according to the desired infection inhibition effect, etc. For the infection inhibitor of the present invention, the content (dry weight) of each of "PTA-1773", "BP-03693", "BP-1051", and "BP-863" can be, for example, 0.001% by mass or more and 100% by mass or less.

[0042] Any additives (microorganisms other than "PTA-1773", "BP-03693", "BP-1051", and "BP-863", solvents (such as water), excipients, buffers, preservatives, etc.) can be incorporated into the infection inhibitor of the present invention within the range that does not impair the functions of "PTA-1773", "BP-03693", "BP-1051", and "BP-863". The types and amounts of such additives can be appropriately set according to the desired effects, etc.

[0043] In a preferred embodiment of the infection inhibitor of the present invention, the constituent microorganism is composed of any one of "PTA-1773", "BP-03693", "BP-1051", and "BP-863".

[0044] The form of the infection inhibitor of the present invention can be liquid, solid, etc. The infection inhibitor of the present invention can be, for example, in the form of containing microbial cells (such as cell culture solutions), or can be the culture supernatant of the microorganism (excluding cells).

[0045] The infection inhibitor of the present invention can be produced by known methods according to its form.

[0046] Since "PTA-1773", "BP-03693", "BP-1051" and "BP-863" are thermophilic, they can be used after sterilization when incorporated into the infection inhibitor of the present invention. By applying a sterilization treatment, it is possible to incorporate "PTA-1773", "BP-03693", "BP-1051" and "BP-863" while preventing the contamination of miscellaneous bacteria.

[0047] The sterilization temperature of "PTA-1773", "BP-03693", "BP-1051" and "BP-863" can be 40 to 100 °C.

[0048] The sterilization time of "PTA-1773", "BP-03693", "BP-1051" and "BP-863" can be 1 to 120 minutes.

[0049] (6) Virus There is no particular limitation on the type of virus that is the object of infection inhibition by the infection inhibitor of the present invention, and it includes any coronavirus and its mutant strains that infect mammals (humans, pets (dogs, cats, etc.), livestock (pigs, etc.)).

[0050] Viruses include both RNA viruses and DNA viruses.

[0051] Examples of RNA viruses include porcine epidemic diarrhea virus belonging to alphacoronavirus, SARS-CoV-2 belonging to betacoronavirus, and avian infectious bronchitis virus (IB) belonging to gammacoronavirus.

[0052] Examples of RNA viruses include seasonal influenza virus, avian influenza (H5N1 type) virus, or classical swine fever virus (genus Pestivirus in the family Flaviviridae).

[0053] Examples of RNA viruses include plant viruses, bacteriophages, etc., and feline calicivirus of the family Caliciviridae that causes feline respiratory diseases, norovirus, norwalk virus that cause infectious gastroenteritis in humans, etc., and rabbit hemorrhagic disease virus that causes hemorrhagic disease in rabbits.

[0054] Examples of coronaviruses include coronaviruses belonging to the subfamily Orthocoronavirinae. These coronaviruses are divided into four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, Deltacoronavirus).

[0055] Examples of coronaviruses belonging to the genus Alphacoronavirus include porcine epidemic diarrhea virus, human coronavirus 229E, etc.

[0056] Examples of coronaviruses belonging to the genus Betacoronavirus include SARS-related coronaviruses (such as SARS-CoV-2), Betacoronavirus 1, murine coronavirus, etc.

[0057] Examples of coronaviruses belonging to the genus Gammacoronavirus include avian infectious bronchitis virus, etc.

[0058] Examples of coronaviruses belonging to the genus Deltacoronavirus include bulbul coronavirus HKU11, etc.

[0059] Examples of influenza viruses include swine influenza (H1N1 type) virus, seasonal influenza, avian influenza (H5N1 type) virus, etc.

[0060] Examples of classical swine fever virus include classical swine fever virus of the genus Pestivirus in the family Flaviviridae.

[0061] Examples of DNA viruses include hepatitis B virus (HBV).

[0062] From the viewpoint of easily obtaining the effects of the present invention, the target of the infection inhibitor of the present invention is preferably porcine epidemic diarrhea virus, SARS-CoV-2, swine influenza virus, hepatitis B virus (HBV), etc. Examples of other preferred viruses include norovirus of the family Caliciviridae, feline calicivirus, norovirus, Norwalk virus, rabbit hemorrhagic disease virus, plant infectious viruses (bean golden mosaic virus, tobacco mosaic virus, nematode-transmitted polyhedrosis virus, tobacco rattle virus), bacteriophages (Cystoviridae), or hepatitis virus.

[0063] (4) Method for inhibiting virus infection using the infection inhibitor of the present invention By contacting the infection inhibitor of the present invention with a virus by any method, virus infection can be inhibited.

[0064] The method of contacting the infection inhibitor of the present invention with a virus is not particularly limited. For example, there may be mentioned a method of spraying the infection inhibitor of the present invention in the air onto an object suspected of having a virus, a method of using a nasal drop, or a method of wiping with an impregnated material (such as thin paper soaked in the infection inhibitor of the present invention), etc.

[0065] The objects contacted by the infection inhibitor of the present invention include not only organisms (for example, the skin surface, inside the nostrils, etc.), but also various articles that may come into contact with organisms, such as daily necessities (for example, the surfaces of furniture, electrical appliances, sundries, etc.) and various facilities (for example, the surfaces of animal breeding facilities such as fences and cages), etc.

[0066] Examples of the social application of the present invention are shown in Figure 6 and Figure 7。 As Figure 6 shown, the infection inhibitor of the present invention can be placed in a container such as a tank and is usually installed in various facilities. In such a scheme, as Figure 7 shown, the entry of foreign animals is detected by an infrared sensor or the like, and the infection inhibitor of the present invention can be sprayed on the foreign animals. As a result, infection sources such as viruses are not easily introduced into the facility. In addition, different from general disinfectants and the like, the infection inhibitor of the present invention is not likely to have an adverse effect on intestinal bacteria. That is, since the present invention relates to the protection of the environmental symbiotic system, it also conforms to the goal of global biodiversity conservation (Nature positive). Examples

[0067] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

[0068] <Experiment 1: Preparation of virus (RNA virus) infection inhibitor> A sample was prepared using mixed microorganisms according to the following method.

[0069] The mixed microorganisms with the ATCC deposit number "PTA-1773" were diluted 100-fold with water and heated at 50 °C for 1 hour or more. After heating, impurities were removed by filtration through a filter with a pore size of 100 μm, and the resulting filtrate was used as a sample (corresponding to the infection inhibitor of the present invention) in the following tests.

[0070] <Experiment 2: Virus (RNA virus) infection inhibition effect test - 1> Using the sample obtained in "Experiment 1", the effect of inhibiting RNA virus infection was investigated. It should be noted that the following tests were conducted by the Food Hygiene and Environment Research Institute Co., Ltd.

[0071] (1) Preparation of virus and cultured cells Porcine epidemic diarrhea virus (PEDV, P-5V strain), which is a porcine infectious coronavirus, was prepared. Vero cells (a cell line derived from the renal epithelium of African green monkey kidneys) were used as the cultured cells.

[0072] (2) Inoculation of virus Based on the following method, the sample was brought into contact with the virus. It should be noted that the following tests were carried out with reference to "Overview of Virus Experimentation Revised 2nd Edition, Maruzen Co., Ltd. Virus Neutralization Test Method".

[0073] (2-1) Preliminary test Before the formal test, first confirm the effect of the test sample on cultured cells (cytotoxicity). First, serially dilute the test sample 10-fold with phosphate buffer, inoculate it into cultured cells, confirm the highest concentration that shows the normal state of the cells after culturing, and determine the virus concentration for the test. As a result, no cytotoxicity was confirmed in the 10-fold dilution. Therefore, set the detection limit in the formal test to 10 1.5 TCID 50 / mL.

[0074] (2-2) Formal test According to the test partition shown in Table 1, respectively take the test sample and phosphate buffer (10 mL each), and add the virus solution (1 mL) according to the concentration determined in the preliminary test. After adding the virus solution, let the resulting test solution stand at room temperature (25 °C) for 3 hours.

[0075] Table 1

[0076] Next, serially dilute each test solution after the reaction is completed 10-fold, and inoculate 100 μL each into the cultured cells in a 96-well plate. After inoculation, culture at 37 °C in a carbon dioxide incubator (5%) for 5 days. Observe the resulting cultured cells under a microscope, confirm the presence or absence of virus proliferation based on the CPE (cytopathic effect) that appears in the cultured cells, and calculate its concentration for each test partition.

[0077] Based on the obtained values, calculate the virus reduction rate (%) of the test area relative to the control area by the following formula. Virus reduction rate (%) = (control area - test area) / control area × 100

[0078] Based on the above results, at the time point 3 hours after the start of sensitization, calculate the reduction rate (%) of the test area relative to the control area, and confirm the infection inhibition effect. The reduction rate is calculated by the following formula. Log reduction value = Log 10 (virus titer of the control area) - Log 10 (virus titer of the test area) Reduction rate (%) = (1 - 1 / 10 对数减少值 ) × 100

[0079] (3) Results The results are shown in Figure 1 and Table 2. In the control group, natural attenuation of the virus amount was observed between the start of the experiment (the time point of virus addition) and 3 hours after the start of the experiment (10 6.1 →10 5.7 TCID 50 / mL). In contrast, in the test group, after 3 hours from the start of the experiment, a significant decrease in the virus amount was observed (<10 1.5 TCID 50 / mL, reduction rate = 99.99% or more). Therefore, it was judged that the thermophilic bacteria fermentation broth of the sample obtained in "Experiment 1" had an inactivating effect of up to 99.99% on porcine epidemic diarrhea virus at 180 minutes of reaction. From the above results, it can be seen that the infection inhibitor of the present invention has an excellent infection inhibitory effect on porcine epidemic diarrhea virus.

[0080] Table 2

[0081] <Test 3: Test - 2 for Inhibitory Effect of Virus (RNA Virus) Infection> Using the sample obtained in "Experiment 1", the inhibitory effect of coronavirus infection was investigated. It should be noted that the following test was conducted by Food Hygiene and Environment Research Institute Co., Ltd.

[0082] (1) Test method The test was conducted under the same conditions as in the above "Experiment 2", except that SARS-CoV-2 coronavirus (delta strain) was used instead of PEDV. It should be noted that the strain used in this example was a human-derived isolate. After isolation and cultivation from saliva using Vero cells, amplification of the SARS coronavirus 2 gene was confirmed by real-time PCR (Notification Law of the Ministry of Health, Labour and Welfare), and changes in N501Y(-) and L452R(+) were confirmed.

[0083] (2) Results The results are shown in Figure 2 and Table 3. In the control group, natural attenuation of the virus amount was observed between the start of the experiment (the time point of virus addition) and 3 hours after the start of the experiment (10 6.7 →10 6.3 TCID 50 / mL). In contrast, in the test group, after 3 hours from the start of the experiment, a significant decrease in the virus amount was observed (<10 1.9 TCID 50 / mL or 10 1.7 TCID 50 / mL, with a reduction rate of 99.99% for all). From the above results, it can be seen that the infection inhibitor of the present invention has excellent inhibitory effect on SARS coronavirus 2.

[0084] Table 3 Test Zone Name Virus Titer at the Start of the Test Virus Titer 3 Hours after the Start of Sensitization Reduction Rate (%) Control Area <![CDATA[10 6.7 > <![CDATA[10 6.3 > Natural Decay Test Area 1 <![CDATA[10 6.7 > <![CDATA[10 1.9 > 99.99 Test Area 2 <![CDATA[10 6.7 > <![CDATA[10 1.7 > 99.99

[0085] <Test 4: Test on the inhibitory effect of virus (RNA virus) infection - 3> Using the sample obtained in "Test 1", the inhibitory effect on swine influenza virus infection was investigated. It should be noted that the following test was conducted by Food Sanitation Research Institute Co., Ltd.

[0086] (1) Test method The test was carried out under the same conditions as in the above "Test 2", except that swine influenza virus H1N1 IOWA strain was used instead of PEDV as the virus, and MDCK cells (a cell line derived from dog kidneys) were used as the cultured cells.

[0087] (2) Results The results are shown in Figure 3 and Table 4. In the control group, natural attenuation of the virus amount was observed between the start of the test (the time point of adding the virus) and 3 hours after the start of the test (10 8.5 →10 7.9 TCID 50 / mL). In contrast, in the test group, a significant decrease in the virus amount was observed 3 hours after the start of the test (<10 1.5 TCID 50 / mL, with a reduction rate of over 99.99% for all). From the above results, it can be seen that the infection inhibitor of the present invention has excellent inhibitory effect on swine influenza virus.

[0088] Table 4 Test Zone Name Virus Titer at the Start of the Test Virus Titer 3 Hours after the Start of Sensitization Reduction Rate (%) Control Area <![CDATA[10 8.5 > <![CDATA[10 6.3 > Natural Decay Test Area <![CDATA[10 8.5 > <![CDATA[10 1.5 > 99.99

[0089] <Test 5: Test on the inhibitory effect of virus (RNA virus) infection - 4> In addition to the above tests, it was also confirmed that it has an inhibitory effect on feline calicivirus, which is an unencapsulated RNA virus of the family Caliciviridae and causes feline respiratory diseases.

[0090] The results are shown in Table 5. In the control group, natural attenuation of the virus amount was observed between the start of the test and 3 hours after the start of the test (10 8.5 →10 7.9 TCID 50 / mL). In the test area, 3 hours after the start of the test, it reached 10 4.3 TCID 50 / mL (reduction rate: 99.97%).

[0091] Table 5 Test Zone Name Virus Titer at the Start of the Test Virus Titer 3 Hours after the Start of Sensitization Reduction Rate (%) Control Area <![CDATA[10 8.5 > <![CDATA[10 7.9 > Natural Decay Test Area <![CDATA[10 8.5 > <![CDATA[10 4.3 > 99.97

[0092] <Test 6: Test on the inhibitory effect of virus (RNA virus) infection - 5> Using the sample obtained in "Test 1", the inhibitory effect on avian infectious bronchitis virus infection was investigated. It should be noted that the following test was conducted by Food Hygiene Research Institute Co., Ltd.

[0093] (1) Preparation of virus and cultured cells An infectious bronchitis virus (infectious bronchitis virus (IBV), H120 strain (Poulvac IB H120, Kyoritsu Pharmaceutical Co., Ltd.)) as an avian infectious virus was prepared.

[0094] (2) Virus inoculation Based on the following method, the sample was brought into contact with the virus. It should be noted that the following test was carried out with reference to "Virology, General Outline Revised 2nd Edition, Maruzen Co., Ltd., Virus Neutralization Test Method".

[0095] (2 - 1) Preparation of test solution The live vaccine was dissolved in purified water to reach 10 5.0 EID 50 or more in 1 mL, thereby preparing a virus solution. Next, according to the test partition shown in Table 6, the sample and phosphate buffer solution (10 mL each) were taken separately, and the virus solution (1 mL) was added. After adding the virus solution, the obtained test solution was allowed to stand at room temperature (25 °C) for a time specified for each test partition.

[0096] Table 6

[0097] (2 - 2) Inoculation into chicken embryos After the sensitization was completed, the test solutions in each test partition were filtered through a membrane filter (0.45 μm) and subjected to 10 - fold serial dilution. Next, 0.1 mL of each dilution was inoculated into the allantoic cavity of 10 - day - old chicken embryos from a chicken flock (Lohmann VALO). It should be noted that five chicken embryos were used for each dilution step. In addition, a control (3) was set up, and only phosphate buffer solution (0.1 mL) was inoculated. After inoculation, incubate at 37 °C for 7 days. Open the chicken eggs 7 days after inoculation and confirm the situation of the embryos in each test section using the control chicken embryos as a standard. Evaluate the chicken embryos that died or showed changes (stunted growth, dwarfing) as "infected", and at the same time calculate the logarithm of the lowest dilution ratio of the test solution showing an infection inhibition effect as the virus titer (Behrens-Karber method).

[0098] Based on the above results, calculate the reduction rate (%) of the test area relative to the control area 3 hours after the start of sensitization, and confirm the infection inhibition effect. Calculate the reduction rate using the following formula. Logarithmic reduction value = Log 10 (Virus titer in the control area) - Log 10 (Virus titer in the test area) Reduction rate (%) = (1 - 1 / 10 对数减少值 ) × 100

[0099] (3) Results The results are shown in Table 7. In the control area, natural attenuation was confirmed until the time point 3 hours after the start of sensitization, and the virus titer changed from 10 5.9 EID 50 / mL to approximately 10 5.3 EID 50 / mL. In contrast, in the test area, it became 10 3.1 EID 50 / mL (reduction rate: 99.37%) at the time point 3 hours after the start of sensitization. From the above results, it can be seen that the thermophilic bacteria fermentation sample obtained in "Test 1" achieved an inactivation effect of 99.37% on avian infectious bronchitis virus after 180 minutes of reaction.

[0100] Table 7 Test Zone Name Virus Titer at the Start of the Test Virus Titer from the Start to 3 Hours after Sensitization Reduction Rate (%) Control Area <![CDATA[10 5.9 > <![CDATA[10 5.3 > Natural Decay Test Area <![CDATA[10 5.9 > <![CDATA[10 3.1 > 99.37

[0101] <Test 7: Test on the Inhibition Effect of Virus (RNA Virus) Infection - 6> Similar to "Test 2", use Porcine epidemic diarrhea virus (PEDV, P-5V strain), which is a porcine infectious coronavirus, to explore the inhibition effect on RNA virus infection. However, in the formal test, the influence of the morphology of the thermophilic bacteria "BP-03693" (including "BP-863") (bacterial cell culture solution or culture supernatant of the bacterial cell culture solution) on the effect was confirmed.

[0102] The specimens were prepared as follows. Specimen 1: Bacterial cell culture solution (test solution of the thermophilic bacterium "BP-03693" prepared in the same manner as in "Test 2") Specimen 2: Specimen containing viable bacteria (vegetative cells) in the bacterial cell culture solution Specimen 3: Specimen in which the vegetative cells of the spore-forming bacteria in the bacterial cell culture solution were reduced by approximately 1 / 100. Specimen 4: Culture supernatant of the bacterial cell culture solution (the test solution bacteria prepared in "Test 2" were centrifuged (14,000 rpm, 10 min), and only the supernatant was collected).

[0103] The results are shown in Table 8. In "Specimen 1", from the start of the test to 3 hours, a natural attenuation of the virus amount was observed (Specimen 1 (first time): 10 6.3 →10 5.7 TCID 50 / mL, Specimen 1 (second time): 10 6.7 →10 6.1 TCID 50 / mL). In "Specimen 2", from the start of the test to 3 hours, it reached 10 2.9 TCID 50 / mL (reduction rate: 99.93%). In "Specimen 3", from the start of the test to 3 hours, it reached 10 3.1 TCID 50 / mL (reduction rate: 99.74%). In "Specimen 4", from the start of the test to 3 hours, it reached 10 1.7 TCID 50 / mL (reduction rate: 99.99%). In summary, compared with the bacterial cells, the fermentation metabolites released outside the bacterial cells themselves are more important for the virus inactivation effect.

[0104] Table 8

[0105] <Test 7: Test on the inhibitory effect of virus (DNA virus) infection> A preliminary test was conducted using hepatitis B virus (HBV) as the DNA virus. As a result, in the presence of "PTA-1773", "BP-03693", "BP-1051", or "BP-863" or their sterilized solutions, an inhibitory effect on the infectivity similar to that of the above RNA virus was confirmed.

[0106] In addition, regarding HBV, it was confirmed that within 30 minutes of exposure to "PTA-1773", "BP-03693", "BP-1051", or "BP-863" or their sterilized solutions (sterilized and filtered through a 0.22-micron filter), the amount of HBV present tended to decrease by approximately 10 3 cells.

[0107] From the above results, it can be seen that the agent of the present invention can control not only RNA viruses but also DNA viruses through the action of metabolites outside the bacterial cells derived from the bacterial bodies.

[0108] It should be noted that Tomato yellow leafcurl virus (TYLCV), which is known as a plant-infectious DNA virus, is known to be mediated by whiteflies. It has been confirmed that in agricultural fields, when applying the fermented product containing "PTA-1773", "BP-03693", "BP-1051", or "BP-863", although it does not necessarily have an adverse effect on whiteflies, there is a tendency for yellow leaf curl disease to be less likely to occur. In addition, root-knot nematodes mediate plant-infectious RNA viruses such as nepovirus and Tobravirus. On the other hand, it has also been found that when applying the fermented product containing "PTA-1773", "BP-03693", "BP-1051", or "BP-863" to soil rich in root-knot nematodes, even if the amount of root-knot nematodes does not decrease, it is difficult to cause crop damage. The phenomena observed in these plants indicate that the agent of the present invention may exert an effect of inhibiting the infectivity of a wide range of DNA viruses and RNA viruses that are infectious to plants. From these results, for example, it has a direct effect of inhibiting virus infection on Begomovirus of the Geminiviridae family, such as TYLCV, a plant-infectious DNA virus; Tobacco mosaic virus of the Virgaviridae family (rod-shaped virus family), known as Tomato mosaic virus; and plant-infectious RNA viruses such as Nepovirus and Tobravirus, and may not have an adverse effect on the symbiotic organisms themselves.

[0109] <Industrial Applicability> The hygiene hypothesis (Strachan DP. Hayfever, hygiene, and household size. BMJ 299:1259 - 1260, 1989) described in the background art has raised concerns about over - hygiene management. Specifically, it is shown that the degree of the hygienic environment in infancy can change the incidence of future allergies, and an unhygienic environment can reduce the incidence of future allergies. Since then, this hypothesis has gradually been widely recognized, and a large amount of data has been accumulated to support it. The relationship between the formation of the gut microbiota and other diseases has also been gradually pointed out. That is, it has been revealed that in terms of animal survival, the construction of an environment in which it co - exists with environmental microbiota is important.

[0110] On the other hand, the form and degree of this co - existence are still a controversial issue, and it is difficult to set strict standards. In the basic technology targeted by this application, it is considered to have a wide range of positive effects on various ecosystems of animals and plants, including activating the immune system, reducing fat accumulation, promoting plant growth, and suppressing the generation of greenhouse gases that cause environmental load, etc. Based on this, it is possible to inhibit virus infection, and this perspective is completely different from traditional virus infection inhibitors.

[0111] For example, bacteriophage Cystoviridae is an RNA virus that can infect actinomycetes, which are soil symbiotic microorganisms involved in plant growth. Therefore, a solution containing "BP - 863" or "BP - 03693" may inhibit the death of actinomycetes and contribute to plant growth. In addition, bacteriophage "Cystoviridae", which also belongs to RNA viruses, can infect "Lactococcus", which is known as a probiotic for fish, etc. Therefore, in order to inhibit the infectivity of bacteriophage "Cystoviridae", which also belongs to RNA viruses, and inhibit the death of "Lactococcus", a solution containing "BP - 863" or "BP - 03693" may increase the production amount of functional molecules derived from "Lactococcus". In fact, when evaluating the gut microbiota of fish, it was confirmed that there is a tendency for an increase in "Lactococcus" in seawater fish and freshwater fish fed with fermented feed containing "BP - 863" or "BP - 03693", and it can be said that the above - mentioned contents are non - contradictory trends.

[0112] In addition, it has been pointed out that insects such as nematodes mediate the transmission of plant - pathogenic viruses, etc. When these viruses are RNA viruses, the adverse effects on plants can be reduced by the products of the present invention. It should be noted that in its proposed mechanism of action, it may be involved in 2-aminoisobutyric acid (α-aminoisobutyric acid, hereinafter referred to as "Aib") and its complexes. Aib is generally referred to as an unusual amino acid. Different from ordinary amino acids, it does not become a constituent element of normal proteins. Due to this property and others, it is considered to be one of the constituent factors for exerting the antibiotic effect such as lantibiotics. The results of synthetic chemistry research suggest that Aib may form a complex with papain (Tsuchiya, K. & Numata, K. Chemoenzymatic synthesis of polypeptides containing the unnatural amino acid 2-aminoisobutyric acid. Chem Commun (Camb) 53, 7318-7321, doi:10.1039 / c7cc03095a (2017)), and may be transformed into a complex with high cell permeability (Terada, K. et al. Artificial Cell-Penetrating Peptide Containing Periodic alpha-Aminoisobutyric Acid with Long-Term Internalization Efficiency in Human and Plant Cells. ACS Biomater Sci Eng 6, 3287-3298, doi:10.1021 / acsbiomaterials.0c00182 (2020)).

[0113] On the other hand, in "BP-03693", "BP-863" is contained, that is, in Caldibacillus hisashii and Paenibacillus No. 36 strain contained in "BP-03693", as an enzyme similar to papain, putative papain-like cysteine peptidase (SEQ ID No. 1) and putative papain-like cysteine peptidase (DUF1796) (SEQ ID No. 2) exist. It is expected that these enzyme groups can participate in the synthesis of lantibiotics with Aib as a constituent factor. Note that after extracting the genomic DNA of these strains, the sequence information of the results of whole-genome analysis (Illumina HiSeq) was determined. Moreover, a related gene (SEQ ID No. 3) of the lantibiotic-related gene (sunA; lantibiotic anti peptide) possessed by Bacillus thermoamylovorans was also detected in the genomic genes of "BP-863". It is expected that these can act together to exert an effect in virus infection inhibition.

[0114] Table 9

[0115] Table 10

[0116] Table 11

[0117] In addition, in animals, in the environment of symbiotic bacteria present in the nose, mouth, skin, bronchus, intestine, etc., and symbiotic bacteria that may be present in soil, drainage, rivers, seawater, sludge, etc. in the natural environment, by suppressing the types and populations of viruses, it is expected to improve the environmental symbiotic function.

[0118] In the future, in building a sustainable society, it is extremely important to consider the perspective of the symbiosis of the entire ecosystem, and it is not ideal to simply deal with pathogens. It is expected that this application will become an important technology in these perspectives.

[0119] As a use of the present invention, one method of use is to slightly dilute the concentration of the virus infection inhibitor for humans, and then, on the basis of minimizing the inhibition of symbiotic microorganisms, add this technology. In addition, from the perspective of a small environmental burden, it can be used in various situations. For example, it is expected to be used for various purposes that require virus infection inhibition without reducing beneficial symbiotic bacteria, such as for inhibiting viruses in soil, drainage, feed, etc., or for applying on the nose or skin, or spraying in a room or livestock house, or for combined use with an air purifier.

Claims

1. A virus infection inhibitor comprising a microorganism of the genus Caldibacillus or its culture.

2. The virus infection inhibitor according to claim 1, wherein The microorganism of the genus Caldibacillus has the NITE accession number "BP-863".

3. The virus infection inhibitor according to claim 1, which is an environmentally symbiotic virus infection inhibitor comprising a mixed microorganism having a symbiotic function for humans, animals, plants, and ecosystems, wherein the mixed microorganism is of the genus Caldibacillus and the genus Paenibacillus.

4. The environmentally symbiotic virus infection inhibitor according to claim 3, wherein, The mixed microorganism has the NITE accession number "BP-03693".

5. The environmentally symbiotic virus infection inhibitor according to claim 4, wherein, The mixed microorganism having the NITE accession number "BP-03693" has the ATCC accession number "PTA-1773".

6. The virus infection inhibitor according to claim 5, wherein, The ATCC accession number "PTA-1773" has the NITE accession number "BP-863" and the NITE accession number "BP-1051".

7. The viral infection inhibitor according to any one of claims 1 to 6, wherein, The virus is porcine epidemic diarrhea virus belonging to alphacoronavirus, SARS-CoV-2 belonging to betacoronavirus, or avian infectious bronchitis virus (IB) belonging to gammacoronavirus.

8. The viral infection inhibitor according to any one of claims 1 to 6, wherein, The virus is seasonal influenza virus, avian influenza (H5N1 type) virus, or classical swine fever virus (genus Pestivirus, Flaviviridae).

9. The viral infection inhibitor according to any one of claims 1 to 6, which does not have an adverse effect on symbiotic organisms, wherein, The virus is norovirus of the family Caliciviridae, feline calicivirus, norovirus, Norwalk virus, rabbit hemorrhagic disease virus, plant infectious virus (bean golden mosaic virus, tobacco mosaic virus, nepovirus, tobacco rattle virus), bacteriophage (Cystoviridae), or hepatitis virus.

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