Analysis system for developing antiviral agent for hones indicated with inhibition

Abstract

The present invention addresses the problem of providing: a method for screening a candidate compound capable of serving as a prophylactic or therapeutic agent for inhibiting individual death of bees caused by virus infection; and a prophylactic or therapeutic agent contributing to inhibiting mass death of bees caused by virus infection. The problem addressed by the present invention is that of determining that the cause of massive death of bees is intestinal injury caused by a protease derived from a virus mediated by mites, and finding that individual death and intestinal injury in a virus-infected group can be suppressed by administering dsRNA of the virus. In addition, it has been found that individual death in the virus-infected group is inhibited by inhibiting a protease derived from a virus by administering E-64 ([(2S, 3S)-3-carboxy oxirane-2-carbonyl]-L-leucine (4-guanidinobutyl) amide hemihydrate).

Description

TECHNICAL FIELD

[0001] The present application relates to a prophylactic and / or therapeutic agent for a disease of honeybees caused by damage to the intestines of honeybees, which can lead to the death of individual honeybees, and a screening method therefor.

[0002] This application claims priority to Japanese Application No. 2023-038426, which is hereby incorporated by reference herein.

[0003] Honeybees are agricultural insects that are required for the pollination of agricultural crops. Honeybees perform pollination of almost all fruits, most vegetables, seedling production, pasture for livestock, and the like, and pollination is essential for their production, and thus the presence of honeybees is indispensable for the site of agricultural production.

[0004] In recent years, a large number of deaths of honeybees have occurred worldwide in association with the occurrence of Varroa destructor, a parasitic mite of honeybees. In recent years, the most important cause of colony death reported by beekeepers in the United States is Varroa destructor (non-patent literature 1). Varroa destructor has spread from Asia to the world with the artificial movement of honeybees, and if such a situation is allowed to occur, most of the agricultural products such as vegetables and fruits cannot be produced. A large number of deaths of honeybees that perform pollen mediation is a big problem in agriculture and beekeeping, and is also an important problem from the viewpoint of biodiversity.

[0005] Recently, it has been reported that a bacterium that has been genetically recombined to have an RNAi function by administration to honeybees reduces the number of individuals of a virus to which honeybees are infected and a parasitic mite (non-patent literature 2). However, in this report, although it is shown that the number of individuals of a virus and a mite can be technically reduced, since a genetically recombined organism is used, it is a technology that cannot be easily used in actual beekeeping fields.

[0006] The solution of the problem of a large number of deaths of honeybees is an important issue in agriculture related to the stable supply of agricultural products. However, the pathogen that has been the cause of a large number of deaths of honeybees so far, the pathogenesis of the pathogen, and its therapeutic agent are not yet fully understood.

[0007] Prior Art Documents Patent Documents Non-patent literature 1: Steinhauer, N., et. al., “United States honey bee colony losses 2022-23: Preliminary results from the Bee Informed Partnership” [online], June 22, 2023, Bee Informed Partnership, [retrieved on March 10, 2024], URL: <https: / / beeinformed.org / 2023 / 06 / 22 / united-states-honey-bee-colony-losses-2022-23-preliminary-results-from-the-bee-informed-partnership / > Non-patent literature 2: Leonard, S. P., et. al., Science. 2020 Jan 31; 367(6477): 573-576 SUMMARY

[0008] PROBLEMS TO BE SOLVED BY THE INVENTION The present application relates to a screening method for a candidate compound for a prophylactic or therapeutic agent for suppressing individual death of honeybees caused by viral infection, and a prophylactic or therapeutic agent for suppressing mass death of honeybees caused by viral infection.

[0009] MEANS FOR SOLVING THE PROBLEMS The present inventors etc. have conducted intensive studies in order to solve the above problems, and as a result, it has been determined that the cause of mass death of honeybees is intestinal damage caused by a virus-derived protease mediated by mites, and it has been found that individual death and intestinal damage in a virus infection group can be suppressed by administering dsRNA of the virus. In addition, it has been found that individual death in the virus infection group is suppressed by inhibiting the virus-derived protease by administering E-64 ([ (2S, 3S) -3-carboxyoxirane-2-carbonyl] -L-leucine (4-guanidinobutyl) amide hemihydrate), and thus the present application has been completed.

[0010] That is, the present application is as follows.

[0011] 1. A screening method for a prophylactic and / or therapeutic agent for a disease of honeybees, which is a disease that causes individual death due to intestinal damage of honeybees, using an inhibitory activity against a virus-derived protease as an index.

[0012] 2. The screening method according to the preceding item 1, wherein the honeybees are honeybees infected with a virus mediated by mites.

[0013] 3. The screening method according to the preceding item 1 or 2, wherein the protease is a cysteine protease.

[0014] 4. The screening method according to the preceding item 1 or 2, wherein the virus is Israeli acute paralysis virus (IAPV) or Deformed wing virus (DWV).

[0015] 5. The screening method according to the preceding item 1, which further uses individual death of the honeybee and / or regeneration of the intestine of the honeybee as an index.

[0016] 6. A prophylactic and / or therapeutic agent for a disease of a honeybee, which comprises a substance that inhibits the activity of a protease derived from a virus, the disease of the honeybee being a disease that causes individual death of the honeybee, which is caused by damage to the intestine of the honeybee.

[0017] 7. The prophylactic and / or therapeutic agent according to the preceding item 6, wherein the substance that inhibits the activity of the protease derived from the virus is E-64 having the following general formula (I) and / or an analog thereof: [Chemical Formula 1] (In the formula, R1 represents OH, an alkoxy group that can be substituted, or an amino group that can be substituted; R2 represents an alkyl group that can be substituted, or a heterocyclic alkyl group that can be substituted and forms a ring via the N to which it is bonded).

[0018] 8. The prophylactic and / or therapeutic agent according to the preceding item 6, wherein the substance that inhibits the activity of the protease derived from the virus is a calpain inhibitor.

[0019] Effects of the Invention According to the present application, by inhibiting the activity of the protease derived from the virus, individual death of the honeybee caused by infection with the virus can be inhibited, and the substance that inhibits the activity of the protease derived from the virus can be used as an effective ingredient of a prophylactic or therapeutic agent for a disease of the honeybee that causes individual death of the honeybee, which is caused by infection with the virus. According to the present application, a screening method that can provide a candidate compound for a prophylactic or therapeutic agent for a disease of the honeybee that causes individual death of the honeybee, which is caused by infection with the virus, can be provided, and a prophylactic or therapeutic agent for a disease of the honeybee that causes individual death of the honeybee, which is based on a novel mechanism of action in which damage to the intestine resulting from inhibition of the activity of the protease derived from the virus is inhibited, can be provided. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a schematic diagram of the steps of sample preparation for MinION analysis. (Example 1) Figure 2is a heat map indicating the results of quantifying each virus amount by MinION analysis. (Example 1) Figure 3 is a quantitative result of viruses of honeybees based on RT-PCR. The horizontal axis indicates sample number (honeybee individual group No.), and the vertical axis indicates the number of virus copies per 1 ng of RNA. (Example 2) Figure 4 is a quantitative result of viral response factors and antibacterial peptides in honeybees. The horizontal axis indicates gene name, and the vertical axis indicates expression ratio (Fold Change; FC). (Example 3) Figure 5 is a quantitative result of intestinal repair factors in honeybees. The horizontal axis indicates gene name, and the vertical axis indicates expression ratio (Fold Change; FC). (Example 3) Figure 6 is a result of RNAi test of viruses in honeybees. It is a graph in which the horizontal axis indicates elapsed days from the start of administration, the vertical axis indicates survival rate, and the number of individuals on day 0 is taken as 100, and the number of individuals on each day is indicated in %. (Example 4) Figure 7 is a result of detection of pH3 antibody-positive cells in honeybees. It is a graph indicating DAPI and immunostaining images of pH3 and a Merge image of microscope images of the midgut of a virus infection group and an uninfected group. (Example 5) Figure 8 is a result of analysis of pH3 antibody-positive cells after dsRNA administration. The horizontal axis indicates administration groups, "None" indicates a control group, "GFPi" indicates a dsRNA administration group of GFP, "IAPVi" indicates a dsRNA administration group of IAPV, and "DWVi" indicates a dsRNA administration group of DWV, and the vertical axis indicates the number of pH3 antibody-positive cells in the midgut per 1 individual. (Example 5) Figure 9 is a result of suppressing individual death of honeybees of virus infection group No. 5 by administration of E-64. It is a graph in which the horizontal axis indicates elapsed days from the start of administration, the vertical axis indicates survival rate, and the number of individuals on day 0 is taken as 100, and the number of individuals on each day is indicated in %. (Example 6) Figure 10 is a result of suppressing individual death of honeybees of virus infection group No. 2 by administration of E-64. It is a graph in which the horizontal axis indicates elapsed days from the start of administration, the vertical axis indicates survival rate, and the number of individuals on day 0 is taken as 100, and the number of individuals on each day is indicated in %. (Example 6) Figure 11is the result of the BPB administration test. In each of the virus infection group and the virus non-infection group, "Sucrose" indicates the result of the sample collected after sucrose administration, and "BPB" indicates the result of the sample collected after BPB administration. The vertical axis indicates the measured value of absorbance at 500 nm. (Example 7) Figure 12 is the result of the measurement of the number of living bacteria in the blood of the honeybee. "Infected" indicates the result of the virus infection group, and "Non-infected" indicates the result of the virus non-infection group. The vertical axis is the number of living bacteria (colony / plate). (Example 8) Figure 13 is the result of the confirmation test of the recovery of the intestinal tract damage of the honeybee by administration of E-64. "None" indicates the control group, "E-64" indicates the E-64 administration group, "Sucrose" indicates the result of the sample collected after sucrose administration, and "BPB" indicates the result of the sample collected after BPB administration. The vertical axis indicates the measured value of absorbance at 500 nm. (Example 9) Figure 14 is the result of the measurement of the protease activity of IAPV protease. In each of the fluorescence activity and the relative activity, "IAPV protease" indicates the result of the enzyme reaction of IAPV protease measured in the absence of E-64, and "IAPV protease + E-64" indicates the result of the enzyme reaction of IAPV protease measured in the presence of E-64. The vertical axis of the left graph indicates the fluorescence intensity. The vertical axis of the right graph indicates the relative value (relative activity) of IAPV protease activity calculated based on the fluorescence intensity, with the fluorescence intensity measured in the enzyme reaction of IAPV protease in the absence of E-64 being taken as 100%. (Example 12) Figure 15 is the result of the analysis on the effect of E-64c and E-64d on the individual death of the honeybee. It is a graph in which the horizontal axis indicates the elapsed days from the start of administration, the vertical axis indicates the survival rate, and the number of individuals on day 0 is taken as 100, and the number of individuals on each day is indicated in %. (Example 13) DETAILED DESCRIPTION

[0021] The present application is based on the insight that in a mass death group of honeybees, the amount of detection of virus mediated by mites, particularly Israeli acute paralysis virus (IAPV) is high; intestinal damage is observed in the virus infection group; individual death and midgut damage can be inhibited by administering dsRNA of the virus; individual death and intestinal damage in the virus infection group can be inhibited by administering dsRNA of the virus. According to these insights, it is first clarified in the present application that the pathogen that becomes the cause of mass death of honeybees is virus mediated by mites, and that intestinal damage caused by a protease derived from the virus can cause mass death of honeybees. The screening method of the present application based on these insights contributes to the development of a new drug for honeybees based on a completely different approach from the past. In addition, the present application is also based on the insight that individual death in the virus infection group can be inhibited by administering E-64 ([ (2S, 3S) -3-carboxyoxirane-2-carbonyl] -L-leucine (4-guanidinobutyl) amide hemihydrate).

[0022] The present application provides a method for screening an effective substance as a prophylactic and / or therapeutic drug for a disease of honeybees that causes individual death due to intestinal damage of honeybees, with an inhibitory activity against a protease derived from a virus as an index, with a substance that inhibits the activity of the protease as a candidate compound.

[0023] The "inhibitory activity against a protease derived from a virus" is an activity that inhibits the activity of a protease derived from a virus, and can be evaluated, for example, by substrate decomposition based on the protease derived from a virus, and the like, and can also be evaluated by a phenomenon caused by inhibition of the activity of the protease derived from a virus in the presence of a test substance.

[0024] As a phenomenon caused by the inhibition of the activity of the virus-derived protease, for example, there can be mentioned the suppression of the death of the individual of the infected honeybee (improvement of the survival rate), the recovery of the intestinal damage, the suppression of the intestinal damage, the reduction of the intestinal regeneration caused by the intestinal damage, and the like. The suppression of the intestinal damage and the reduction of the intestinal regeneration can be evaluated by the number of anti-phosphorylated histone H3 antibody (pH3 antibody) positive cells. In more detail, in the intestinal damage caused by the virus-derived protease, the repair (regeneration) of the damaged intestinal tract occurs, and at this time, the mitosis of the cells can be observed. Since the pH3 antibody can recognize the phosphorylated histone H3 specific to the mitosis, by obtaining a positive image based on the pH3 antibody, the mitotic cells can be detected. When the intestinal damage is suppressed, the number of the pH3 antibody positive cells in the midgut per one individual of the honeybee decreases, because the mitotic cells observed at the time of the regeneration of the damaged intestinal tract decrease. The recovery and the suppression of the intestinal damage can be evaluated by the amount of Bromophenol Blue (BPB) in the blood of the honeybee after the administration of the BPB. Since the BPB is taken into the body due to the intestinal damage, the amount of the BPB detectable in the blood after the administration of the BPB increases, and the amount of the BPB detectable in the blood after the administration of the BPB decreases due to the recovery of the intestinal damage. By suppressing the intestinal damage, the increase in the amount of the BPB detectable in the blood after the administration of the BPB is suppressed.

[0025] Therefore, the screening method of the present application can evaluate the inhibitory activity of the test substance on the virus-derived protease from more angles, in addition to the inhibitory activity on the virus-derived protease, by evaluating the phenomenon as an index.

[0026] The test substance for the screening method of the present application is not particularly limited, and can be any publicly known compound and novel compound, for example, a biologically produced substance, a chemically synthesized compound, and the like, and more specifically, for example, there can be mentioned a nucleic acid, a nucleic acid analogue, a saccharide, a lipid, a protein, a peptide, an antibody, an antibody fragment, an organic low molecular compound, a compound library, and the like.

[0027] In one embodiment, the screening method of the present application comprises the following steps: (A) a step of bringing a virus-derived protease into contact with a test substance; (B) a step of measuring the activity of the virus-derived protease brought into contact with the test substance; and (C) comparing the activity of the virus-derived protease that has contacted the test substance with the activity of the virus-derived protease that has not contacted the test substance, whereby, in the case where the activity of the virus-derived protease that has contacted the test substance is lower than the activity of the virus-derived protease that has not contacted the test substance, the test substance is selected as a candidate for a prophylactic and / or therapeutic agent for a disease of honeybees that is a disease caused by damage to the intestines of honeybees and that can cause death of the individual honeybees.

[0028] In the process (A) of the above method, the virus-derived protease and the test substance are placed under contact conditions. The virus-derived protease can be a protease synthesized in a publicly known protein synthesis system, such as an Escherichia coli protein synthesis system, an insect cell protein synthesis system, an animal cell protein synthesis system, a cell-free protein synthesis system, and the like.

[0029] The measurement of the activity of the virus-derived protease in the process (B) of the above method can be performed using a publicly known or novel protease activity measurement method, such as a protease activity measurement method that utilizes the decomposition of a substrate (e.g., casein, a synthetic substrate, and the like). The protease activity measurement method that utilizes the decomposition of a substrate can be performed, for example, by adding the virus-derived protease to a substrate in the presence of the test substance, and then, after a certain period of time, quantifying the undecomposed substrate and / or the decomposed substrate. The quantification of the undecomposed substrate and / or the decomposed substrate can be performed, for example, by a method using a fluorescently labeled substrate, an electrophoresis method, absorbance measurement, or the like. In the case where the method that utilizes the decomposition of a substrate is used in the process (B), the contact of the test substance with the virus-derived protease in the process (A) can be performed in a protease substrate solution, or the virus-derived protease that has contacted the test substance can be further contacted with a substrate in another solvent. The more the amount of the undecomposed substrate, the lower the protease activity, and the more the amount of the decomposed substrate, the higher the protease activity.

[0030] In the process (C) of the above method, the activity of the virus-derived protease that has contacted the test substance is compared with the activity of the virus-derived protease that has not contacted the test substance. The comparison of the activities of the proteases is preferably performed based on whether there is a statistically significant difference.

[0031] In one embodiment, the screening method of the present application further comprises the following processes (a) and (b), the following processes (a) and (c), or the following processes (a), (b), and (c) in addition to the above processes (A) to (C): (a) a process of administering the test substance to a group of individual honeybees and rearing the individual honeybees; (b) a step of determining the survival rate of the group of the honeybee individuals, comparing the survival rate with that of the control group reared without the administration of the test substance, whereby, in the case where the survival rate of the group of the honeybee individuals reared with the administration of the test substance is higher than that of the control group reared without the administration of the test substance, the test substance is selected as a candidate substance for a prophylactic and / or therapeutic agent for a disease of the honeybee, which is a disease that causes death of the honeybee individuals due to damage to the intestinal tract of the honeybee; (c) a step of counting the number of anti-phosphorylated histone H3 antibody (pH3 antibody)-positive cells of the midgut of each of the individuals of the group of the honeybee individuals, comparing the average value thereof with the average value of the number of the pH3 antibody-positive cells of the control group reared without the administration of the test substance, whereby, in the case where the average value of the number of the pH3 antibody-positive cells of the group of the honeybee individuals reared with the administration of the test substance is lower than that of the control group reared without the administration of the test substance, the test substance is selected as a candidate substance for a prophylactic and / or therapeutic agent for a disease of the honeybee, which is a disease that causes death of the honeybee individuals due to damage to the intestinal tract of the honeybee; or, a step of administering bromophenol blue (BPB) to the group of the honeybee individuals reared with the administration of the test substance and the control group reared without the administration of the test substance, determining the concentration of BPB in the blood after the administration of BPB, whereby, in the case where the increase in the concentration of BPB in the blood due to the administration of BPB is inhibited (decreased) as compared with the control group, the test substance is selected as a candidate substance for a prophylactic and / or therapeutic agent for a disease of the honeybee, which is a disease that causes death of the honeybee individuals due to damage to the intestinal tract of the honeybee.

[0032] In the step (a) of the above method, the number of the individuals at the start of rearing of each of the group of the honeybee individuals reared with the administration of the test substance and the control group reared without the administration of the test substance is not particularly limited, and can be, for example, 1 to 200, 10 to 100, preferably 20 to 50, more preferably 30 to 35. The steps (a) to (c) are preferably performed in a sterilized environment. The rearing temperature of each group is not particularly limited, and is preferably 15 to 35°C, more preferably 20 to 35°C, more preferably 25 to 35°C, more preferably 27 to 33°C.

[0033] In the step (a) of the above method, the method of administration of the test substance is not particularly limited, and it is preferable to contain the test substance in a bait (e.g., a sugar-containing solution such as a sucrose solution) and allow the honeybees to freely ingest it. The concentration of the test substance is not particularly limited, and can be, for example, 1 to 1000 μM, preferably 10 to 500 μM, more preferably 10 to 300 μM, more preferably 20 to 250 μM, more preferably 50 to 200 μM, more preferably 50 to 150 μM, in the case of E-64 or an analog thereof or a calpain inhibitor. The group of the honeybee individuals of the step (a) is not particularly limited, and is preferably a group of the honeybee individuals derived from a population infected with a virus.

[0034] The comparison of the survival rate in step (b) of the above method is performed after a certain period of time from the administration in step (a). The period of time is not particularly limited, and is preferably 1 day or more, more preferably 2 days or more, more preferably 3 days or more, and more preferably 4 days or more, after the administration. For example, it can be 1 to 30 days, 1 to 20 days, 1 to 15 days, 2 to 14 days, 3 to 13 days, 4 to 12 days, 5 to 11 days, 6 to 10 days, 7 to 10 days, 8 to 10 days, 9 to 10 days, or 10 days, after the administration. In step (b), the number of times of measuring the survival rate can be one, but it is preferably measured at intervals of a certain period of time. The interval is not particularly limited, and for example, it can be 1 hour to 3 days, preferably 12 hours to 2 days, more preferably 18 to 36 hours, and more preferably 1 day. When the survival rate is measured at intervals of a certain period of time, the comparison of the survival rate is preferably performed at a time point at which the survival rate of the control group measured at intervals of a certain period of time is less than 100%, for example, at a time point of 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. The comparison of the survival rate is preferably performed based on whether there is a statistically significant difference.

[0035] The counting of the number of pH3 antibody-positive cells in the midgut in step (c) is performed after a certain period of time from the administration in step (a). The period of time is not particularly limited, and is preferably 1 day or more, more preferably 2 days or more, more preferably 3 days or more, and more preferably 4 days or more, after the administration. For example, it can be 1 to 30 days, 1 to 20 days, 1 to 15 days, 2 to 12 days, 3 to 11 days, 4 to 10 days, 5 to 9 days, or 6 to 8 days, after the administration. In step (c), the number of individuals in which the number of pH3 antibody-positive cells in the midgut is counted is not particularly limited, and can be all of the surviving individuals of the group of bee individuals at the time of counting, or can be a part thereof (for example, 1 to 99%, 10 to 90%, 20 to 80%, 30 to 70%, 40 to 60%, or the like, of all of the surviving individuals). The timing of the administration of BPB in step (c) is not particularly limited as long as it is after the administration in step (a), and for example, the survival rate of the control group that is not administered with the test substance can be reduced to 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, based on the number of individuals of the bees before the rearing in step (a) as 100%.

[0036] The present application provides a prophylactic and / or therapeutic agent for a disease of a honeybee, which comprises a substance that inhibits the activity of a virus-derived protease, the disease of the honeybee being a disease that causes death of an individual of the honeybee due to damage to the intestinal tract of the honeybee.

[0037] The prophylactic and / or therapeutic agent of the present application can be mixed into water and / or feed (e.g., a sugar-containing solution such as sucrose, pollen, honey, artificial feed, etc.) in a manner that an effective amount of the substance that inhibits the activity of a virus-derived protease is achieved, and administered to the honeybee by water absorption, ingestion, etc. The effective amount of the substance that inhibits the activity of a virus-derived protease in water absorption, ingestion, etc. can be 1 to 1000 μM, preferably 10 to 500 μM, more preferably 10 to 300 μM, more preferably 20 to 250 μM, more preferably 50 to 200 μM, more preferably 50 to 150 μM, in the case of E-64, its analogs, and / or a calpain inhibitor. In addition, the prophylactic and / or therapeutic agent of the present application can be used by spraying, dipping, coating, and / or mixing to a honeybee, a beehive, a plant (including its soil and the like growing environment) that is a honey source of the honeybee, and / or a suitable material. The material can be administered, for example, by being installed in or around a beehive and allowing the honeybee to come into contact therewith.

[0038] The dosage form of the prophylactic and / or therapeutic agent of the present application is not particularly limited, and can be mixed with a pharmaceutically acceptable carrier, additive, and formulated into a liquid, a suspension, a wettable powder, an emulsion, an aerosol, a powder, a granule, a tablet, etc., and is preferably a liquid. The method of producing these dosage forms is not particularly limited, and the formulation can be produced according to the techniques that can be used in a conventional agricultural chemical formulation. The content of the substance that inhibits the activity of a virus-derived protease in the formulation is not particularly limited, and can be, for example, 0.000000001 to 99 w / v%, 0.00000001 to 50 w / v%, 0.0000001 to 1 w / v%, 0.0000001 to 0.1 w / v%, 0.001 to 50 w / v%, 0.1 to 10 w / v%, etc. Examples of the pharmaceutically acceptable carrier, additive can include sugars such as sucrose, dextrin, glucose, etc.; surfactants such as sodium lauryl sulfate, laurylamine acetate, polyoxyethylene lauryl alcohol, etc.; lubricants such as talc, calcium stearate, etc.; stabilizers such as tocopherol, ascorbic acid, vitamins, etc.; thickening agents such as starch, xanthan gum, polyvinylpyrrolidone, etc., and are not particularly limited.

[0039] The virus in the present application is preferably a virus mediated by mites, for example, Israeli acute paralysis virus (IAPV), Deformed wing virus (DWV), Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Chronic bee paralysis virus (CBPV), Sacbrood virus (SBV), Varroa destructor virus-1 (VDV-1), Kashmir bee virus (KBV), Lake Sinai virus (LSV), and the like, preferably Israeli acute paralysis virus, Deformed wing virus, and more preferably Israeli acute paralysis virus.

[0040] The virus-derived protease in the present application is preferably a virus-derived cysteine protease, more preferably a cysteine protease derived from IAPV or DWV, and more preferably a cysteine protease derived from IAPV. Examples of the cysteine protease derived from IAPV include a polypeptide having the amino acid sequence shown in SEQ ID NO: 51, the amino acid sequence shown in SEQ ID NO: 52, and the amino acid sequence shown in SEQ ID NO: 53, and a polypeptide having protease activity in which 1 to 40, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, and more preferably 1 to 3, amino acid residues are deleted, added, inserted, or substituted in the amino acid sequence shown in SEQ ID NO: 51, 52, or 53. Examples of the base sequence of the cDNA encoding the cysteine protease derived from IAPV include the base sequence shown in SEQ ID NO: 54, the base sequence shown in SEQ ID NO: 55, and the base sequence shown in SEQ ID NO: 56, and a base sequence in which 1 to 50, preferably 1 to 20, more preferably 1 to 5, and more preferably 1 to 3, bases are deleted, substituted, and / or added in the base sequence shown in SEQ ID NO: 54, 55, or 56. Furthermore, the base sequence of the cDNA encoding the cysteine protease derived from IAPV includes a mutant having a nucleotide sequence having 50% or more, for example, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, or 99.9% or more homology with the base sequence shown in SEQ ID NO: 54, 55, or 56. The homology of the base sequence can be determined using a known algorithm such as BLAST or FASTA. Furthermore, the base sequence of the cDNA encoding the cysteine protease derived from IAPV includes a mutant having a base sequence that hybridizes to the base sequence shown in SEQ ID NO: 54, 55, or 56 under stringent conditions. Examples of the stringent hybridization conditions include, for example, as a washing condition after hybridization, "5 x SSC, 1% SDS, 37°C", "1 x SSC, 0.1% SDS, 37°C", "0.5 x SSC, 0.1% SDS, 42°C", "5 x SSC, 1% SDS, 65°C", and the like. In the present application, a polypeptide in which the amount of the substrate (casein or a synthetic substrate) that is decomposed is larger (the amount of the undecomposed substrate is smaller) than that of a control in which only the substrate is dissolved in a solvent, is determined to "have protease activity" by dissolving the polypeptide and the substrate in a solvent and incubating at 37°C for 1 hour.

[0041] The substance that inhibits the activity of the IAPV-derived cysteine protease in the present application is not particularly limited, and examples thereof include organic low-molecular compounds, nucleic acids, saccharides, lipids, proteins, peptides, and the like. As the organic low-molecular compounds, E-64 ([ (2S, 3S) -3-carboxyoxirane-2-carbonyl] -L-leucine (4-guanidinobutyl) amide), an analog of E-64, a calpain inhibitor, and the like, hydrates, hemihydrates, acid anhydrides, mixtures thereof, and the like can be used. As the nucleic acids, siRNA, antisense oligonucleotides, expression vectors thereof, and the like against the IAPV-derived cysteine protease gene can be exemplified. In the present specification, "siRNA" is an abbreviation of short interfering RNA, and is an RNA molecule having a double-stranded RNA portion composed of about 10 to 40 bases (hereinafter sometimes referred to as dsRNA). The siRNA has a function of cleaving the mRNA of a target gene having a sequence complementary to the antisense strand thereof, and inhibiting the expression of the target gene.

[0042] As the analog of E-64 in the present application, E-64-c ([ (2S, 3S) -3-carboxyoxirane-2-carbonyl] -L-leucine (3-methylbutyl) amide), E-64-d ([ (2S, 3S) -3-ethoxycarbonyloxirane-2-carbonyl] -L-leucine (3-methylbutyl) amide), JPM-OEt ((2S, 3S) -3-[[[ (1S) -1-[[[2- (4-hydroxyphenyl) ethyl] amino] carbonyl] -3-methylbutyl] amino] carbonyl] -2-oxirane carboxylic acid ethyl ester), CA-074 ([ (2S, 3S) -3-propylcarbamoyloxirane-2-carbonyl] -L-isoleucyl-L-proline), CA-074Me ([ (2S, 3S) -3-propylcarbamoyloxirane-2-carbonyl] -L-isoleucyl-L-proline methyl ester), Ep-459, Ep-460, Ep-479, Ep-429, and the like can be exemplified. Ep-459, Ep-460, Ep-479, and Ep-429 have the structures shown below, respectively.

[0043] According to one embodiment of the present application, the substance that inhibits the activity of the virus-derived protease is E-64 and / or an analog thereof, which has the following general formula (I).

[0044] [Chemical Formula 3] (In the formula, R1 represents OH, an alkoxy group which can be substituted, or an amino group which can be substituted; R2represents an alkyl group which can be substituted, or a heterocycloalkyl group which can be substituted, forming a ring via the N to which it is bonded. The term "alkyl" refers to a fully saturated hydrocarbon of the formula C x H 2x+1 (wherein x is a number of 1 or more). Generally, the alkyl groups of the present application contain 1 to 20, preferably 1 to 10, more preferably 1 to 4 carbon atoms. The alkyl group can be linear or branched, and can be substituted. Where a subscripted character is used in this specification, the subscripted character refers to the number of carbon atoms that the specified group can contain. Thus, for example, C 1-4 An alkyl group refers to an alkyl group of 1 to 4 carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, butyl and its isomers (e.g. n-butyl, isobutyl and t-butyl); pentyl and its isomers, hexyl and its isomers, heptyl and its isomers, octyl and its isomers, nonyl and its isomers; decyl and its isomers. C1-C6 alkyl includes all linear, branched or cyclic alkyl groups having 1 to 6 carbon atoms, thus, including methyl, ethyl, n-propyl, isopropyl, butyl and its isomers (e.g. n-butyl, isobutyl and t-butyl); pentyl and its isomers, hexyl and its isomers, cyclopentyl, 2-, 3- or 4-methylcyclopentyl, cyclopentylmethyl and cyclohexyl.

[0045] The term "alkoxy" refers to a group consisting of an oxygen atom bonded to a linear or branched alkyl group, a cycloalkyl group, an aralkyl group or a cycloalkylalkyl group, which can be substituted, either by itself or as part of another substituent. Non-limiting examples of suitable alkoxy groups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy and hexyloxy.

[0046] The term "heterocycloalkyl" refers to a cyclic alkyl group, either by itself or as part of another substituent, comprising at least 2 different elements in the ring (preferably carbon and nitrogen), i.e. a monovalent, saturated or unsaturated hydrocarbon group having 1, 2 or 3 cyclic structures. The cycloalkyl group includes all saturated or partially saturated (containing 1 or 2 double bonds) hydrocarbon groups containing 1 to 3 rings, including monocyclic, bicyclic or polycyclic alkyl groups. The cycloalkyl group can contain more than 3 carbon atoms in the ring, and generally, according to the present application, contains 3 to 15 atoms. Further rings of the polycyclic cycloalkyl group can be condensed, cross-linked and / or bonded via one or more spiro atoms.

[0047] The term "aryl" means a polyunsaturated, aromatic hydrocarbon group typically containing 6 to 10 atoms of which at least one ring is aromatic, formed by one ring (i.e., phenyl) or multiple aromatic rings fused together (e.g., naphthalene or anthracene) or covalently linked. The aromatic ring(s) can optionally contain from 1 to 3 additional rings fused to it (any of cycloalkyl, heterocycloalkyl, or heteroaryl). Aryl is also intended to include partially hydrogenated derivatives of the carbon ring systems enumerated in this specification. Non-limiting examples of aryl groups include phenyl, biphenyl, 5- or 6-tetralinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-azulenyl, 1- or 2-naphthyl, 1-, 2-, or 3-indenyl, 1-, 2-, or 9-anthryl, 1-, 2-, 3-, 4-, or 5-acephenanthryl, 3-, 4-, or 5-acenaphthyl, 1-, 2-, 3-, 4-, or 10-phenanthryl, 1- or 2-alkylidene, 1-, 2-, 3-, or 4-fluorenyl, 4- or 5-indanylyl, 5-, 6-, 7-, or 8-tetrahydronaphthyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl, dibenzo[a,d]cycloheptenyl, and 1-, 2-, 3-, 4-, or 5-pyrenyl. The aryl ring can be substituted with one or more substituents.

[0048] The term "halo" or "halogen" is a generic term for fluorine, chlorine, bromine, or iodine.

[0049] The term "may be substituted" means that at any available bonding site, there can be one or more substituents (e.g., 1 to 4 substituents, such as 1, 2, 3, or 4 substituents, or 1 to 2 substituents). Non-limiting examples of such substituents include alkyl, halo, hydroxyl, carbonyl, nitro, amino, oxime, imine, azide, hydrazine, cyano, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, alkoxy, thiol, alkylthio, carboxyl, carboxylalkyl ester, acylamino, alkyl ester, carbamate, thioamide, urea, sulfonamide, guanidine, and the like.

[0050] Preferred E-64 and / or analogs thereof of the present application can be exemplified by compounds represented by the following formulas (II) to (VII).

[0051] [Chemical Formula 4] As the calpain inhibitor in the present application, for example, dazcapistat, alicapistat, TNCA (Benzo[b]thiophene-2-carboxylic Acid), and the like can be exemplified. Dazcapistat, alicapistat, and TNCA have the structures shown below, respectively.

[0052] [Chemical Formula 5]

[0053] Example Hereinafter, in order to deepen the understanding of the present invention, the present invention will be described in detail with reference to examples. However, it goes without saying that these examples are not intended to limit the scope of the present invention.

[0054] Example 1 MinION-based quantification of honey bee viruses (Bees as the subject) The subjects were 14 groups (bee individual groups No. 1 to 14), including colonies dying from mites and healthy colonies at 13 bee farms in Japan.

[0055] (Sample Preparation) The midgut of honey bees was collected and total RNA was extracted using ISOGEN II (manufactured by Nippon Gene). E. coli Poly(A) polymerase (manufactured by New England BioLabs Japan (NEB)) was allowed to act on 30 μg of RNA to add poly A. TM -dT30 <super>polyA attached RNA was purified with an mRNA purification kit (manufactured by Takara Bio). In addition, excess rRNA was removed with NEB Next rRNA Depletion Kit v2 (Human / Mouse / Rat) with RNA sample purification beads (manufactured by NEB), and a sample RNA containing viral RNA was obtained. A schematic diagram of the steps of sample preparation for MinION analysis is shown in Figure 1 .

[0056] (Method of MinION analysis) For the prepared RNA, a library was prepared using Direct cDNA Sequencing Kit (manufactured by Oxford Nanopore Technologies), Native Barcoding Expansion 1-12 (PCR-free) (manufactured by Oxford Nanopore Technologies), and the RNA converted to cDNA was quantified with a MinION flow cell (manufactured by Oxford Nanopore Technologies: FLO-MIN106D). The sequence information of the detected viruses was compared with the library information of the viruses, and the quantitative values of the amounts of each virus were calculated. The quantitative results recorded in the heat map are shown in Figure 2 .

[0057] As shown in Figure 2 , it was found that Israeli acute paralysis virus (IAPV) and Deformed wing virus (DWV) were mainly present.

[0058] Example 2 Quantification of viruses in honeybees based on RT-PCR (Method of sample preparation and RT-PCR analysis) The midgut of honeybees in honeybee individual groups No. 1 to 14 was collected, and total RNA was extracted using ISOGEN II (manufactured by Nippon Gene). From the total RNA, the viruses contained in the midgut RNA of honeybees were quantified using One Step PrimeScript TM III RT-qPCR Mix (manufactured by Takara Bio) and the probes and primers shown in Table 1. In the quantification, DNA fragments based on the sequences of each RNA virus were used, and according to the relationship between the viral copy number and the CT value of RT-PCR, the copy number of the viruses mixed in the midgut RNA of honeybees was calculated. The results are shown in Figure 3 . The base sequence (DNA) of the positive control virus is shown in Table 2.

[0059] As Figure 3 shown in Table 1, the amount of virus detection in the bee individual groups No. 1, 2, 3 and 5 from the dying population due to mites was high, and in the virus infection groups No. 2, No. 3 and No. 5, mainly IAPV and DWV, the amount of detection of IAPV was more than 10 times that of DWV, which was a very high value.

[0060] Table 1 Table 2 Example 3 RNA-seq analysis of biological factors of bees and quantification of each factor (Sample preparation and RT-PCR analysis method) The midgut of the individuals of the virus infection groups No. 1, 2, 3 and 5 of bees and the individuals of the virus non-infection groups No. 6, 7, 9 and 14 was collected, and total RNA was extracted using ISOGEN II (manufactured by Nippon Gene Co., Ltd.). After purifying mRNA with oligo dT and synthesizing cDNA with random primers, a sequencing library was prepared. Sequencing analysis (DNBSEQ-G400 100 bp paired-end analysis) was performed from the obtained library. Data analysis was performed based on the sequence information of the analysis results, and the amount of mRNA in the midgut of the bees of the bee individual groups No. 1, 2, 3, 5, 6, 7, 9 and 14 was quantified. The number of factors that increased and decreased in gene expression in the virus infection groups No. 1, 2, 3 and 5 of bees compared to the gene expression of the virus non-infection groups No. 6, 7, 9 and 14 was tabulated. The list of expression variation genes is shown in Table 3. The quantification results of the virus response factors and the antimicrobial peptides are shown in Figure 4 , and the quantification results of the intestinal repair factors are shown in Figure 5 . The quantification values of each factor shown in the figures were calculated from the quantification values obtained by RNA-seq analysis.

[0061] As shown in Table 3, in the biological body of the virus-infected bees, a decrease in the expression of immune factors, metabolism-related factors, and an increase in the expression of apoptosis-related factors were observed, and it was known that IAPV or DWV caused a decrease in immune function and metabolic function, cell death, and tissue decomposition by expression variation of these factors, and induced the death of the individual bees. In addition, it was known from the analysis of histochemical analysis including Figure 4 , Figure 5 and Example 5 described later that these viruses allowed bacteria to mix into the body by opening holes in the intestine, and caused a decrease in immune function and metabolic function, cell death, and tissue decomposition by inducing sepsis.

[0062] Table 3

[0063] Example 4 RNAi test of viruses of honeybees (dsRNA adjustment method and RNAi test method) After cloning Israel Acute Paralysis Virus (IAPV) using two sets of primers (IAPV cloning primer 1-F, R (SEQ ID NO: 37, 38) and IAPV cloning primer 2-F, R (SEQ ID NO: 39, 40)) and Deformed Wing Virus (DWV) using one set of primers (DWV cloning primer-F, R (SEQ ID NO: 41, 42)), dsRNAs were synthesized using each virus primer (IAPV T7 primer 1-F, R (SEQ ID NO: 43, 44) and IAPV T7 primer 2-F, R (SEQ ID NO: 45, 46) and DWV T7 primer-F, R (SEQ ID NO: 47, 48) based on the DNA fragment using T7 RiboMAX Express Large Scale RNA Production System (manufactured by Promega) and the dsRNAs were synthesized as IAPV dsRNA (a mixture of IAPV dsRNA 1 and IAPV dsRNA 2) and DWV dsRNA. For GFP, GFP dsRNA was synthesized using a vector (pEGFP-C3) to which GFP was linked and primers (GFP T7 primer-F, R (SEQ ID NO: 49, 50)). Each dsRNA was dissolved in a 50% sucrose solution at a concentration of 100 μg / ml and administered to the honeybees of the group No. 5 infected with the virus. The 50% sucrose solution containing the dsRNA was replaced every day and the number of individuals of the honeybees was measured every day. The control group was administered with a 50% sucrose solution not containing the dsRNA. The sterile in vivo drug evaluation rearing method was used. TM Express Large Scale RNA Production System (manufactured by Promega) and the dsRNAs were synthesized as IAPV dsRNA (a mixture of IAPV dsRNA 1 and IAPV dsRNA 2) and DWV dsRNA. For GFP, GFP dsRNA was synthesized using a vector (pEGFP-C3) to which GFP was linked and primers (GFP T7 primer-F, R (SEQ ID NO: 49, 50)). Each dsRNA was dissolved in a 50% sucrose solution at a concentration of 100 μg / ml and administered to the honeybees of the group No. 5 infected with the virus. The 50% sucrose solution containing the dsRNA was replaced every day and the number of individuals of the honeybees was measured every day. The control group was administered with a 50% sucrose solution not containing the dsRNA. The sterile in vivo drug evaluation rearing method was used.

[0064] (sterile in vivo drug evaluation rearing method) A new sterile in vivo drug evaluation rearing method of honeybees was established. The incubator and the rearing cage were sterilized with 70% ethanol after being washed. 30 to 35 honeybees were put in every 1 beehive and a 50% sucrose solution (sterilely dissolved with sterilized water) and a solution in which pollen was dissolved in a 50% sucrose solution were given. The rearing temperature was managed at 30°C. The 50% sucrose solution was replaced every day and the number of individuals was measured at the time of replacement. The drug was administered after being dissolved in a 50% sucrose solution. At the time point when the survival rate became 50% or less of the initial, total RNA collection, histological analysis of the midgut, etc. were performed on the surviving individuals. Pharmacological analysis was performed from the collected RNA, tissues, blood.

[0065] As Figure 6 As shown, the results of the RNAi test were that the RNAi of IAPV inhibited the individual death of the honeybees and septicemia after the mite infection, but the RNAi of DWV had no effect. From these results, it was known that the pathogen that became the cause of the mass death of the honeybees was IAPV.

[0066] Example 5 Analysis of the intestinal regeneration of the midgut of the honeybee (Sample preparation method and detection method) After the dissection of the midgut of the honeybee, the fixation with 4% paraformaldehyde for 30 minutes, the washing with PBS containing 0.2% Triton X-100 (hereinafter sometimes referred to as PBX) for 20 minutes at room temperature for 3 times, the reaction with 2% BSA / PBX for 1 hour at room temperature, the blocking was performed. Next, the reaction with 0.5% BSA / PBX containing Phospho-Histone H3 (pH3) antibody (manufactured by Cell Signaling Technology) for 8 hours at 4°C. After the washing with PBX for 20 minutes at room temperature for 3 times, the reaction with 0.5% BSA / PBX containing secondary antibody for 8 hours at 4°C. After the washing with PBX for 20 minutes at room temperature for 3 times, the mounting on a glass slide, the counting of the number of pH3 antibody positive cells was performed with a fluorescence microscope. The microscopic images were observed with a confocal laser microscope. In addition, the counting of the number of pH3 antibody positive cells in the midgut of each 1 individual of the 7th day of rearing was performed with the administration of the dsRNA of IAPV, DWV or GFP of Example 4.

[0067] The microscopic images of the midgut of the virus infection group (honeybee individual group No. 5) and the uninfected group are shown in Figure 7 . In addition, the number of pH3 antibody positive cells of the midgut of each group of the 7th day of rearing with the administration of the dsRNA of IAPV, DWV or GFP is shown in Figure 8 . The intestinal regeneration cells were detected by pH3 antibody. Due to the intestinal damage, the intestinal regeneration was observed, and the result meant that the intestine was damaged. As shown in Figure 7 , the intestinal regeneration cells were detected in the virus infection group, and thus the midgut damage was confirmed, and it was presumed that the protease of the infection virus was involved. As shown in Figure 8 , it was confirmed that the midgut damage was inhibited by the dsRNA of IAPV.

[0068] Example 6 Inhibition of the individual death of the honeybee by the administration of E-64 (Experimental method and administration method) E-64 ([ (2S, 3S) -3-carboxyoxirane-2-carbonyl] -L-leucine (4- guanidinobutyl) amide hemihydrate, manufactured by Peptide Institute, Inc.) was dissolved in a 50% sucrose solution at 200 μM, 100 μM, 50 μM, and administered to the bees of the groups No. 2 and No. 5 in which the viral infection was observed. To the control group (none), only 50% sucrose was administered. The solution containing E-64 was replaced every day, and the change in the number of individuals of the bees was measured at the time of replacement. The results are shown in Figure 9 and Figure 10 . The bees were reared using the aseptic in vivo drug evaluation rearing method described above.

[0069] Since the protease of IAPV is a cysteine protease called 3C-like protease, as shown in Figure 9 and Figure 10 , the cysteine protease inhibitor E-64 was administered to the dual system from the population that was dying due to the mites, and as a result, it was found that the death of the bees caused by the mite infection was inhibited, and the proliferation of the pathogenic virus and the cause of the intestinal damage were due to the protease of the virus. According to these results, E-64 can be effectively used as an antiviral agent for inhibiting the mass death of bees by inhibiting the protease of IAPV.

[0070] Example 7 BPB administration test (Method of experiment) Virus-infected bees and uninfected bees were administered with 50% sucrose and reared for 10 days. During this rearing period, bromophenol blue (BPB) (Fuji Photo Film and Otsuka Pharmaceutical) was dissolved in a 50% sucrose solution at 10 mg / ml, and administered to the virus-infected bees and uninfected bees for 24 hours. The number of individuals of the bees before rearing was taken as 100%, and when the survival rate of the virus-infected group decreased to about 50%, BPB was administered to the bees of each group. After the administration of BPB, the blood of the bees (more than 30 individuals) was collected, and the BPB concentration in the blood was measured using a microspectrophotometer NanoDrop One (ThermoFisher Scientific) at an absorbance of 500 nm.

[0071] When the blood was measured at an absorbance of 500 nm, it was possible to detect substances other than BPB, so the blood at the time of sucrose administration was also collected as a control to perform the measurement. As a result, the BPB concentration in the blood increased in the virus-infected group by the administration of BPB compared to the sucrose administration, and in contrast, no increase due to the administration of BPB was observed in the uninfected group Figure 11 Since the intestines of uninfected bees are normal, BPB is not ingested and no BPB is detected in the blood. However, in bees with intestinal damage caused by viral infection, BPB is ingested and detected in the blood. The results of this example confirm that the intestinal damage occurs in bees infected with the virus.

[0072] Example 8 Determination of viable bacteria count in honey bee blood (Experimental Method) Blood was collected from infected bees (100 or more individuals) and uninfected bees (100 or more individuals) and the number of viable bacteria in the blood was measured using MRS culture medium (Biokar Diagnostics). The collected blood was spread onto the MRS culture medium and the number of viable bacteria was measured 24 hours later.

[0073] Normally, there are no bacteria in bee blood. Blood collection was performed after thorough disinfection, but since bees were raised outdoors, a small amount of bacteria was inevitably detected in the blood of bees that were not infected with the virus. However, the blood of bees infected with the virus contained about 10 times more bacteria than the blood of bees that were not infected with the virus. Figure 12 This result suggests that the bees' intestines were damaged by the virus, allowing intestinal bacteria to enter the bloodstream, where bacteria were detected. It is believed that the detected bacteria non-specifically degraded bee-derived mRNA, causing the bees' death.

[0074] Example 9 Confirmation experiment of recovery of intestinal damage in honey bees by E-64 administration (Experimental Method) Virus-infected honey bees were divided into a control group (none) administered with 50% sucrose and a group (E-64) administered with a 100μM solution of E-64 (Peptide Research Institute) dissolved in 50% sucrose. During this period, bromophenol blue (BPB) (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in 50% sucrose at 10mg / ml was administered to each group for 24 hours. The number of bees before feeding was taken as 100%. When the survival rate of the control group dropped to approximately 50%, BPB was administered to each group. After BPB administration, blood was collected from at least 30 bees, and blood BPB concentrations were measured using a NanoDropOne microspectrophotometer (ThermoFisher Scientific) at an absorbance of 500nm.

[0075] As a result, the blood concentration of BPB increased by administering BPB compared to the control group administered sucrose, and, on the contrary, the blood concentration of BPB decreased to the same level as the value after the administration of BPB in the virus-uninfected group of Example 7 (Fig. 6) by administering E-64. From these results, it was found that the intestinal damage of the honeybee caused by the virus was recovered to the normal level by administering E-64. Figure 11 Figure 13

[0076] Example 10 Protein expression of IAPV protease (Experimental method) ​​RNA was extracted from the midgut of the virus-infected honeybee using ISOGEN II (Nippon Gene), and reverse transcription was performed on the RNA using PrimeScript RT reagent Kit (Takara) to obtain midgut-derived cDNA. Next, the midgut-derived cDNA was subjected to PCR using IAPV protease primer-F1 (SEQ ID NO: 59) and IAPV protease primer-R (SEQ ID NO: 60) with KOD FX Neo (TOYOBO) (30 cycles of 98°C for 30 seconds, 60°C for 30 seconds, and 68°C for 2 minutes), and a cDNA fragment of IAPV protease (composed of the base sequence of SEQ ID NO: 57 encoding the amino acid sequence of SEQ ID NO: 58) was obtained. The cDNA fragment of IAPV protease was subjected to restriction enzyme treatment with BamHI and HindIII, and the expression vector pET21a (Merck) was also subjected to restriction enzyme treatment with BamHI and HindIII. The cDNA fragment of IAPV protease and the DNA fragment of pET21a that had been subjected to restriction enzyme treatment were subjected to a ligation reaction at 16°C for 1 hour using Ligation High Ver.2 (TOYOBO) to ligate the vector and the cDNA fragment, and transformed into Escherichia coli. Colony PCR was performed to obtain an expression vector having the cDNA fragment of IAPV protease. The expression vector of IAPV protease was transformed into Escherichia coli Rosetta-gamiB (Merck), pre-cultured in 50 ml of LB medium containing ampicillin, and then inoculated into 500 ml of LB medium containing ampicillin, after which the culture was incubated until the absorbance at 600 nm reached 0.5, isopropyl β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM, and the culture was incubated at 15°C for 24 hours. The culture solution was centrifuged (8000 rpm, 10 minutes, 4°C), and after recovering the Escherichia coli bacteria, the bacteria were dissolved in 10 ml of sonication buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM DTT) and disrupted by sonication. The disrupted solution was centrifuged (12000 rpm, 10 minutes, 4°C) to obtain the supernatant of the bacteria disruption solution. Purification of IAPV protease was performed from the supernatant to obtain a purified protein solution.Purification was performed by cation exchange chromatography using a buffer containing 20 mM Tris-HCl (pH 7.5) and 1 M NaCl and HiPrep SP XL 16 / 10 (Cytiva) and gel filtration chromatography using a buffer containing 0.15 M NaCl and 50 mM phosphate buffer (pH 7.0) and HiLoad 16 / 600 Superdex 200 (Cytiva) and 20 mM Tris-HCl (pH 7.5).

[0077] Example 11 Purified enzyme-based IAPV protease activity assay (Method) As the substrate for the IAPV protease activity assay, Dnp-PNDIVDVTMQK(Nma)-NH2 or Nma-TMQMWKDQVAK(Dnp)-NH2, which is a synthetic peptide and a fluorescent substrate conjugate, was used. The activity assay substrate emits fluorescence when it is cleaved by the protease. The activity assay substrate was adjusted to 20 μM with an assay buffer (50 mM citrate buffer, pH 6.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT). The purified protein solution of the IAPV protease obtained in Example 10 was used as an enzyme solution. The assay buffer containing the activity assay substrate 50 μl and the enzyme solution 50 μl were mixed in the presence or absence of each of the inhibitors shown in Table 4, and the fluorescence intensity after 30 minutes of reaction at 37°C in a 96-well black plate was measured using a fluorescence plate reader TECAN (Tekan Japan) at an excitation wavelength of 340 nm and a fluorescence wavelength of 440 nm. Each of the inhibitors was adjusted to a final concentration of 1 mM, and the enzyme reaction was performed after 1 hour of pre-incubation at 37°C.

[0078] The fluorescence intensity measured in the absence of the inhibitors (Table 4, "none") was taken as 100% of the relative activity, and the relative activity of the IAPV protease calculated from the fluorescence intensity measured in the presence of each of the inhibitors is shown in Table 4. As shown in Table 4, each of E-64, E-64c, E-64d, CA074 methyl ester (CA-074Me), JPM-OEt, and CA074, which are E-64 analogs, and dazcapistat, alicapistat, and TNCA, which are calpain inhibitors, inhibited the IAPV protease activity. These results support the mechanism that E-64 and its analogs and calpain inhibitors act on the individual death of honeybees via IAPV protease inhibition.

[0079] Table 4

[0080] E-64, E-64c and E-64d were manufactured by Peptide Institute, CA074 methyl ester (CA-074Me), JPM-OEt, CA074, dazcapistat and alicapistat were manufactured by Med Chem Express, and TNCA was manufactured by BLD pharmtec.

[0081] Example 12 Protein expression and protease activity assay of IAPV protease (Experimental methods) The cDNA derived from the midgut of the virus-infected honeybee obtained by the same method as in Example 10 was subjected to a PCR reaction (98°C for 30 seconds, 60°C for 30 seconds, 68°C for 2 minutes, 30 cycles) using IAPV protease primer-F2 (SEQ ID NO: 61) and IAPV protease primer-R (SEQ ID NO: 60) with KOD FX Neo (TOYOBO), to obtain a cDNA fragment of IAPV protease (consisting of the base sequence of SEQ ID NO: 57 encoding the amino acid sequence of SEQ ID NO: 58). The cDNA fragment of IAPV protease was subjected to restriction enzyme treatment with BamHI and HindIII, and the expression vector pMAL-c6T (New England Biolabs) was also subjected to restriction enzyme treatment with BamHI and HindIII. The cDNA fragment of IAPV protease subjected to the restriction enzyme treatment and the DNA fragment of pMAL-c6T were subjected to a ligation reaction at 16°C for 1 hour using Ligation High Ver.2 (TOYOBO), to ligate the vector and the cDNA fragment, and transformed into E. coli. Colony PCR was performed to obtain an expression vector having the cDNA fragment of IAPV protease. The expression vector of IAPV protease was transformed into E. coli Rosetta-gamiB (Merck), pre-cultured in 50 ml of LB medium containing ampicillin, and then inoculated into 500 ml of LB medium containing ampicillin, and cultured until the absorbance at 600 nm reached 0.5, to which IPTG was added to a final concentration of 1 mM, and cultured at 15°C for 24 hours. The culture solution was centrifuged (8000 rpm, 10 minutes, 4°C), and after recovering the E. coli cells, the cells were dissolved in 10 ml of sonication buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM DTT), and the cells were disrupted by sonication. The disrupted solution was centrifuged (12000 rpm, 10 minutes, 4°C) to obtain the supernatant of the cell disrupted solution. As a result of protein analysis based on SDS-PAGE, the expression fusion protein of Maltose Binding Protein (MBP)-IAPV protease expressed from pMAL-c6T reached about 40 to 50% of the total protein.

[0082] Next, the crude enzyme solution of the expression fusion protein of IAPV protease was used as an enzyme solution, and the IAPV protease activity was measured by the same method as in Example 11 in the presence or absence of E-64.

[0083] The fluorescence intensity measured in the absence of E-64 was reduced by the addition of E-64. When the fluorescence intensity measured in the absence of E-64 was taken as 100% of the relative activity, the relative activity of the IAPV protease calculated from the fluorescence intensity measured in the presence of E-64 was 9% (Example 2). Figure 14 Thus, using the IAPV protease synthesized in the E. coli protein synthesis system, the IAPV protease inhibitory activity of E-64 was confirmed.

[0084] Example 13 Analysis of the effect of E-64c and E-64d on the individual death of honeybees (Methods) E-64c (manufactured by Peptide Institute) or E-64d (manufactured by Peptide Institute) was dissolved in a 50% sucrose solution at 100 μM and administered to the honeybees of the group No. 2 in which viral infection was observed. To the control group (none), only 50% sucrose was administered. The solution containing E-64c or E-64d was replaced every day, and the change in the number of individuals of the honeybees at that time was measured. The aforementioned aseptic in vivo drug evaluation feeding method was used for the feeding.

[0085] As a result, the survival rate of the E-64c administration group and the E-64d administration group was higher than that of the control group. Thus, it was confirmed that E-64c and E-64d can inhibit the disease of honeybees caused by mite infection, which can lead to the death of individuals of the honeybees (Example 3). Figure 15 Based on these results, E-64c and E-64d can inhibit the activity of IAPV protease and can be effectively used as a prophylactic and / or therapeutic agent against the disease of honeybees, which is a disease that can lead to the death of individuals of the honeybees caused by damage to the intestinal tract of the honeybees.

[0086] Industrial applicability According to the present application, by screening of compounds with the inhibition of the protease of the virus as an index, an important technology for developing an antiviral agent that contributes to the inhibition of the mass death of honeybees can be provided, and the development of an agent that is effective for the preservation of honeybees and has high safety can be expected.< / super>

Claims

1. A method for screening a preventive and / or therapeutic drug for a honey bee disease, wherein the method uses the inhibitory activity against a virus-derived protease as an indicator, wherein the honey bee disease is caused by damage to the intestinal tract of honey bees and can lead to individual death.

2. The screening method according to claim 1, wherein Honey bees are bees infected with a virus mediated by mites.

3. The screening method according to claim 1 or 2, wherein The protease is a cysteine ​​protease.

4. The screening method according to claim 1 or 2, wherein The virus is either Israeli acute paralysis virus (IAPV) or deformed wing virus (DWV).

5. The screening method according to claim 1, further using individual death of honey bees and / or intestinal regeneration of honey bees as indicators.

6. A preventive and / or therapeutic agent for a honey bee disease, comprising a substance that inhibits the activity of a virus-derived protease, wherein the honey bee disease is caused by damage to the honey bee's intestines and can lead to the death of individual honey bees.

7. The preventive and / or therapeutic drug according to claim 6, wherein The substance that inhibits the activity of virus-derived proteases is E-64 and / or its analogs having the following general formula (I): [Chemical Formula 1] In the formula, R1 represents OH, an alkoxy group which may be substituted, or an amino group which may be substituted; R2 represents an alkyl group which may be substituted, or a heterocycloalkyl group which may be substituted and forms a ring via the nitrogen to which it is bonded.

8. The preventive and / or therapeutic drug according to claim 6, wherein Substances that inhibit the activity of virus-derived proteases are calpain inhibitors.

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