Use of capsaicin in the preparation of feed additives and / or drugs for the prevention and treatment of viral infections in fish

Abstract

This invention belongs to the field of aquaculture technology and relates to a novel use of capsaicin, particularly its use in the preparation of drugs and / or feed additives for the prevention and treatment of fish viral infections. The core of this invention lies in the significant protective effect of capsaicin against carp spring viremia virus (SVCV), grass carp reovirus (GCRV), and carp herpesvirus type 2 (CyHV-2) infections, effectively inhibiting the transcription of SVCV, GCRV, and CyHV-2 viral genes and the expression of viral proteins. Feed additives and / or drugs containing capsaicin can be used for the prevention and treatment of fish viral diseases, offering advantages such as high protective efficiency and environmental friendliness.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and to a new use of capsaicin, particularly to the use of capsaicin in the preparation of feed additives and / or drugs for the prevention and treatment of viral infections in fish. Background Technology

[0002] Spring viraemia of carp virus (SVCV) is a single-stranded, negative-sense RNA virus belonging to the family Rhabdoviridae and the genus Vesicularvirus, primarily infecting cyprinid fish. The SVCV genome is approximately 11 kb in length, encoding five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase (L). It exhibits high pathogenicity and mortality. SVCV typically causes large-scale outbreaks in spring when water temperatures are between 10-25°C, leading to systemic hemorrhage, organ inflammation, and acute death in infected fish, resulting in significant economic losses to aquaculture. Currently, there are no commercially available vaccines or specific drugs for SVCV. The main control measures rely on virus detection, isolation and culling, and environmental disinfection, but these have limited effectiveness and are insufficient to handle sudden outbreaks.

[0003] Grass carp reovirus (GCRV) belongs to the genus *Aquaticreovirus* of the family Reoviridae. It is a double-stranded RNA virus that causes severe hemorrhagic disease in grass carp. Based on nucleotide clustering of VP6, GCRV is mainly classified into three genotypes: GCRV-I, GCRV-II, and GCRV-III. Among them, GCRV-II is the dominant circulating strain in major grass carp farming areas in my country (Central China, South China, etc.). Currently, research on the prevention and control of grass carp hemorrhagic disease mainly focuses on vaccine development, selection of disease-resistant strains, application of immune activators, and the construction of ecologically healthy aquaculture models. However, the development of drugs to combat grass carp hemorrhagic disease has been hampered.

[0004] Crucian carp, a major freshwater economic fish in my country, is an important source of high-quality animal protein for residents. However, disease has long been a key bottleneck restricting the healthy development of crucian carp aquaculture. Among them, hematopoietic organ necrosis disease caused by Cyprinidherpesvirus 2 (CyHV-2) is a major disease of farmed crucian carp, characterized by rapid onset, high infectivity, and high mortality, seriously jeopardizing the industry's development. Currently, although significant progress has been made in vaccine development and disease-resistant breeding for crucian carp hematopoietic organ necrosis disease, there are still no effective drugs for it. Summary of the Invention

[0005] The EPC cells used in this invention are carp epithelial cell lines, purchased from the China Center for Type Culture Collection (Wuhan University), catalog number GDC0174, and are available through commercial channels. The CIK cells are grass carp kidney cell lines, purchased from the China Center for Type Culture Collection (Wuhan University), catalog number GDC0086, and are available through commercial channels. The GiCB cells are crucian carp brain tissue cell lines (Ma, J., Ma, J., Jiang, N., Lapatra, SE, Jin, L., & Xu, J., et al. (2015). Establishment of a novel and highly permissive cell line for the efficient replication of cyprinid herpesvirus 2 (cyhv-2)), originally donated by Researcher Zeng Lingbing of the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. The relevant germplasm resources are available to the public from the applicant and may only be used for replicating the experiments of this invention and may not be used for other purposes.

[0006] The purpose of this invention is to provide a novel use for capsaicin, specifically relating to the use of capsaicin-containing feed additives and / or drugs for the prevention and control of viral diseases in fish, with advantages of high protective efficiency and environmental friendliness. To achieve the above-mentioned objective, this invention provides the following technical solution:

[0007] Use of capsaicin in the preparation of feed additives and / or drugs for the prevention and treatment of viral infections in fish.

[0008] Preferably, the fish virus is one or more of carp spring viremia virus, grass carp reovirus, and carp herpesvirus type 2.

[0009] Preferably, the feed additive and / or drug exerts its preventive and therapeutic effect by inhibiting the gene transcription and protein expression of the aforementioned fish virus.

[0010] More preferably, when the fish virus is a carp spring viremia virus, the genes are the n gene and the p gene, and their nucleotide sequences are as shown in SEQ ID NO. 1-2, respectively:

[0011] n-gene:

[0012]

[0013] p gene:

[0014] ATGTCTCTACATTCGAAATTGTCAGAAAGTCTTAAAGCTTATGCTAATTTAGAGAAGACGGTTAAAGAAATAGAAGAACAGGTATCGACTATGGAAGAGCCCATTCCAAAGACAGTAAAATATGTTACCTTTGAGGAGGACTCATCTGAGGGGGACTGGGAATCAGATTCGGGGGATGATGATGAGGATTCAATCGATGAATCTGTGATCCCCGATTACCTCAGAGAAAGTAGCAGCATCACAGTGGATGAAGATGAAGAAGATCAGAAAGCAGATAAAGAAGAATATCTTCCGACAGTCAGTTGGGAAGAAGAATCCACAGGAATAGATCTAGGGTTTGGACCTGGGATAGTGATGCCGTCTGTGTCGAACCATGAGGGAGGTACATATGTTCGTTATAACGGCCTCGGTAACGTAGACCCAAATTATAAGAACTTGATTTCTAAAATGATGAGAAGCCTGATTGGGCAAATTGGAAACAAGTACGGATATGACATTGATCTATTTGATTATCAGGGAGATTTCCTGGAGGTGTTCTTACCCCACAAACCAAGCAAGGAGGATGTCCGACCTGGCATACGTGCAGAGAAAAAACACGAGGAAAGCCCCAGTAAGCAGGTCTCCAAACCTGAGAAAAAGGAAAAAACCATCCAGAAGACGGGGGATGAATGCGGAAGATTTCCTATGGATAAAGAGGCCAAGAGAAGAGAACCAGAGGGATTATGGGAGATCATGAAGGTCTTGTCCGTCCAGTTTGATCCCTGGAAAGAAGATGAGCCTCCACTGAACATGACCATCCGAGATCTATTCATAAGCGAATCTGAGTTTTGTCTGCACTGTAATCACAGCCAAACAGAGCGAGAAATGGCCTTGGTTGGGATCAAACTGAGGAGATTGTACAATAAGTTGTATCAAAAATATAGGTTGTAA(SEQ ID NO.2).

[0015] More preferably, when the fish virus is grass carp reovirus, the genes are s6 and s9 genes, and their nucleotide sequences are as shown in SEQ ID NO. 3-4 respectively:

[0016] s6 gene:

[0017]

[0018] s9 gene:

[0019]

[0020] More preferably, when the fish virus is carp herpesvirus type 2, the genes are orf72 and orf80 genes, and their nucleotide sequences are as shown in SEQ ID NO. 5-6, respectively:

[0021] orf47 gene:

[0022]

[0023] orf80 gene:

[0024]

[0025] More preferably, when the fish virus is carp spring viremia virus, the proteins are N protein and P protein, and their amino acid sequences are as shown in SEQ ID NO.7-8 respectively:

[0026] N protein:

[0027] MSVIRIKTNATVAAVLPANEDQADYPSTFFEGGNEIRLYVNREEKLDVLRQYVYMGLVEKNCKIQHVNAYLYAVLKXERELLEADWNSFGHKIGIQGDKIGPFN LVRVEDIPDGLPDGKLNTEVSAEDDAWLPLFLLGLYRVGRASETAYRTLLMESLIKQCKAIKSDWVSPVTATHKYFDVWGNDGNYLKIVACVDMFYNHFKKSIK ATFRWGTIVSRFKDCAALATLGHVVKITGLTIEEVFTWVLQTEVADELVKMMKPGQEIDNSTSYMPYLIDMGISAKSPYSTIKNPSFHFWGQLVAALCRSKRAL NARQPDEIDSMSISNASLLMAYALGSSPDIEQQFSTGDTYRKPPKETSYLVSEEPKNRSVVEWIAWYSDVDNKPTDDMLMMAKRVAGTISSGPRDNSVGKWI(SEQ ID NO.7);

[0028] P protein:

[0029] MSLHSKLSESLKAYANLEKTVKEIEEQVSTMEEPIPKTVKYVTFEEDSSEGDWESDSGDDDEDSIDESVIPDYLRESSSITVDEDEEDQKADKEEYLPTVSWEEESTGIDLGFGPGIVMPSVSNHEGGTYVRYNGLGNVDPNYKNLISKMMRSL IGQIGNKYGYDIDLFDYQGDFLEVFLPHKPSKEDVRPGIRAEKKHEESPSKQVSKPEKKEKTIQKTGDECGRFPMDKEAKRREPEGLWEIMKVLSVQFDPWKEDEPPLNMTIRDLFISESEFCLHCNHSQTEREMALVGIKLRRLYNKLY(SEQ ID NO.8).

[0030] More preferably, when the fish virus is grass carp reovirus, the protein is VP7 protein, and its amino acid sequence is as follows: MPLHMIPQVAHAMVRAAAAGRLTLYTKTKTETTNFDHAEYVTCGRYTICAFCLTTLAPHANVKTIQDSHACSRQPNEAIRSLVEVSDKAQIALVGSRTVDYHELDVKAGFVAPTADETVVPSKDIVELPFRTCDLDDSSATACVRNHCQAGHDGVTHLPILSGDFKLPNEHPTKPLDDTHPHDKVLTRCPKTGLLLVHDTHAHATAVVATAATRAILMHDLLTSANVDDGHQARSACYGPTFSNLTFACHSTCASDMAHFDCGQIVGLDLH (SEQ ID NO. 9).

[0031] More preferably, when the fish virus is carp herpesvirus type 2, the protein is ORF47 protein and ORF80 protein, and their amino acid sequences are as shown in SEQ ID NO. 10-11 respectively:

[0032] ORF47 protein:

[0033] MLERVRDPYWREAGVFIKDDVEYASNCLMVSFPAEALEIHLYSGGFGRLWRADLAPNGITSFYGYDKYFHRQVCSRYMLEDPDALLVKLETCKKFLLCVLMGGPFVHPSFTYLNMCSSCLMETEHGVEFMEIRLTDVICGHYCFVHPAHVSPPELGEQEKRLDSMDGAQAASTVHGGVSLMADDAMEDLQAGRKNVLDIKSAREAQSAKKADNVEQHAFRTQNMNVLIECARAANKIRKTLARARDLKDQLGKLRASLKEQEAPKYMHEVPLYKWYLAYASLIDLSRLKRDFIVRPSRINVHETLPDSHLYTLRGSRVTVKRAHEYLGYATATDPALSAGVIKQAGIMKNVSTRSLRVSVYNEYLNTIVPPIVKLNLNGYIVPRYGFFKYYESMQFSPGQLTQHRHHLNANIDLKDILGCTFEQGRKLIGANTLGWQYHWLYANLEAYRIMHKLAGHRVGGNPLGISTRSTSLAKYFLVFGITHVALKLKHVFVIEPIQNLYRLFLCWERDVDDDEDDTSTITTVHTEAASESASSVHSSSTTTTHDDDAPSASQIHYTQAAHSTTTAAATTASSAAAVLPEEPD(SEQ ID NO.10);

[0034] ORF80 protein:

[0035] MSTVCNQSTSYLSALPRVSVEPSREVGITKSLSRLYPVALKSSKYSRQQQRVCIKLDTDESQQHDLSERLAKSLQGSIWTKPQLRRHLLQQLPVVCRDRGLDLVESGAQALMNAGPFTVHCVRQALIGSQFFLRLGSLIDQCVKHEFEYRKGPIVQTLYATYGWKPLASGVLVSS RRARGHVCNHCRNVCDVQGVSAHGTELDAVATDSNGGLVLLEIKTHSGSTVTKTLLNRYKTQTWLGELMFRNTYGLCSWTKLHSYIVFVDPSRYTVDSVIQVPSVPKRIHPRLFSAFPSLQTLCFVRNNILAKKKRAPQISKTKTTDPLKISKKRQISKSKTTTTATAQDE(SEQ ID NO.11).

[0036] Preferably, the concentration of capsaicin in the feed additive and / or drug is 0.1-1 mol / L.

[0037] More preferably, the feed additive also includes feed-grade acceptable excipients.

[0038] It should be noted that the feed additives described in this invention can be supplemented with different types of excipients to promote fish growth and enhance their immunity. These excipients include, but are not limited to, the following types: vitamins, minerals, fat sources, and / or carbohydrate sources. Further, the vitamins are selected from vitamin A, vitamin B1, vitamin B2, vitamin C, vitamin D, and / or vitamin E; the minerals are selected from calcium dihydrogen phosphate, calcium carbonate, magnesium sulfate, ferrous sulfate, zinc sulfate, copper sulfate, manganese sulfate, potassium iodate, sodium selenite, and / or cobalt chloride; the fat sources are selected from fish oil and / or vegetable oil; and the carbohydrate sources are selected from starch and / or cellulose.

[0039] More preferably, the drug further includes pharmaceutically acceptable excipients.

[0040] It should be noted that the drugs described in this invention can be prepared into suitable drug types by adding different types of pharmaceutically acceptable excipients. These drug types include, but are not limited to, the following: oral tablets (including but not limited to coated tablets), oral granules, oral powders, and injections (including but not limited to lyophilized powders for injection or emulsions for injection). These pharmaceutically acceptable excipients include, but are not limited to, diluents, wetting agents, binders, lubricants, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, preservatives, local analgesics, pH adjusters, isotonic and / or isotonic regulators, etc. Further, the diluent is selected from starch, sucrose, and / or cellulose, etc.; the wetting agent is selected from water and / or ethanol, etc.; the binder is selected from starch paste, sugar, cellulose, and / or povidone, etc.; the lubricant is selected from talc, magnesium stearate, magnesium dodecyl sulfate, and / or micronized silica, etc.; the solvent is selected from water, ethanol, glycerol, propylene glycol, fatty oil, and / or ethyl acetate, etc.; the emulsifier is selected from glycerol fatty acid esters, higher fatty acid salts, sulfates, phospholipids, gelatin, pectin, agar, sodium alginate, and / or bentonite, etc.; the antioxidant is selected from sulfites, metabisulfites, and / or ascorbic acid, etc.; and the preservative is selected from parabens, sodium benzoate, quaternary ammonium compounds, alcohols, and / or phenols, etc.

[0041] More preferably, the drug dosage form is an oral dosage form or an injectable dosage form.

[0042] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0043] This invention provides the use of capsaicin in the preparation of drugs and / or feed additives for the prevention and treatment of viral infections in fish. It has been verified that capsaicin can significantly inhibit the transcription of SVCV, GCRV, and CyHV-2 viral genes and the expression of viral proteins. Feed additives and / or drugs containing capsaicin can be used for the prevention and treatment of viral diseases in fish, and have the advantages of high protective efficiency and environmental friendliness. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 This is a comparison of the protective effects of capsaicin on SVCV virus-infected cells.

[0046] Figure 2 This is a comparison of the protective effects of capsaicin on GCRV virus-infected cells.

[0047] Figure 3 This is a comparison of the protective effects of capsaicin on CyHV-2 virus-infected cells.

[0048] Figure 4 This is a graph showing the quantitative analysis of the transcriptional level of the n gene in SVCV virus.

[0049] Figure 5 This is a graph showing the quantitative analysis of the p gene transcription level in SVCV virus.

[0050] Figure 6 This is a graph showing the quantitative analysis of the transcriptional level of the s6 gene in GCRV virus.

[0051] Figure 7 This is a graph showing the quantitative analysis of the transcriptional level of the s9 gene in GCRV virus.

[0052] Figure 8 This is a graph showing the quantitative analysis of the transcriptional level of the orf47 gene in CyHV-2 virus.

[0053] Figure 9 This is a graph showing the quantitative analysis of the transcriptional level of the orf80 gene in CyHV-2 virus.

[0054] Figure 10 This is a Western blotting image showing the expression of N and P proteins in SVCV virus.

[0055] Figure 11 This is a Western blot image showing the expression of the VP7 protein in GCRV virus.

[0056] Figure 12 This is a Western blot image showing the expression of ORF47 and ORF80 proteins in CyHV-2 virus.

[0057] Figure 13 This is a schematic diagram comparing the survival of zebrafish infected with SVCV within 7 days with capsaicin.

[0058] Figure 14 This is a schematic diagram comparing the survival of grass carp infected with GCRV within 15 days after the application of capsaicin.

[0059] Figure 15 This is a schematic diagram comparing the survival of crucian carp infected with CyHV-2 within 15 days after the application of capsaicin. Detailed Implementation

[0060] The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0061] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of embodiments. Furthermore, in the description of this application, the term "comprising" means "including but not limited to". Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single digits within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0062] Capsaicin is a natural alkaloid with the molecular formula C2. 18 H 27 NO3, with a molecular weight of 293.4, has the following chemical structural formula: Capsaicin is the main pungent component of chili peppers. It is primarily found in the placenta and seeds of chili peppers. By binding to TRPV1 receptors in human nerve endings, it mimics the burning sensation of high temperatures, triggering the transmission of pain and heat signals. This property makes capsaicin widely used, such as in medicine to relieve chronic pain like neuralgia and arthritis. In recent years, capsaicin has been found to possess antioxidant, anti-inflammatory, and potential anti-cancer activities; appropriate intake can promote metabolism.

[0063] This invention provides the use of capsaicin in the preparation of feed additives and / or drugs for the prevention and treatment of viral infections in fish.

[0064] Specifically, the fish virus is one or more of the following: carp spring viremia virus, grass carp reovirus, and carp herpesvirus type 2.

[0065] Specifically, feed additives and / or drugs exert their preventive and therapeutic effects by inhibiting the gene transcription and protein expression of the aforementioned fish viruses.

[0066] More specifically, when the above-mentioned fish virus is carp spring viremia virus, the above-mentioned genes are the n gene and the p gene, and their nucleotide sequences are shown in SEQ ID NO.1-2, respectively.

[0067] More specifically, when the above-mentioned fish virus is grass carp reovirus, the above-mentioned genes are s6 gene and s9 gene, and their nucleotide sequences are shown in SEQ ID NO.3-4, respectively.

[0068] More specifically, when the above-mentioned fish virus is carp herpesvirus type 2, the above-mentioned genes are orf72 gene and orf80 gene, and their nucleotide sequences are shown in SEQ ID NO.5-6 respectively.

[0069] More specifically, when the above-mentioned fish virus is carp spring viremia virus, the above-mentioned proteins are N protein and P protein, and their amino acid sequences are shown in SEQ ID NO.7-8, respectively.

[0070] More specifically, when the above-mentioned fish virus is grass carp reovirus, the above-mentioned protein is VP7 protein, and its amino acid sequence is shown in SEQ ID NO.9.

[0071] More specifically, when the above-mentioned fish virus is carp herpesvirus type 2, and the above-mentioned proteins are ORF47 protein and ORF80 protein, their amino acid sequences are shown in SEQ ID NO.10-11, respectively.

[0072] Specifically, the concentration of capsaicin in feed additives and / or drugs is 0.1-1 mol / L.

[0073] More specifically, the aforementioned feed additives also include feed-grade acceptable excipients.

[0074] More specifically, the aforementioned drugs also include pharmaceutically acceptable excipients.

[0075] More specifically, the above-mentioned drug dosage forms are oral dosage forms or injectable dosage forms.

[0076] The following specific embodiments further illustrate the use of capsaicin of the present invention in the preparation of feed additives and / or drugs for preventing and treating viral infections in fish. This section further illustrates the content of the present invention in conjunction with specific embodiments, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0077] In the following examples, the Multiplicity of Infection (MOI) represents the ratio of the number of viruses capable of infecting cells to the total number of cells in a system. The specific calculation formula is as follows:

[0078]

[0079] Among them, virus titer (TU / mL) refers to the number of infectious virus particles contained in each milliliter of solution, virus volume (mL) refers to the volume of virus solution added, and total cell count refers to the total number of target cells used for infection.

[0080] In the following examples, the upstream and downstream primer nucleotide sequences for qPCR detection of the transcriptional levels of the n and p genes in SVCV virus, the s6 and s9 genes in GCRV virus, and the orf47 and orf80 genes in CyHV-2 virus are shown in SEQ ID NO. 12-23:

[0081] SVCV-n-FP:TGAGTGCTGAGGACGAT (SEQ ID NO. 12);

[0082] SVCV-n-RP:TTTGTGAGTGCCGTTA (SEQ ID NO. 13);

[0083] SVCV-p-FP:TTGGACCTGGGATAGTGA (SEQ ID NO. 14);

[0084] SVCV-p-RP: CTTGCTTGGTTTGTGGG (SEQ ID NO. 15);

[0085] GCRV-s6-FP:GTGTTGACCCTGGATGTGAG (SEQ ID NO. 16);

[0086] GCRV-s6-RP:GTTAGCAGCGGTAGTGACTTG (SEQ ID NO. 17);

[0087] GCRV-s9-FP:GCCGCTCGTGATTTGTTA (SEQ ID NO. 18);

[0088] GCRV-s9-RP:GGGTAGGTGTCGGGTAGTTC (SEQ ID NO. 19);

[0089] CyHV2-orf47-FP:TCCCTCGTTCACCTATCTC (SEQ ID NO. 20);

[0090] CyHV2-orf47-RP:GAATCCAACCGCTTCTCC (SEQ ID NO. 21);

[0091] CyHV2-orf80-FP:CTGGTGTACTGGTCAGCTCC (SEQ ID NO. 22);

[0092] CyHV2-orf80-RP:GGGTCTTTGTGACGGTGGAA (SEQ ID NO. 23).

[0093] Example 1: Effects of capsaicin on SVCV, GCRV, and CyHV-2-infected cells

[0094] Antiviral assays were performed in 24-well cell culture plates. EPC cells were used to detect SVCV cytopathic effects, CIK cells were used to detect GCRV cytopathic effects, and GiCB cells were used to detect CyHV-2 cytopathic effects. Cells in good growth condition were passaged and seeded into 24-well plates and cultured overnight at 28°C in a 5% CO2 incubator with M199 medium (Thermo Fisher Scientific Inc., Catalog No. 31100035) containing 1% penicillin-streptomycin solution (100X penicillin-streptomycin solution, Thermo Fisher Scientific Inc., Catalog No. 15070063) and 10% fetal bovine serum (10099141C, Thermo Fisher Scientific Inc., Catalog No. 10099141C). SVCV (MOI 1) was seeded into EPC cells, GCRV (MOI 1) into CIK cells, and CyHV-2 (MOI 1) into GiCB cells, with 1 mol / L capsaicin added for treatment. After seeding, cells were cultured at 28°C for 72 h. Cells were then fixed with 4% paraformaldehyde for 1 h and stained overnight with 1% crystal violet. The next day, the stained culture plates were photographed to observe the cytopathic effect.

[0095] The protective effects of capsaicin against cells infected with SVCV virus, GCRV virus, and CyHV-2 virus are as follows: Figure 1-3 As shown in the figure, "Control" represents the control group, "Capsaicin" represents the capsaicin-treated group, "Null" represents uninfected cells, and "SVCV" represents cells infected with SVCV virus. It is evident that viral infection induces cytopathic effects in cells; compared to the control group, capsaicin treatment significantly reduces the cytopathic effects produced by virus-infected cells.

[0096] Example 2: Effects of capsaicin on gene transcription of SVCV, GCRV, and CyHV-2 viruses

[0097] Cells infected with the virus were treated with 1 mol / L capsaicin, and the transcription level of viral genes in the cells was detected. EPC cells, GCO cells, and GiCB cells in good growth condition were passaged and cultured overnight at 28°C, using the same method as in Example 1. Virus inoculation and drug treatment were performed according to the method described in Example 1; after culturing for another 24 hours, the cell culture medium was discarded, and TRIzol was used... TM Reagents (purchased from Thermo Fisher Scientific Inc., catalog number: 15596026CN) were used to extract total RNA after cell lysis. The specific steps were as follows: First, 200 μL of chloroform was added, vortexed for 20 seconds, allowed to stand for 2-3 minutes, and centrifuged at 12000g for 15 minutes at 4°C. Then, 200 μL of supernatant was gently transferred to a new 1.5 mL enzyme-free EP tube, 200 μL of anhydrous ethanol was added, and the mixture was stirred. Then, an RNase-free adsorption column CR3 (purchased from Beijing Bio-Lab Technology Co., Ltd., catalog number: WH0057) was added, and the tube was centrifuged at 12000g for 1 minute. Next, the tube was washed twice with 600 μL of wash buffer RW (purchased from Beijing Bio-Lab Technology Co., Ltd., catalog number: WH0057), and centrifuged at 12000g for 1 minute. Finally, total RNA was eluted with 30 μL of LEPC H2O, centrifuged at 12000g for 2 minutes, and collected into a new sterile centrifuge tube. cDNA was obtained by reverse transcription using the GoScript reverse transcription kit (purchased from Promega (Beijing) Biotech Co., Ltd., catalog number: A5001). The transcriptional level of viral genes was detected by qPCR. The specific steps were as follows: A CFX96TM Real-Time PCR instrument (Bio-Rad Laboratories, Inc.) was used. The amplification system consisted of 5 μL of 2×SYBR Green master Mix (purchased from GlpBio Technology., catalog number: GK10002), 2.5 μL of H2O, 2 μL of cDNA, and 0.25 μL each of upstream and downstream primers (the nucleotide sequences of the upstream and downstream primers for detecting the transcriptional level of each viral gene are shown in SEQ ID NO.12-23; where "FP" represents the upstream primer and "RP" represents the downstream primer); the amplification conditions were: 95℃ for 5 min, 95℃ for 10 s, 60℃ for 20 s, and 72℃ for 20 s, for 40 cycles. The plate was read at 72℃, and melting curves were plotted at 65-95℃.

[0098] The expression levels of the n and p genes in SVCV virus, the s6 and s9 genes in GCRV virus, and the orf47 and orf80 genes in CyHV-2 virus are shown in the following figures. Figure 4-9As shown in the figure, the vertical axis “Rel mRNA Level” represents the relative mRNA level, “Control” represents the control group, and “Capsaicin” represents the capsaicin treatment group. The experimental results show that treatment with 0.1 mol / L and 1 mol / L capsaicin significantly reduced the transcription levels of SVCV-related n and p genes, GCRV-related s6 and s9 genes, and CyHV-2-related orf47 and orf80 genes in cells.

[0099] Example 3: Effects of capsaicin on the expression of SVCV, GCRV, and CyHV-2 viral proteins

[0100] EPC, GCO, and GiCB cells in good growth condition were seeded into 6-well plates and cultured overnight at 28°C, using the same method as in Example 1. Following the method described in Example 1, the cells were inoculated with virus and capsaicin, cultured for another 24 hours, then the culture medium was discarded. The cells were washed with phosphate-buffered saline (PBS, Thermo Fisher Scientific Inc., catalog number: 20012027) and frozen at -80°C. 400 μL of RIPA lysis buffer (Thermo Fisher Scientific Inc., catalog number: 89900) was added to the frozen cells, and the cells were incubated at 4°C for 1 hour to lyse them. The cell lysates were then transferred to new 1.5 mL EP tubes, and an appropriate amount of SDS loading buffer (Merck KGaA, catalog number: S3401) was added. The tubes were then incubated in a boiling water bath at 100°C for 15 minutes and then placed on ice for later use. The expression levels of SVCV protein (N and P), GCRV protein (VP7), and CyHV-2 protein (ORF47 and ORF80) after capsaicin treatment were detected by Western blotting. The internal control protein Actin antibody was α-Smooth Muscle Actin (ACTA2) Rabbit mAb (purchased from Wuhan Aibote Biotechnology Co., Ltd., catalog number A17910).

[0101] Figure 10-12The expression levels of SVCV proteins (N and P), GCRV proteins (VP7), and CyHV-2 proteins (ORF47 and ORF80) detected by Western blotting are shown. In the figure, "Capsaicin" represents the capsaicin treatment group, hollow triangles indicate the order of capsaicin concentration from lowest to highest (0.1 mol / L and 1 mol / L), "IB:N" represents N protein, "IB:P" represents P protein, "IB:VP7" represents VP7 protein, "IB:ORF47" represents ORF47 protein, "IB:ORF80" represents ORF80 protein, and "IB:Actin" represents the internal control protein Actin. The results show that treatment with 0.1 mol / L and 1 mol / L capsaicin significantly reduced the levels of SVCV viral proteins N and P, and the protein expression of GCRV viral protein VP7, and CyHV-2 viral proteins ORF47 and ORF80.

[0102] Example 4: Effect of capsaicin on the survival rate of zebrafish, grass carp, and crucian carp infected with the virus.

[0103] Zebrafish (Danio rerio, purchased from the National Zebrafish Resource Center), grass carp (Ctenopharyngodonidella, sourced from the Liangzihu Base of the Institute of Hydrobiology, Chinese Academy of Sciences for aquaculture research), and crucian carp (Carassius auratu, sourced from the Liangzihu Base of the Institute of Hydrobiology, Chinese Academy of Sciences for aquaculture research) were temporarily held in the laboratory for one month for health assessment. The zebrafish, grass carp, and crucian carp were then intraperitoneally injected using a Nanoject III microinjector (Drummond Scientific Company) (SVCV for zebrafish, GCRV for grass carp, and CyHV-2 for crucian carp). The infected fish were then cultured in an appropriate amount of water with the aeration system turned on. Six hours after infection, capsaicin was added to the water to a final concentration of 1 nmol / L. Mortality was observed and the lethality rate was calculated daily.

[0104] Figure 13-15 The survival rate of fish infected with SVCV, GCRV, and CyHV-2 viruses within 15 days after capsaicin treatment is shown. The horizontal axis represents the number of days of culture, and the vertical axis represents the percentage of survival. In the figure, "Control" represents the control group, and "Capsaicin" represents the capsaicin-treated group. The results show that the mortality rate of zebrafish, grass carp, and crucian carp in the capsaicin-treated group was significantly lower than that in the control group, indicating that capsaicin has a protective effect on zebrafish, grass carp, and crucian carp infected with the viruses.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of capsaicin for the preparation of a feed additive and / or a medicament for the prevention and / or treatment of viral infections in fish, characterized in that, The fish virus is one or more of spring viremia of carp virus, grass carp reovirus and carp herpesvirus 2.

2. Use according to claim 1, characterized in that, The feed additive and / or the medicine play a preventive and therapeutic role by inhibiting gene transcription and protein expression of the fish virus.

3. The use according to claim 2, characterized in that: When the fish virus is a spring viremia of carp virus, the gene is n a gene and p a gene, the nucleotide sequences of which are shown in SEQ ID NO. 1-2, respectively; When the fish virus is grass carp reovirus, the gene is s6 gene and s9 genes, the nucleotide sequences of which are respectively shown as SEQ ID NO. 3-4; When the fish virus is a herpesvirus, the gene is orf47 a gene and orf80 a gene, the nucleotide sequences of which are represented by SEQ ID NO. 5-6, respectively.

4. The use according to claim 2, characterized in that: when the fish virus is spring viremia of carp virus, the proteins are N protein and P protein, and the amino acid sequences are shown as SEQ ID NO. 7-8, respectively; when the fish virus is grass carp reovirus, the protein is VP7 protein, and the amino acid sequence is shown as SEQ ID NO. 9; when the fish virus is carp herpesvirus 2, the proteins are ORF47 protein and ORF80 protein, and the amino acid sequences are shown as SEQ ID NO. 10-11, respectively.

5. The use according to claim 1, characterized in that, The concentration of the capsaicin in the feed additive and / or the medicine is 0.1-1 mol / L.

6. The use according to claim 1, characterized in that, The feed additive further comprises a feed-acceptable excipient.

7. The use according to claim 1, characterized in that, The medicine further comprises a pharmaceutically-acceptable excipient.

8. Use according to claim 7, characterized in that, The medicine dosage form is an oral dosage form or an injection dosage form.

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