Application of capsaicin in preparation of feed additive and / or medicine for preventing and treating fish virus infection

Abstract

The invention belongs to the technical field of aquaculture, and relates to a new application of capsaicin, in particular to an application of capsaicin in preparation of drugs and / or feed additives for preventing and treating fish virus infection. The core of the invention lies in that the capsaicin has an obvious protection effect on infection of spring viraemia of carp virus (SVCV), grass carp reovirus (GCRV) and herpesvirus type 2 (CyHV-2), and can obviously inhibit transcription of SVCV, GCRV and CyHV-2 virus genes and expression of virus proteins. The capsaicin-containing feed additive and / or medicine can be used for preventing and treating fish virus diseases, and has the advantages of high protection efficiency and environmental friendliness.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, and particularly to a new use of capsaicin, and in particular to the use of capsaicin in preparing feed additives and / or medicines for preventing and treating viral infections in fish. Background Art

[0002] Spring viraemia of carp virus (SVCV) is a single-stranded, negative-sense RNA virus belonging to the Rhabdoviridae family and the genus Vesiculoviridae, primarily infecting cyprinid fish. The SVCV genome is approximately 11 kb long and encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase (L). It is highly pathogenic and has a high mortality rate. SVCV often causes large-scale outbreaks in spring when water temperatures range from 10-25°C, causing systemic hemorrhage, organ inflammation, and acute death in diseased fish, resulting in significant economic losses to the aquaculture industry. Currently, there is no commercially available vaccine or specific treatment for SVCV. The main prevention and control measures rely on virus detection, isolation and culling, and environmental disinfection, but these are limited in effectiveness and cannot cope with sudden outbreaks.

[0003] Grass carp reovirus (GCRV), a double-stranded RNA virus belonging to the genus Aquatic Reovirus in the family Reoviridae, causes severe grass carp hemorrhagic disease. Based on VP6 nucleotide cluster typing, GCRV is divided into three main genotypes: GCRV-I, GCRV-II, and GCRV-III. GCRV-II is the predominant strain in major grass carp aquaculture areas in my country (Central and Southern China). Currently, research on the prevention and control of grass carp hemorrhagic disease focuses on vaccine development, breeding of disease-resistant germplasm, application of immune activators, and the establishment of ecologically healthy aquaculture models. However, the development of anti-grass carp hemorrhagic disease drugs has been hindered.

[0004] Crucian carp, a major freshwater fish species in my country, is a vital source of high-quality animal protein for residents. However, disease has long been a key bottleneck restricting the healthy development of the crucian carp aquaculture industry. Hematopoietic necrosis, caused by Cyprinid herpesvirus 2 (CyHV-2), is a major disease of aquaculture crucian carp. Its rapid onset, high infectiousness, and high mortality rate pose a serious threat to the industry. Despite significant progress in vaccine development and disease-resistant breeding, there is still no effective treatment for hematopoietic necrosis. Summary of the Invention

[0005] The EPC cells used in the present invention are carp epithelial cell lines, purchased from the China Type Culture Collection Wuhan University Collection Center, numbered GDC0174, and can be purchased through commercial channels; CIK cells are grass carp kidney cell lines, purchased from the China Type Culture Collection Wuhan University Collection Center, numbered GDC0086, and can be purchased through commercial channels; 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)), the original source of which is a gift from Researcher Zeng Lingbing of the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. The public can obtain relevant germplasm resources from the applicant, which can only be used for repeating the experiments of the present invention and cannot be used for other purposes.

[0006] The present invention aims to provide a new use of capsaicin, specifically a feed additive and / or drug containing capsaicin that can be used to prevent and treat viral diseases in fish, with the advantages of high protection efficiency and environmental friendliness. To achieve the above-mentioned purpose, the present invention provides the following technical solutions:

[0007] Use of capsaicin in preparing feed additives and / or medicines for preventing and treating 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 additives and / or drugs play a preventive and therapeutic role by inhibiting gene transcription and protein expression of the above-mentioned fish viruses.

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

[0011] n gene:

[0012]

[0013] p基因:

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

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

[0016] s6 gene:

[0017]

[0018] s9 gene:

[0019]

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

[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:

[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 the amino acid sequence thereof is as shown in SEQ ID NO. 9: MPLHMIPQVAHAMVRAAAAGRLTLYTKTKTETTNFDHAEYVTCGRYTICAFCLTTLAPHANVKTIQDSHACSRQPNEAIRSLVEVSDKAQIALVGSRTVDYHELDVKAGFVAPTADETVVPSKDIVELPFRTCDLDDSSATACVRNHCQAGHDGVTHLPILSGDFKLPNEHPTKPLDDTHPHDKVLTRCPKTGLLLVHDTHAHATAVVATAATRAILMHDLLTSANVDDGHQARSACYGPTFSNLTFACHSTCASDMAHFDCGQIVGLDLH (SEQ ID NO. 9).

[0031] More preferably, when the fish virus is grass carp reovirus, the protein is VP7 protein, and the amino acid sequence thereof is as shown in SEQ ID NO. 9: MPLHMIPQVAHAMVRAAAAGRLTLYTKTKTETTNFDHAEYVTCGRYTICAFCLTTLAPHANVKTIQDSHACSRQPNEAIRSLVEVSDKAQIALVGSRTVDYHELDVKAGFVAPTADETVVPSKDIVELPFRTCDLDDSSATACVRNHCQAGHDGVTHLPILSGDFKLPNEHPTKPLDDTHPHDKVLTRCPKTGLLLVHDTHAHATAVVATAATRAILMHDLLTSANVDDGHQARSACYGPTFSNLTFACHSTCASDMAHFDCGQIVGLDLH (SEQ ID NO. 9).

[0032] ORF47 protein:

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

[0034] ORF80蛋白:

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

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

[0037] More preferably, the feed additive further comprises feed science acceptable adjuvants.

[0038] It should be noted that the feed additives described herein can be supplemented with various types of supplementary materials to promote fish growth and enhance their immunity, including but not limited to vitamins, minerals, fat sources, and / or carbohydrate sources. Furthermore, vitamins may be selected from vitamin A, vitamin B1, vitamin B2, vitamin C, vitamin D, and / or vitamin E; minerals may be 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; fat sources may be selected from fish oil and / or vegetable oil; and carbohydrate sources may be selected from starch and / or cellulose.

[0039] More preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0040] It should be noted that the medicament of the present invention may be prepared into a suitable pharmaceutical form by adding different types of pharmaceutically acceptable excipients, including but 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 powder injections or injectable emulsions). These pharmaceutically acceptable excipients include but are not limited to diluents, wetting agents, adhesives, lubricants, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, preservatives, local analgesics, pH adjusters, isotonic and / or isotonic modifiers, etc. Furthermore, the diluent is selected from starch, sucrose and / or cellulose; the wetting agent is selected from water and / or ethanol; the binder is selected from starch slurry, sugar, cellulose and / or povidone; the lubricant is selected from talc, magnesium stearate, magnesium lauryl sulfate and / or micro-powdered silica; the solvent is selected from water, ethanol, glycerol, propylene glycol, fatty oil and / or ethyl acetate; the emulsifier is selected from glycerol fatty acid esters, higher fatty acid salts, sulfates, phospholipids, gelatin, pectin, agar, sodium alginate and / or bentonite; the antioxidant is selected from sulfites, pyrosulfites and / or ascorbic acid; the preservative is selected from parabens, sodium benzoate, quaternary ammonium compounds, alcohols and / or phenols.

[0041] More preferably, the pharmaceutical dosage form is an oral dosage form or an injection dosage form.

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

[0043] The present invention provides the use of capsaicin in the preparation of medicines and / or feed additives for preventing and treating 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 medicines containing capsaicin can be used for the prevention and treatment of viral diseases in fish, and have the advantages of high protection efficiency and environmental friendliness. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

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

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

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

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

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

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

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

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

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

[0054] Figure 10 This is a Western Blot detection diagram of the expression of N protein and P protein in SVCV virus.

[0055] Figure 11 This is a Western Blot detection image of VP7 protein expression in GCRV virus.

[0056] Figure 12 This is a Western Blot detection diagram of the expression of ORF47 protein and ORF80 protein in CyHV-2 virus.

[0057] Figure 13 Schematic diagram comparing the effect of capsaicin on the survival of zebrafish infected with SVCV within 7 days.

[0058] Figure 14 This is a comparative diagram of the effect of capsaicin on the survival of grass carp infected with GCRV within 15 days.

[0059] Figure 15 This is a comparative diagram of the effect of capsaicin on the survival of crucian carp infected with CyHV-2 within 15 days. DETAILED DESCRIPTION

[0060] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts 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 the embodiments. In addition, in the description of this application, the term "including" means "including but not limited to". Various embodiments of the present invention may be 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 understood 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 the range. For example, the range description from 1 to 6 should be considered to have 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., as well as single numbers within the numbered ranges, such as 1, 2, 3, 4, 5 and 6, regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0062] Capsaicin is a natural alkaloid with the molecular formula C 18 H 27 NO3, molecular weight is 293.4, and its chemical structure is Capsaicin is the primary pungent ingredient in chili peppers. Found primarily in the placenta and seeds of chili peppers, it binds to TRPV1 receptors on human nerve endings, mimicking the sensation of high-temperature burning and triggering pain and heat signaling. This property has led to its widespread use in medicine, for example, to relieve chronic pain such as neuralgia and arthritis. In recent years, capsaicin has been found to possess antioxidant, anti-inflammatory, and potential anticancer activities, and appropriate intake can boost metabolism.

[0063] The embodiments of the present invention provide the use of capsaicin in preparing a feed additive and / or a medicine for preventing and treating viral infection in fish.

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

[0065] Specifically, the feed additives and / or drugs play a preventive and therapeutic role by inhibiting the gene transcription and protein expression of the above-mentioned fish viruses.

[0066] More specifically, when the fish virus is carp spring viremia virus, the genes are n gene and p gene, and their nucleotide sequences are shown as 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 as SEQ ID NO.3-4 respectively.

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

[0069] More specifically, when the fish virus is carp spring viremia virus, the 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 cyprinid herpesvirus type 2, the above-mentioned proteins are ORF47 protein and ORF80 protein, and their amino acid sequences are shown as SEQ ID NO.10-11 respectively.

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

[0073] More specifically, the above feed additives also include feed science-acceptable adjuvants.

[0074] More specifically, the above-mentioned medicine further includes pharmaceutically acceptable excipients.

[0075] More specifically, the above-mentioned drug dosage form is an oral dosage form or an injection dosage form.

[0076] The following further illustrates the use of capsaicin of the present invention in preparing feed additives and / or medicines for preventing and treating viral infections in fish with specific examples. This section further illustrates the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means adopted in the examples 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 this area.

[0077] In the following examples, the multiplicity of infection (MOI) represents the ratio of the number of viruses that can infect cells to the total number of cells in a system. The specific calculation formula is as follows:

[0078]

[0079] Among them, the virus titer (TU / mL) refers to the number of infectious virus particles contained in each milliliter of solution, the virus volume (mL) refers to the volume of the added virus solution, and the total number of cells 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 transcription levels of n gene and p gene in SVCV virus, s6 gene and s9 gene in GCRV virus, and orf47 gene and orf80 gene in CyHV-2 virus are as 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 Effect of capsaicin on SVCV, GCRV and CyHV-2 infected cells

[0094] Antiviral experiments were performed in 24-well cell culture plates. EPC cells were used to detect cytopathic effects of SVCV, CIK cells were used to detect lesions of GCRV, and GiCB cells were used to detect lesions of CyHV-2. The cells with good growth were passaged and inoculated into 24-well plates. M199 medium (purchased from Thermo Fisher Scientific Inc., catalog number: 31100035) containing 1% double antibody (100X penicillin-streptomycin solution, purchased from ThermoFisher Scientific Inc., catalog number: 15070063) and 10% fetal bovine serum (purchased from ThermoFisher Scientific Inc., catalog number: 10099141C) was used to culture overnight at 28°C in a CO2 incubator with a volume fraction of 5%. EPC cells were inoculated with SVCV at an MOI of 1, CIK cells with GCRV at an MOI of 1, and GiCB cells with CyHV-2 at an MOI of 1. 1 mol / L capsaicin was added to the cells. Following inoculation, the cells were cultured at 28°C for 72 hours. The cells were fixed with 4% paraformaldehyde for 1 hour and then stained with 1% crystal violet overnight. The stained plates were photographed the following day to observe cytopathic effects.

[0095] The protective effects of capsaicin on 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 can be seen that viral infection induces a cytopathic effect in cells, and capsaicin treatment significantly reduces the cytopathic effect in virus-infected cells compared to the control group.

[0096] Example 2 Effect of capsaicin on SVCV, GCRV and CyHV-2 viral gene transcription

[0097] The cells infected with the virus were treated with 1 mol / L capsaicin and the transcription level of the viral genes in the cells was detected. EPC cells, GCO cells and GiCB cells with good growth status were passaged and inoculated into 6-well plates and cultured overnight at 28°C. The culture method was the same as that in Example 1. Virus was inoculated and drug treatment was carried out according to the method described in Example 1. After 24 hours of continuous culture, the cell culture medium was discarded and TRIzol TM After lysing the cells with reagent (purchased from Thermo Fisher Scientific Inc., catalog number: 15596026CN), total RNA was extracted. The specific steps were as follows: first, 200 μL of chloroform was added, shaken for 20 seconds, allowed to stand for 2-3 minutes, and centrifuged at 12000g in a 4°C centrifuge for 15 minutes; 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 mixed, and then an RNase-free adsorption column CR3 (purchased from Beijing Biolab Technology Co., Ltd., catalog number: WH0057) was added and centrifuged at 12000g for 1 minute; secondly, 600 μL of rinse solution RW (purchased from Beijing Biolab Technology Co., Ltd., catalog number: WH0057) was washed twice and centrifuged at 12000g for 1 minute; finally, total RNA was eluted with 30 μL DEPC 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 transcription level of viral genes was detected by qPCR. The specific steps were as follows: using a CFX96TM Real-Time PCR instrument (Bio-Rad Laboratories, Inc.), the amplification system: 2×SYBR Green master mix (purchased from GlpBio Technology., catalog number: GK10002) 5 μL, H2O 2.5 μL, cDNA 2 μL, upstream primer and downstream primer 0.25 μL each (the nucleotide sequences of the upstream and downstream primers for detecting the transcription level of each viral gene are shown in SEQ ID NO.12-23; wherein, "FP" represents the upstream primer and "RP" represents the downstream primer); amplification conditions: 95°C for 5 min, 95°C for 10 s, 60°C for 20 s, 72°C for 20 s, for 40 cycles, reading the plate at 72°C, and drawing the melting curve at 65-95°C.

[0098] The expression level detection results of n gene and p gene in SVCV virus, s6 gene and s9 gene in GCRV virus, and orf47 gene and orf80 gene in CyHV-2 virus are as follows: 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-treated group. The experimental results showed that 0.1 mol / L and 1 mol / L capsaicin treatment can significantly reduce 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 Effect of capsaicin on SVCV, GCRV and CyHV-2 viral protein expression

[0100] EPC cells, GCO cells, and GiCB cells in good growth condition were inoculated into 6-well plates and cultured overnight at 28°C. The culture method was the same as in Example 1. Virus inoculation and capsaicin treatment were performed according to the method in Example 1. After further culture for 24 hours, the culture medium was discarded, washed with phosphate buffered saline (PBS, purchased from Thermo Fisher Scientific Inc., Catalog No.: 20012027), and then frozen in a -80°C refrigerator. 400 μL RIPA lysis buffer (purchased from Thermo Fisher Scientific Inc., Catalog No.: 89900) was added to the frozen cells, and the cells were placed at 4°C for cell lysis for 1 hour. The cell lysate was then aspirated into a new 1.5 mL EP tube, and an appropriate amount of SDS loading buffer (purchased from Merck KGaA, Catalog No.: S3401) was added. The tube was incubated in a boiling water bath at 100°C for 15 minutes and then placed on ice for later use. Western blot was used to detect the expression levels of SVCV protein (N and P), GCRV protein (VP7), and CyHV-2 protein (ORF47 and ORF80) after capsaicin treatment. The internal reference protein Actin antibody was α-Smooth Muscle Actin (ACTA2) Rabbit mAb (purchased from Wuhan Aibote Biotechnology Co., Ltd., catalog number A17910).

[0101] Figure 10-12Western blot analysis of the expression levels of SVCV proteins (N and P), GCRV protein (VP7), and CyHV-2 proteins (ORF47 and ORF80) is shown. "Capsaicin" indicates the capsaicin-treated group, and hollow triangles indicate the order of capsaicin concentrations from low to high (0.1 and 1 mol / L). "IB:N" indicates N protein, "IB:P" indicates P protein, "IB:VP7" indicates VP7 protein, "IB:ORF47" indicates ORF47 protein, "IB:ORF80" indicates ORF80 protein, and "IB:Actin" indicates the internal control protein Actin. The results showed that after treatment with 0.1 mol / L and 1 mol / L capsaicin, the intracellular levels of SVCV viral proteins N and P, GCRV viral protein VP7, and CyHV-2 viral proteins ORF47 and ORF80 were significantly reduced.

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

[0103] Zebrafish (Danio rerio, purchased from the National Zebrafish Resource Center), grass carp (Ctenopharyngodonidella, sourced from the Liangzi Lake Base of the Institute of Hydrobiology, Chinese Academy of Sciences for research purposes), and crucian carp (Carassius auratus, sourced from the Liangzi Lake Base of the Institute of Hydrobiology, Chinese Academy of Sciences for research purposes) were housed in the laboratory for one month for health assessment. Zebrafish, grass carp, and crucian carp were intraperitoneally injected with SVCV, GCRV, and CyHV-2 using a Nanoject III microinjector (Drummond Scientific Company). The challenged fish were housed in appropriate aeration water. Six hours after challenge, capsaicin was added to the water to a final concentration of 1 nmol / L. Mortality was observed daily, and the mortality rate was calculated.

[0104] Figure 13-15 The survival of fish infected with SVCV, GCRV, and CyHV-2 viruses within 15 days after capsaicin treatment is shown. The horizontal axis is the number of culture days, and the vertical axis is the fish survival rate (Percentage of survival). In the figure, "Control" represents the control group and "Capsaicin" represents the capsaicin-treated group. The results showed 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, that is, capsaicin had a protective effect on zebrafish, grass carp, and crucian carp after infection with the virus.

[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 in the scope of protection of the present invention.

Claims

1. Use of capsaicin in the preparation of feed additives and / or medicines for preventing and treating viral infections in fish.

2. The use according to claim 1, characterized in that The fish virus is one or more of carp spring viremia virus, grass carp reovirus and carp herpes virus type 2.

3. The use according to claim 1 or 2, characterized in that The feed additive and / or drug plays a preventive and therapeutic role by inhibiting the gene transcription and protein expression of the fish virus as claimed in claim 2.

4. The use according to claim 3, characterized in that: When the fish virus is carp spring viremia virus, the genes are n gene and p gene, and their nucleotide sequences are shown as SEQ ID NO.1-2 respectively; When the fish virus is grass carp reovirus, the genes are s6 gene and s9 gene, and their nucleotide sequences are shown in SEQ ID NO.3-4 respectively; When the fish virus is cyprinid herpesvirus type 2, the genes are orf72 gene and orf80 gene, and their nucleotide sequences are shown as SEQ ID NO.5-6 respectively.

5. The use according to claim 3, characterized in that: When the fish virus is carp spring viremia virus, the proteins are N protein and P protein, and the amino acid sequences are shown in SEQ ID NO.7-8 respectively; When the fish virus is grass carp reovirus, the protein is VP7 protein, and its amino acid sequence is shown in SEQ ID NO.9; When the fish virus is cyprinid herpesvirus type 2, the proteins are ORF47 protein and ORF80 protein, and their amino acid sequences are shown as SEQ ID NO.10-11 respectively.

6. The use according to claim 1 or 2, characterized in that The concentration of capsaicin in the feed additive and / or medicine is 0.1-1 mol / L.

7. The use according to claim 1, characterized in that The feed additive also includes feed science acceptable adjuvants.

8. The use according to claim 1, wherein The drug also includes pharmaceutically acceptable excipients.

9. The use according to claim 7, characterized in that The pharmaceutical dosage form is an oral dosage form or an injection dosage form.

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