CN111297851B - Application of Coumarin in Antiviral Infection of Honeybees - Google Patents
Application of Coumarin in Antiviral Infection of Honeybees Download PDFInfo
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- CN111297851B CN111297851B CN202010255727.XA CN202010255727A CN111297851B CN 111297851 B CN111297851 B CN 111297851B CN 202010255727 A CN202010255727 A CN 202010255727A CN 111297851 B CN111297851 B CN 111297851B
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Abstract
The invention relates to the technical field of bee breeding, in particular to application of coumarin in antiviral infection of bees. The coumarin is found to be capable of inhibiting the proliferation of the bee sacbrood virus (CSBV) in the bee body and reducing the copy number of the virus; meanwhile, the expression of endogenous antibacterial peptide can be induced, and the innate immunity defense capacity of bees is improved. The coumarin is capable of remarkably improving the survival rate of Chinese bee larvae infected with Chinese bee sacbrood virus (CSBV) and reducing the death rate of the Chinese bee larvae. The coumarin can be used for preparing medicines or feeds for preventing and treating CSBV infection, has important significance for preventing and treating Chinese bee sacbrood, and has good market application value.
Description
Technical Field
The invention relates to the technical field of prevention and control of bee virus diseases, in particular to application of coumarin in bee antiviral infection.
Background
Bees are important pollination insects in nature, about one third of crops in the world need bee pollination to a certain extent, and some fruits, vegetables and the like completely depend on bee pollination. Bee pollination can significantly improve the yield and quality of crops, thus having important economic value in the aspect of agricultural production. In addition, the bee can also produce various peak products, such as honey, royal jelly, propolis, bee pollen and the like. The quality of bee products is closely related to the health state of bees, potential safety hazards exist in the bee products produced by susceptible bees, and medicines for treating bee diseases can also be remained in the bee products to further cause the safety problems of the bee products.
The Chinese honeybee (Apis cerana) has the advantages of strong anti-mite capability, low temperature resistance and good collection of sporadic honey powder source, plays an important role in the bee-keeping industry and plays an extremely important role in maintaining the balance of a natural ecosystem. However, apis cerana are often harmed by a variety of pathogens, of which apis cerana are most seriously harmed by middle bee sacvirus (CSBV). CSBV mainly infects 1-3 day old larva, causes the larva in the bee colony to die in a large amount and can not pupate, leads to the number of worker bees newly emerged from the house to be insufficient, and then leads to the situation of the middle bee colony to be weakened, the resistance of the bee colony is reduced, and the bee colony is more easily invaded by other viruses, thereby showing the phenomenon of multi-virus common infection. CSBV can be transmitted within bee colonies by means of food mutual feeding, faecal contamination, mating behaviour and reproduction of gametes. The drone can act as a viral vector to transmit the virus to offspring through the reproductive gametes. The non-group-boundary property of the male bees can directly determine the genetic diversity of the bee colony, so that the disease resistance of the bee colony can be greatly influenced. At present, the Chinese bee sacbrood disease is mainly prevented and controlled by comprehensive prevention and control measures of changing the prince-ova, strictly disinfecting, strengthening management, preventing secondary infection by using antiviral drugs and antibiotics, but the prevention and control effect is not ideal, the harm and the spread of the virus are difficult to be effectively controlled by the common antiviral drugs, and drug residues in bee products can be caused.
Endogenous antimicrobial peptides play a key role in immune regulation and pathogen defense as an important component of innate immunity. Endogenous antimicrobial peptides also have immunoregulatory activity, participate in regulating innate and adaptive immune responses, are important mediators of the body's defense against foreign substance invasion, and are also immunologically active molecules produced by the body's adaptation to the environment.
When bees are infected by microorganisms or other exogenous substances, hemolymph produces a certain amount of antibacterial peptides including hymenoptera antibacterial peptide (hymenoptera antibacterial peptide), bee Defensin (Defensin), bee antibacterial peptide (Apidaecin) and bee moth antibacterial peptide (abeecin). These peptides are synthesized from fat bodies and secreted into haemolymph, which is a rapid and effective defense mechanism and can be used to rapidly kill or eliminate exogenous microorganisms. The defense mechanism is different from the immune response of animals, insects have no strict immune response mechanism, the antibacterial peptides produced by insect hemolymph can be invaded by one or more microorganisms and have no specificity, and the induction products are not only produced by specific invaded microorganisms but also have resistance to non-invaded microorganisms generally. The antibacterial peptides have different molecular weights, chemical structures and antibacterial mechanism effects, and different antibacterial peptides can resist different bacteria, fungi, viruses, tumor cells and the like, wherein the bee antibacterial peptide is the main antibacterial peptide of bees, and the hymenoptera antibacterial peptide is the most important supplementary peptide in the bee antibacterial peptide and mainly plays an inhibiting role on part of gram-negative bacteria generating resistance to the bee antibacterial peptide; the bee moth antibacterial peptide is used as a backup peptide of the bee antibacterial peptide, and only plays a role when the bactericidal abilities of the bee antibacterial peptide, the bee antibacterial peptide and the hymenoptera antibacterial peptide are lost. The bee defensin has the least expression amount and is the only antibacterial peptide for inhibiting gram-positive bacteria in bee hemolymph. The content of the bee antibacterial peptide is influenced by different factors, including bacteria, fungi, baby bugs, viruses, bee mites, medicaments and the like. The antibacterial peptide plays a role in resisting bee viruses, and researchers find that: when the bee is inoculated with Acute Bee Paralysis Virus (ABPV), the expression level of the antibacterial peptide gene is obviously increased; in the midgut epithelial tissue of the bee, the infection level of the bee residual wing virus (DWV) and the expression level of the bee antibacterial peptide gene are linearly related; after the bee is infected by the virus, the expression levels of the bee antibacterial peptide, the hymenoptera antibacterial peptide and the bee moth antibacterial peptide in the bee body are obviously reduced. Bee mites can also affect the expression of bee antimicrobial peptides. In the aspect of medicine, researchers find that the acaricide can significantly influence the expression of the bee antibacterial peptide gene, after bees contact the flumethrin vinegar, the expression level of the hymenoptera antibacterial peptide is significantly up-regulated, and the coumaphos can down-regulate the expression of the hymenoptera antibacterial peptide and the bee moth antibacterial peptide gene. In conclusion, the bee antibacterial peptide is a key part of humoral immunity of bees, and plays a role in coordination to resist invasion of various pathogenic microorganisms.
Chinese patent CN103524602A discloses the treatment of bee sacbrood by administering exogenous antibacterial peptide, but the administration of exogenous antibacterial peptide still faces the potential safety risk of antibacterial peptide drugs, the mass production of antibacterial peptide is limited, the production efficiency is low and the production cost is high (linchengde, penhongjuan, wang derived sea. the application and existing problems of antibacterial peptide. tropical medical journal 2007,1 (7): 86-90.). Therefore, the development of a low-cost and high-efficiency prevention and treatment method for the Chinese sacbrood is of great significance to the bee breeding industry.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide the application of coumarin in the antiviral infection of bees.
The coumarin is a compound known in the prior art, is known as benzopyrone, is lactone of cis-o-hydroxycinnamic acid, and is a parent nucleus of a large class of coumarin compounds existing in the plant world. It is classified into simple coumarin, furocoumarin, pyranocoumarin and other coumarins. The coumarin drug has the function of inhibiting the synthesis of blood coagulation factors in the liver. The natural coumarin is widely distributed in the nature (animals, plants and microorganisms), and has small relative molecular mass, simple and easy operation of a synthesis method, and a pi-pi conjugate system and a rigid condensed ring contained in the structure, so that the natural coumarin can act on various active sites in a living body in a non-covalent action mode, and the compounds have good biological activity, such as antibiosis, antioxidation, antitumor, AIDS resistance, anti-inflammation, anticancer, antivirus and the like. Coumarin exists in plant in free state or combined with sugar to form glycoside, and the glycoside can be cyclized into free lactone state by enzymolysis. The coumarin has a parent nucleus structure as follows:
the simple coumarins are coumarins with substituents only on benzene rings, most of the coumarins are known to have oxygen-containing functional groups at C-7, and the 7-hydroxycoumarins can be regarded as the parent of the coumarins with few exceptions, so that 7-hydroxycoumarins are selected as a representative in the examples of the invention to discuss the control effect of the coumarins in bee virus infection.
The invention provides application of coumarin, structural analogues or derivatives thereof in preparing a medicament for treating or preventing bee virus infection.
The invention provides an application of coumarin, structural analogues or derivatives thereof in preparing feed for treating or preventing bee virus infection.
The invention provides application of coumarin, structural analogues or derivatives thereof in inhibiting bee virus proliferation.
The application of the coumarin, the structural analogue or the derivative thereof in inhibiting the bee virus proliferation can be used for inhibiting the virus proliferation in apparently healthy bees and can also be used for inhibiting the virus proliferation in virus disease bees.
According to one embodiment of the invention, the intervention of 7-hydroxycoumarin is found to effectively reduce the virus copy number in bodies infected with CSBV larvae in the concentration range of 0.75-75 mu g/mL, so that the fatality rate of the larvae is reduced.
In the application, the coumarin is 7-hydroxycoumarin, and the virus copy number in bodies of CSBV bee larvae is effectively inhibited within the concentration range of 0.75-75 mug/mL.
The virus of the present invention is preferably Chinese bee sacbrood disease virus (CSBV). Experiments prove that the survival rate of Chinese bee larvae infected with CSBV can be remarkably improved by the application of coumarin, on one hand, the coumarin can directly act on viruses to inhibit the copy number of the viruses, and on the other hand, the coumarin can also induce the expression of endogenous antibacterial peptides of the bees to enhance the innate immunity defense of organisms to resist the viruses, so that the incidence rate of CSBV infected larvae is reduced. Based on the application, the invention provides the application of coumarin, structural analogues or derivatives thereof in preparing medicines for improving the immunity of bees.
Experiments prove that the coumarin can induce the transcription and translation of endogenous antibacterial peptide genes, improve the expression level of endogenous antibacterial peptides (such as bee antibacterial peptide, bee defensin, bee moth antibacterial peptide and hymenoptera antibacterial peptide), and further improve the immunity of organisms.
Therefore, the invention provides the application of coumarin, structural analogues or derivatives thereof in preparing medicines for improving the immunity of bees. The invention also provides application of the coumarin, the structural analogue or the derivative thereof in preparing feed for improving the immunity of bees.
The improvement of the bee immunity is realized by improving the expression quantity of endogenous antibacterial peptide of the bee.
The endogenous antibacterial peptide can be any one or more of hymenoptera antibacterial peptide, bee antibacterial peptide and bee moth antibacterial peptide.
The coumarin of the invention comprises simple coumarin, furocoumarin, pyranocoumarin and other coumarins; the other coumarins are coumarins having a substituent on the α -pyrone ring. The structural analogs of coumarin include isocoumarin and dicoumarin.
The active ingredient of the medicine or feed comprises one or more selected from coumarin, structural analogues thereof and derivatives thereof.
The invention also provides a medicament for treating or preventing bee virus infection, wherein the active component of the medicament comprises one or more selected from coumarin, structural analogues thereof and derivatives thereof.
The active ingredient of the medicament of the invention may comprise only coumarin, a structural analogue or derivative thereof; may also comprise a plurality selected from coumarin, structural analogs thereof and derivatives thereof; other active ingredients besides coumarin, structural analogues and derivatives thereof (e.g. other active ingredients capable of enhancing bee immunity or antiviral ability) may also be included.
The medicine of the invention can also contain auxiliary materials allowed in the field of pharmacy; the dosage form of the medicament can be any dosage form allowed in the field of pharmacy.
The feed of the present invention may comprise coumarin, a structural analogue or derivative thereof, and may also comprise a plurality selected from coumarin, a structural analogue or derivative thereof; can also contain other active ingredients besides coumarin, structural analogues and derivatives thereof (such as other active ingredients capable of improving bee immunity or antiviral ability); in addition to the above active ingredients, the feed according to the invention may also contain nutrients required by the bees, such as: pollen, royal jelly, fructose, glucose, sucrose, soybean flour, yeast powder, skimmed milk powder, inorganic salt, vitamins, water, etc.
The feed of the invention can be solid feed, semi-solid feed or liquid feed.
In the above application of the coumarin provided by the invention, the dose of the coumarin administered to bees is preferably not less than 15 ng/bee/time. More preferably 0.1 to 1. mu.g/piece. The time interval of each coumarin administration is preferably 22-24 h.
Specifically, for 3-day-old bees, 15 ng-1.5 mug/bee/time; 22.5 ng-2.25 mug of bees at the age of 4 days per bee; 37.5 ng-3.75 mug of bees in 5 days old per bee per time; 6-day-old bees 60 ng-6 mug/bee/time.
The invention has the beneficial effects that: the invention discovers for the first time that the coumarin can obviously improve the survival rate of the Chinese bee larva infected with CSBV and reduce the death rate. On one hand, the coumarin can inhibit the replication of CSBV and reduce the copy number of viruses, and the intervention of 7-hydroxycoumarin effectively reduces the copy number of viruses infecting CSBV larvae in the concentration range of 0.75-75 mug/mL, so that the death rate of the larvae is reduced; on the other hand, the coumarin can also induce the expression of endogenous antibacterial peptide and improve the innate immunity defense of bees. The experimental result of the invention shows that the mortality of Chinese bee larvae infected with CSBV is greatly reduced (can be reduced by 8.2 times at most) after the intervention of coumarin, and the survival larvae can pupate and emerge into adult bees; the virus copy number in the larvae of the Chinese bees infected with CSBV after the coumarin is dried is greatly reduced (the virus copy number can be reduced by 62300 times at most), and the quantitative-effect relationship is presented, and meanwhile, the coumarin intervention can improve the virus infection degree of apparent healthy larvae and is beneficial to the normal growth and development of the larvae; the expression levels of Apidacetin, Abaecin and Hymenoptaecin in Chinese bee larvae infected with CSBV after coumarin intervention are all obviously up-regulated.
The new function of the coumarin provided by the invention on bee virus infection provides a basis for the research of bee immune defense mechanism. The coumarin can be applied to preparation of medicines or feeding materials for preventing and treating Chinese bee sacbrood, provides a new method for preventing and treating Chinese bee sacbrood, and has good market application prospect.
Drawings
FIG. 1 is a graph showing the statistical results of survival rate of 6-day-old larvae after the dry prognosis of 7-hydroxycoumarin in Experimental example 1; wherein CK represents the control group, 0.75 represents the low concentration group of the preparation group, 7.5 represents the medium concentration group of the preparation group, 75 represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV +0.75 represents the low concentration group of the intervention group, CSBV +7.5 represents the medium concentration group of the intervention group, and CSBV +75 represents the high concentration group of the intervention group.
FIG. 2 is a statistical result of mortality of 4-6 day old larvae after the dried 7-hydroxycoumarin in Experimental example 1; wherein CK represents the control group, 0.75 represents the low concentration group of the preparation group, 7.5 represents the medium concentration group of the preparation group, 75 represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV +0.75 represents the low concentration group of the intervention group, CSBV +7.5 represents the medium concentration group of the intervention group, and CSBV +75 represents the high concentration group of the intervention group. The top of the legend corresponds to the columns of the histogram from the left to the right. This arrangement is the same for fig. 3-7 below.
FIG. 3 shows the results of the detection of the number of copies of 7-hydroxycoumarin dried larvae of 4-6 days old viruses in Experimental example 2; wherein CK represents the control group, 0.75 represents the low concentration group of the preparation group, 7.5 represents the medium concentration group of the preparation group, 75 represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV +0.75 represents the low concentration group of the intervention group, CSBV +7.5 represents the medium concentration group of the intervention group, and CSBV +75 represents the high concentration group of the intervention group.
FIG. 4 shows the amount of expressed bee antibacterial peptide (Apidaecin) after infection of CSBV and 7-hydroxycoumarin in Experimental example 3; wherein CK represents a control group, low represents a low concentration group of the preparation group, medium represents a medium concentration group of the preparation group, high represents a high concentration group of the preparation group, CSBV represents an infection group, CSBV + low represents a low concentration group of the intervention group, CSBV + medium represents a medium concentration group of the intervention group, and CSBV + high represents a high concentration group of the intervention group.
FIG. 5 shows the expression levels of Hymenoptera antimicrobial peptide (Hymenoptaecin) after infection with CSBV and 7-hydroxycoumarin in Experimental example 3 of the present invention; wherein CK represents a control group, low represents a low concentration group of the preparation group, medium represents a medium concentration group of the preparation group, high represents a high concentration group of the preparation group, CSBV represents an infection group, CSBV + low represents a low concentration group of the intervention group, CSBV + medium represents a medium concentration group of the intervention group, and CSBV + high represents a high concentration group of the intervention group.
FIG. 6 shows the expression levels of the honeybee moth antibacterial peptide (Abaecin) after the infection of CSBV and 7-hydroxycoumarin in Experimental example 3 of the present invention; wherein CK represents a control group, low represents a low concentration group of the preparation group, medium represents a medium concentration group of the preparation group, high represents a high concentration group of the preparation group, CSBV represents an infection group, CSBV + low represents a low concentration group of the intervention group, CSBV + medium represents a medium concentration group of the intervention group, and CSBV + high represents a high concentration group of the intervention group.
FIG. 7 shows the amount of bee Defensin (Defensin) expressed after infection of CSBV and 7-hydroxycoumarin in Experimental example 3 of the present invention; wherein CK represents a control group, low represents a low concentration group of the preparation group, medium represents a medium concentration group of the preparation group, high represents a high concentration group of the preparation group, CSBV represents an infection group, CSBV + low represents a low concentration group of the intervention group, CSBV + medium represents a medium concentration group of the intervention group, and CSBV + high represents a high concentration group of the intervention group.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Statistical analysis of the data in the following examples was performed using SPSS 22.0 (SPSS corporation, chicago, usa). The effect of the nutritional supplement on the Chinese bee larvae is expressed by Mean + -SE. And comparing the change of the virus copy number infected with CSBV in the Chinese bee larva by adopting an analysis of variance and an LSD multiple analysis of variance method.
Example 1 intervention of healthy Apis cerana larvae and infection with CSBV larvae Using 7-Hydroxycoumarin
1. Sample supply book
The Apis cerana Fabricius is from Apis cerana Fabricius research institute of China academy of agricultural sciences. In order to obtain 2-day-old larvae, after queen bees are closed on the son spleens to lay eggs for 96 hours, the son spleens containing the 2-day-old larvae are taken out from the bee colony and put into a 24-well plate, and the plate is placed in a constant temperature and humidity incubator with the temperature of 32 +/-1 ℃ and the relative humidity of 75 +/-5% RH for pre-culture.
The presence or absence of CSBV and other viral infections (including BQCV, DWV, IAPV, KBV, ABPV, CBPV) in the larvae was determined by RT-PCR, and healthy larvae without viral infections were used as samples in this example.
The primer sequences provided in the above-mentioned detection primer references for various viruses are as follows: CSBV detection primers were referenced to Grabenstein, E., W.Ritter, M.J.Carter, S.Davison, H.Pechhalker, J.Kolodziejek, O.Boeckking, I.Derakshifar, R.Moosbeckhofer, E.Licek, and N.Nowotny.Sacbrood virus of the honeybee (Apis mellea): rapid identification and phylogenetic analysis conversion-PCR.clin.Diagen. Lab.Immunol.2001,8: 93-104; BQCV detection primer references M, Benjeddou, N, Leat, M, Allsop, S, Davison.detection of acid bean analysis virus and black queen cell virus from the host genes by reverse transcription transcriptional pcr.applied and environmental microbiology.2001,67(5): 2384-; DWV detection primer refer to Tentcheva, D., L.Gauthier, S.Jouvee, L.Canaday-Rochelle, B.Dainat, F.Counters, M.E.Colin, B.V.Ball, and M.Berginin.Polymeraschen reaction detection of formed with virus (DWV) in Apis melifera and Varroa detector. idiology.2004, 35: 431-; IAPV detection primers are referred to Eyal, Maori, Shai, Lavi, Rita, Mozes-Koch, Yulia, Gantman, Yuv al, Peretz, Orit, Edelbaum, Edna, Tanne, Ilan, Sela. isolation and characterization of analysis resources, a direct improvement of strategies in an array, identification for direction product to intra-and inter-site recognition, the Journal of general vision, 2007,88(Pt12): 3428-38; KBV detection primers were Stoltz, D., X.R.Shen, C.Boggis, and G.Sisson.molecular diagnostics of Kashmir bean virus infection.J.apic.Res.1995,34: 153-; ABPV detection primer refer to Bakonyi, T., R.Farkas, A.Szendroi, M.Dobos-Kovacs, and M.Rusvai.detection of acid bean analysis virus by RT-PCR in the gene bean and Varroa derivative field samples, screening of sensing human diagnosis experiments.Apidiologue.2002, 33: 63-74; CBPV detection primers are referred to Ribie're, M., C.Triboultot, L.Mathieu, C.Aurie ' res, J.P.Faucon, and M.Pe ' pin.molecular diagnostic of molecular be medical science in Apidiologie, 2002,33: 339-.
The detection method specifically comprises the following steps: RNA was extracted using RNeasy mini kit (edley) according to the product instructions (the method includes a step of removal of DNA, thus ensuring that only RNA is present in the final extract). Total RNA was eluted in 30. mu.L of elution buffer and used directly for RT-PCR. First strand cDNA was generated immediately using the extracted RNA using the Quantitec reverse transcription kit (Takara) (according to the product instructions) and the cDNA was stored at-20 ℃. The PCR amplification reaction is a 50 mu L system; PCR reaction conditions included pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 15 seconds, and 35 cycles; extension for 5min at 72 ℃. The results showed that no CSBV and other viruses were detected.
2. Preparation of syrup
Preparation and feeding of the syrups are carried out according to the Standard for feeding Apis mellifera in 2016 and adjusted on this basis (Karl Crailsheim, Robert Brodschneider, Pierrick Aupinel, Dieter Behrens, Elke Genersch, Jutta Vollmann & Ulrike Riessberger-Gall. Standard methods for identifying the real area re-addressing of Apis mellea. journal of Apimulturral Research,2013,52(1): 1-15.). The formula, ratio and dosage of the syrup fed to the larvae of different ages of days are shown in table 1.
TABLE 1 syrup formulation, ratio (w/w%) and dosage to feed
3. Preparation of virus liquid
Chinese bee sacbrood with typical sacbrood morphology is selected from another Chinese bee farm of the bee institute of Chinese agricultural academy of sciences, and the existence of CSBV in the Chinese bee body is confirmed by RT-PCR. To obtain CSBV, 210 larvae infected with CSBV were taken, placed in a sterile triturator and 2100 μ L sterile Phosphate Buffered Saline (PBS) was added and triturated. Grinding, centrifuging at 8000rpm at 4 deg.C for 30min, repeating for 2 times, collecting supernatant, performing RT-PCR detection with cDNA reverse transcribed after RNA extraction as template, and storing the supernatant at-80 deg.C as original venom without other viruses.
Virus detection Using the Absolute quantitative PCR method (K.M.Hong, H.Najjar, M.Hawley, R.D.Press.quantitative real-time PCR with automatic sample preparation for diagnosis and monitoring of genomic infection in bone matrix genomic infection. clinical chemistry.2004,50(5):846 856; HU Zhi Gang, CHEN Ke Ping, YAO Qin, GAO Gui Tian, XU Jia-Ping, CHEN Hui-Qing.cloning and Characterification of genomic movement PP-BP, Gene Induced by Viral infection. acta.acta.acta.2005, En.48: 875)
The PCR reaction is carried out in a BIOER lineGene9600 real-time PCR system, and the upstream and downstream primers are respectively: 5'-ccttggagtttgctatttacg-3', and 5'-cctacatccttgggtcag-3'. The real-time fluorescent quantitative PCR reaction system is 15 mu L, comprises 7.5 mu L of reaction solution of 0.3 mu L, SYBR of each forward primer and reverse primer, 1 mu L of template and water till 15 mu L. The qPCR reaction conditions adopt a two-step method: the first step comprises a constant temperature section (denaturation at 95 ℃ for 3min) and a circulation section (denaturation at 95 ℃ for 5 s; annealing at 60 ℃ for 30s, 40 cycles); the second step is a melting section (9)Denaturation at 5 ℃ for 15 s; annealing at 60 ℃ for 5 s; denaturation at 95 ℃ for 15 s. ) Taking 1 mul of original venom to carry out qPCR, and obtaining the copy number of the original venom to be 6.74 multiplied by 104copies/. mu.L for subsequent vaccination experiments.
Preparing the toxic syrup: IC based on Pre-test Virus infection50And (3) detecting results, selecting the feed venom according to the proportion of the original venom: syrup (syrup composition see table 1) ═ 1: 3, preparing a mixture of 5 μ L virus stock solution and 15 μ L syrup, i.e. CSBV of 1.685 × 104copies/. mu.L of toxic syrup. The toxicant-containing syrup was fed to 3-day-old test larvae.
4. Preparation of 7-hydroxy coumarin preparation
7-Hydroxycoumarin (7-Hydroxycoumarin) is white needle crystal. Melting point 225 ℃ 228-. Easily soluble in alcohol, chloroform and acetic acid, soluble in dilute alkali solution, insoluble in ether, and slightly soluble in boiling water (1g/100 ml). The solution showed blue fluorescence. Can be sublimated. The structural formula of the 7-hydroxycoumarin is as follows:
the 7-hydroxycoumarin has activity of inhibiting the growth of a series of tumor cells of a human body, and has activity of inhibiting prostate cancer, malignant melanoma and metastatic renal carcinoma cells clinically, and the 7-hydroxycoumarin can inhibit the growth of tumor cells and the metastasis of infectious viruses.
In this example, a 7-hydroxycoumarin standard (source leaf product) was purchased, prepared into a 1mg/mL stock solution with high-purity water, and filtered and sterilized for future use. The test was conducted by diluting the test solution with 1.2 fold-by-fold of each day-old syrup (divided into low concentration 0.75. mu.g/mL, medium concentration 7.5. mu.g/mL and high concentration 75. mu.g/mL).
Preparation of the toxin-containing preparation: the original venom was mixed with 7-hydroxycoumarin preparation (syrup composition see table 1) at a ratio of 1: 3 to obtain a mixture of 5 μ L virus stock solution and 15 μ L7-hydroxycoumarin preparation, i.e. CSBV of 1.685 × 104copies/. mu.L of the toxin-containing preparation. The toxicant-containing formulation was fed to 3-day-old test larvae.
5. 7-Hydroxycoumarin intervention test
The larvae after preculture for 1 day (3 days old) were taken out of the incubator, and the larvae with good health status were placed in 48-well plates, and randomly divided into a control group, an infected group, a preparation group and an intervention group, each group consisting of 24 samples, and the steps were repeated three times. Grouping and feeding conditions are detailed in table 2.
Table 2 coumarin intervention test groups
The optimal time for infecting CSBV by the bee larvae is 2-3 days old, so that CSBV infection and 7-hydroxycoumarin intervention treatment are carried out when the larvae are 3 days old, and the prevention and treatment effects of 7-hydroxycoumarin on the CSBV infection of the bees are analyzed. Feeding the infection group with the syrup containing toxin (preparation method is described in the above 3), and feeding the intervention group with the preparation containing toxin (preparation method is described in the above 4); the larvae aged 4-6 days were fed with syrup or 7-hydroxycoumarin preparations, respectively, and only the larvae fed with syrup were used as CK control (the feeding amount is shown in Table 1). The above fed food is placed on one side of the bottom of the culture plate to avoid contacting with larvae. Feeding was done every 24h and feeding and mortality of larvae were observed and recorded.
Forceps were sterilized with 75% ethanol (prepared with DEPC-treated water) for 5min and rinsed three times with DEPC-treated water, 5 each of the differently treated live larvae were removed each day, immediately treated with liquid nitrogen for freezing, and then stored in a-80 ℃ freezer for subsequent viral copy number detection.
Example 2 survival and mortality analysis of coumarin after intervention in healthy larvae and infection with CSBV larvae
1. Survival rate
The survival rate of each group of bee larvae in example 1 is counted, the result is shown in fig. 1, and the result shows that the survival rate of the larvae of the CK group and the coumarin preparation group fed conventionally can reach more than 88%, no statistical difference exists, and the result shows that 7-hydroxycoumarin intervention cannot cause abnormal death of the larvae and does not influence the growth and development of healthy larvae.
The survival rate of the CK group larvae at 6 days of age is 94.43%, while the group infected with CSBV is dead in a large number, the survival rate is only 43.05%, and the statistics show a very significant difference. The survival rate of the infected CSBV larva is greatly improved after the intervention of 7-hydroxycoumarin, the survival rate of the infected CSBV larva reaches 87.5-93.06% at the age of 6 days, and the results show that the intervention of 7-hydroxycoumarin can antagonize the influence of CBSV infection on the survival of the larva, so that more than 50% of infected CSBV larva can be prevented from dying of illness, the infected CSBV bee colony is prevented from collapsing, and the method plays an important role in the recovery and the multiplication of the bee colony.
2. Mortality rate
Mortality statistics were performed on each group of bee larvae in example 1, and the mortality of the larvae was determined as follows: the death numbers of the larvae in the pre-culture (2 days old) and the virus inoculation day (3 days old) are not counted and analyzed to eliminate the influence of mechanical death of the larvae in the experimental process, the death numbers of the larvae are counted at regular time every day from the next day (4 days old) of virus inoculation, the death larvae are removed, and the steps are repeated until the 6 days old is finished.
Larval mortality per day-number of larvae dead per day/number of larvae alive per day x 100%;
overall larval mortality rate-number of larval deaths/total number of samples x 100%.
The statistical results of the mortality of all groups are shown in fig. 2, and the results show that the mortality of 4-6-day-old larvae in CK groups fed conventionally is lower than 3%, and the mortality of 4-6-day-old larvae after being dried by 7-hydroxycoumarin with various concentrations is not greatly increased every day, which indicates that the growth and development of healthy larvae are not influenced by coumarin intervention, and the feed can be used for auxiliary feeding of larvae.
CSBV is a typical induction source, and the death rate of larvae at the next day (4 days old) after 3 days old larvae are infected with CSBV is greatly increased and is up to 25 percent, the death rate at 5 days old and 6 days old is reduced, but the death rate per day is more than 20 percent, and only 43.05 percent of experimental larvae survive by 6 days old (figure 1). The study of Liang Qin et al (Liang Qin, Chendafu, bee protection science [ M ]. China agricultural Press, 2009.2-3.) found that CSBV is most susceptible to 2-3 days old larvae, the infected larvae cannot pupate, and the larvae die largely in the later stage of the larvae, which is consistent with the experimental results of the present invention.
The death rate of larvae in all groups infected by CSBV and interfered by 7-hydroxycoumarin is greatly reduced, and the death rate of CSBV infected groups is 56.95%; the mortality rate of the 0.75 mu g/mL concentration group is 12.5 percent and is reduced by 4.56 times; the mortality rate of the 7.5 mu g/mL concentration group is 6.94 percent and is reduced by 8.21 times; the mortality rate of the 75 mu g/mL concentration group is 11.11 percent and is reduced by 5.13 times; the death rate of larvae at each day age is greatly reduced, particularly the death rate of larvae at 4 days age is less than 5%, the survival rate of the larvae at 6 days age is up to more than 87%, and the detailed result is shown in figure 1.
Example 3 detection of viral copy number following coumarin intervention in healthy larvae and infection with CSBV larvae
CSBV test was performed on the 4-6 day old Apis cerana larva (CK group) with healthy appearance in example 1, and the number of Virus copies per day old larva was found to be 66-70, which indicated that apparently healthy larva also had Virus Infection but showed no obvious symptoms, indicating that CSBV generally had hidden Infection in bee colony, and that the larva could live with Virus in a certain concentration, which is consistent with the results of study on Liu Shan, L., Liuhao, W., Jun, G., Yujie, T., Yanping, C., Jie, W., Jilian, L., Chinese Sacbroud infestations in Asian Honey Bees (iris Apis cerana cerana) and Host response to the Virus Infection, Journal of neurology.2017, and dot htx.20125.09/357.006. The virus copy number of the internal bodies (preparation groups) of the 4-6-day-old apis cerana larvae with apparent health after the 7-hydroxycoumarin is dried is reduced to below 7, which shows that the intervention of the 7-hydroxycoumarin can improve the virus infection degree of the apparent health larvae and is beneficial to the normal growth and development of the larvae.
The virus copy number in the larvae of the infected group grows in an explosive manner, and the virus copy number in the larvae of 4 days old is as high as 12.1 multiplied by 104Is 1700 times of CK group, so the massive virus increment causes the infected CSBV larva to be died, the death rate is up to 25 percent, the virus copy number in the bodies of 5 days old and 6 days old is reduced along with the growth and development, but still is maintained at 2.4 multiplied by 104Above, it was shown that the single-day larval mortality rate was still above 20% (fig. 3).
The copy number of the single-day virus in the larvae with 4-6 days old of each concentration of 7-hydroxycoumarin intervention group is reduced to below 7 (figure 3), and the detection of CSBV can be regarded as no CSBV, which shows that after the feeding of coumarin, the infection of CSBV larvae by the virus generates strong antagonistic virus reaction, the infection degree of the larvae by the virus is obviously reduced, and the fatality rate of the infected CSBV larvae is reduced. The virus copy data is specifically as follows:
CSBV infection group: 4 days old 121000; 47100 days old; 23800 is 6 days old.
Concentration group of 0.75. mu.g/mL: 4 days old 1.94; 3.39 at 5 days old; 4.35 at 6 days old; compared with the CSBV infected group, the 4-day-old reduction is 62371 times, the 5-day-old reduction is 13893 times, and the 6-day-old reduction is 5471 times.
Concentration group of 7.5. mu.g/mL: 4 days old 2.53; 2.76 at 5 days old; 6 days old 3.64; compared with the CSBV infected group, the 4-day age is reduced by 47826 times, the 5-day age is reduced by 17065 times, and the 6-day age is reduced by 6538 times.
Concentration group of 75. mu.g/mL: 4 days old 2.48; 3.96 at 5 days old; 6.32 days old; compared with the CSBV infected group, the reduction of the 4-day age is 48790 times, the reduction of the 5-day age is 11893 times, and the reduction of the 6-day age is 3766 times.
Example 4 Effect of CSBV infection and coumarin intervention on the expression level of genes involved in larval immunity
To analyze the effect of CSBV Infection and 7-hydroxycoumarin intervention on the Immune system of bee larvae, the expression levels of the antimicrobial peptide genes in each group of bee larvae in example 1 were examined by real-time fluorescent quantitative PCR using β -actin as an internal reference gene (Shan, L., Liuhao, W., Jun, G., Yujie, T., Yanping, C., Jie, W., Jilian, L., Chinese Sacbrood Virus Infection in Asian Home Bees (Apis cerana cerana) and Host Immune Responses to the Virus Infection, Journal of Inverture Pathology.2017, doi http:// dx.doi.org/10.1016/j.j.j.2017.09.006). The detection result is specifically as follows:
1. expression detection of bee antibacterial peptide (Apidaecin) gene
The detection result is shown in fig. 4, the relative expression quantity of the Apidaecin of the larva of the Control (CK) group is only 1.24-1.55, after the larva is infected with CSBV for 24 hours, the relative expression quantity of the Apidaecin of the bee is increased in the larva body to 21.45, the 5-day-old bee reaches the maximum 22.21, and the 6-day-old bee falls back to 20.12.
The 7-hydroxycoumarin intervention has the effect of remarkably improving the expression level of Apidacin in both a CK group and an infected group, and for the CK group, the 7-hydroxycoumarin intervention effectively increases the expression level of Apidacin, and the expression level of Apidacin increases with the increase of age of days, and reaches 61.58-81.16 with the age of 6 days as the highest.
For an infected group, after 7-hydroxycoumarin intervention, the expression level of Apidacin is firstly reduced and then increased, the 6-day age reaches the highest value and reaches 53.41-92.07, meanwhile, the survival rate of larvae is increased from 43.05 percent of a CSBV infected group to 87.50-93.06 percent, and the death of the larvae caused by CBSV infection is obviously antagonized.
The invention intervenes the exogenous way through 7-hydroxycoumarin to strongly induce the expression capacity of the endogenous antimicrobial peptide Apidacetins, enhances the innate immunity defense of the larvae to resist CSBV invasion, and reduces the fatality rate. The result shows that the 7-hydroxycoumarin has the capacity of strongly inducing the high expression of the endogenous antimicrobial peptide Apidaecins of healthy larvae and infected CSBV larvae.
2. Hymenoptera antibacterial peptide (Hymenoptaecin)
As shown in FIG. 5, the relative expression level of hymenoptera antimicrobial peptides of the larvae of the Control (CK) group was only 1.31-1.56, and after 24 hours of infection with CSBV, the expression level of Hymenoptaecin in the larvae was increased, and the relative expression level of the larvae aged 4-6 days was maintained at about 20.47-21.86 without significant change.
The intervention of 7-hydroxycoumarin on healthy larvae can cause the expression level of Hymenoptaecin to be remarkably increased, and the expression level of Hymenoptaecin of 5-day-old larvae is increased to 331.17 at the highest.
The intervention of 7-hydroxycoumarin on infected CSBV larvae also greatly increases the expression level of Hymenoptacin, and the expression level of 5-day-old larvae is the highest and can reach 788.18.
The results show that the exogenous approach intervenes by 7-hydroxycoumarin to strongly induce the expression capacity of endogenous antibacterial peptide Hymenoptacin, enhance the innate immunity defense of larvae to resist CSBV invasion and reduce the fatality rate. The 7-hydroxycoumarin is proved to have the capacity of strongly inducing the high expression of the endogenous antibacterial peptide Hymenoptaecin of healthy larvae and infected CSBV larvae.
3. Bee moth antibacterial peptide (Abaecin)
As shown in FIG. 6, the expression level of Abaecin in larvae of healthy Control (CK) group is only 1.90-3.69, and after 24 hours of CSBV infection, the expression level of Abaecin in larvae is remarkably increased to 73.25, but the expression level of Abaecin in 5-6 days drops back to 25.4-65.73.
Administration of 7-hydroxycoumarin intervention to healthy larvae caused an upregulation of abeecin expression, with only moderate concentrations being slightly downregulated to 1.44 at 6 days of age. After the 7-hydroxycoumarin is intervened for 24 hours, the Abaecin expression level in the larvae is up-regulated to 34.16-43.12, the 5-day-old larvae reach 72.14-190.52 at the maximum, the 6-day-old larvae fall back to be equivalent to CK, and the Abaecin expression level is 1.44-16.44.
The intervention of 7-hydroxycoumarin is given to infected CSBV larvae, the Abaecin expression of 5-day-old larvae can be greatly increased, the expression level is up to 119.22, the survival rate of the larvae is increased from 43.05% of an infected group to 87.50-93.06%, the 7-hydroxycoumarin intervention is shown to induce the high expression of the Abaecin gene of the bee antibacterial peptide, and the death of the larvae caused by CBSV infection is resisted.
4. Bee Defensin (Defensin)
The detection result is shown in fig. 7, the Defensin expression level of the larvae in the healthy Control (CK) group is only 1.32-1.62, the Defensin expression level in the larvae is increased to 46.26 after the larvae are infected with CSBV for 24 hours, the maximum Defensin expression level of 5 days is 52.59, and the Defensin expression level of 6 days is fallen back to 22.28.
The intervention of 7-hydroxycoumarin on healthy larvae can cause the expression level of the Defensin to be up-regulated, and the maximum expression level is up-regulated to about 50; however, the intervention of 7-hydroxycoumarin in infected CSBV larvae only limited up-regulated the amount of Defensin expression in 6-day-old infected CSBV larvae.
According to the invention, the dynamic change of the expression of the Immune related genes in larvae of 4-6 days old of Chinese Bees infected with CSBV is analyzed, so that the expression of the antibacterial peptide genes in the larvae infected with CSBV is higher than that of healthy larvae on the whole, and the result shows that the Infection of CSBV can promote the expression of the Immune related genes in the larvae of the Bees, and is matched with the result of research on Liu-Shan (Shan, L., Liuhao, W., Jun, G., Yujie, T., Yanping, C., Jie, W., Jiian, L., Chinese Sacbrood Virus Infection in Asian House Bees (Apis cerana cerana) and Host Immune Responses to the Virus Infection, Journal of Invertebpath Pathology.2017, doi: http:// dx.dog/10.1016.006.2017.09).
The invention takes Chinese bee infected with CSBV and healthy Chinese bee as research objects, and utilizes a fluorescent quantitative PCR method to quantitatively detect and compare the expression conditions of 4 antibacterial peptide genes in the disease-sensitive and healthy bee larva development stages, thereby defining the immune response condition of the Chinese bee after CSBV infection; and quantitatively detecting and comparing the expression conditions of 4 antibacterial peptide genes after 7-hydroxycoumarin is given to infected and healthy bee larvae: the intervention of 7-hydroxycoumarin strongly induces the expression capacity of endogenous antimicrobial peptide hymenoptera antimicrobial peptide (Hymenoptaecin), bee moth antimicrobial peptide (Abaecin) and bee antimicrobial peptide (Apidaecin) of infected CSBV larvae. The high expression of the three antibacterial peptides for one day or more resists the invasion of CSBV to larvae, reduces the fatality rate of the larvae, promotes the larvae to pupate and emerge into adult bees, and is an effective method for enhancing the immunity of the larvae of the Chinese bees and resisting CSBV infection; meanwhile, the 7-hydroxycoumarin intervention can also up-regulate the expression level of the healthy bee larva antibacterial peptide to form a rapid and effective defense mechanism for rapidly killing or eliminating exogenous microorganisms and protecting the growth and development of larvae.
In conclusion, the coumarin compounds including the 7-hydroxycoumarin can inhibit the replication of CSBV and reduce the copy number of CSBV in the bee larva body; can also induce the expression of the endogenous antibacterial peptide of the bee, improve the innate immunity defense of the bee larva, resist the invasion of CSBV to the larva and prevent the further infection of CSBV and other pathogenic microorganisms, thus being used for preventing and treating the sacbrood disease of the bee.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> bee institute of Chinese academy of agricultural sciences
Application of <120> coumarin in resisting virus infection of bees
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Claims (9)
- Use of 7-hydroxycoumarin in the manufacture of a medicament for the treatment or prevention of vesicular virus infection in bees.
- Use of 7-hydroxycoumarin in the preparation of a feed for the treatment or prevention of vesicular virus infection in bees.
- 3.7-hydroxycoumarin in preparing medicine for inhibiting bee sacbrood virus proliferation in bee.
- 4.7-hydroxycoumarin in preparing medicine for improving bee immunity.
- Application of 7-hydroxycoumarin in preparing feed for improving immunity of bees is disclosed.
- 6. The use of claim 4 or 5, wherein the improvement of bee immunity is achieved by increasing the expression level of endogenous antimicrobial peptides of bees;the endogenous antibacterial peptide is any one or more of hymenoptera antibacterial peptide, bee antibacterial peptide and bee moth antibacterial peptide.
- 7. The use according to any one of claims 1 to 5, wherein the 7-hydroxycoumarin is effective in reducing the number of copies of a virus infecting CSBV bee larvae in a concentration range of 0.75 to 75 μ g/mL.
- 8. The use according to any one of claims 1, 3 or 4, wherein the active ingredient of the medicament comprises 7-hydroxycoumarin.
- 9. Use according to claim 2 or 5, wherein the active ingredient of the feed comprises 7-hydroxycoumarin.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1220263A (en) * | 1997-10-02 | 1999-06-23 | 普利瓦药物，化学，食品，化妆品工业公司 | Novel hydroxy and polyhydroxy derivatives of coumarin, preparation thereof and antiviral action thereof |
| WO2015089078A1 (en) * | 2013-12-10 | 2015-06-18 | Beeologics, Inc. | Compositions and methods for virus control in varroa mite and bees |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1220263A (en) * | 1997-10-02 | 1999-06-23 | 普利瓦药物，化学，食品，化妆品工业公司 | Novel hydroxy and polyhydroxy derivatives of coumarin, preparation thereof and antiviral action thereof |
| WO2015089078A1 (en) * | 2013-12-10 | 2015-06-18 | Beeologics, Inc. | Compositions and methods for virus control in varroa mite and bees |
Non-Patent Citations (2)
| Title |
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| Antimicrobial properties of Fomitopsis officinalis in the light of its bioactive metabolites: a review;Carolina Girometta;《Mycology》;20181025;第10卷(第1期);第32-39页 * |
| 蒙药小白蒿化学成分的研究;王青虎;《中草药》;20091031;第40卷(第10期);第1540-1543页 * |
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