Application of Manganese-Containing Compound in Preparation of Drug for Preventing and Treating Bombyx mori Nuclear Polyhedrosis Virus

Abstract

The present invention belongs to the field of agricultural biotechnology. Specifically, the present invention relates to the application of manganese-containing compounds in the preparation of drugs for preventing and controlling Bombyx mori nucleopolyhedrovirus. Through a large number of studies, the present invention discovers that manganese-containing compounds can be used to prevent and control Bombyx mori nucleopolyhedrovirus (BmNPV), and at the same time, can also increase the body weight of Bombyx mori and silkworm cocoons themselves. The discovery of the present invention provides a new idea for the prevention and control of BmNPV infection in Bombyx mori, and also lays a foundation for the prevention and control of virus infection in sericulture production. The compounds provided by the present invention have low drug costs, are energy-saving and environmentally friendly, and have obvious prevention and control effects.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and specifically, the invention relates to application of a manganese-containing compound in preparing a drug for preventing and treating Bombyx mori nuclear polyhedrosis virus. Background Art

[0002] Bombyx mori nuclear polyhedrosisvirus (BmNPV) is one of the main pathogens of silkworm breeding. The disease often breaks out in the silkworm breeding countries in the world. It has the characteristics of strong infectivity and no specific medicine, which will cause serious economic losses every year. Among the pathogens of silkworm viral diseases, baculovirus is a toxic pathogen that infects a variety of insects. BmNPV is a typical baculovirus, belonging to group I, the main envelope protein, and BmNPV causes infection through the specific interaction between the structural protein on the ODV envelope and the host midgut cell surface receptor. At present, the prevention and control of BmNPV mainly relies on "good varieties" + "good methods", that is, selecting silkworm varieties with excellent resistance and scientific and standardized feeding methods. At present, some varieties with strong resistance to BmNPV have been obtained through molecular biology assisted breeding. At the same time, attention should be paid to the disinfection of the breeding environment and raw materials when breeding silkworms, strengthening the prevention and control of mulberry garden pests and diseases, improving the quality of mulberry leaves, strengthening the management of silkworm breeding, improving the climate environment of the silkworm room, and improving the physique of silkworms. These are measures that cannot be ignored in the prevention and control of BmNPV disease. Further research is still needed to improve the disease resistance of silkworms or to discover drugs to control BmNPV in silkworms. Summary of the invention

[0003] In the course of studying the immunology of silkworms, the inventors unexpectedly discovered through extensive testing that manganese-containing compounds can be used to prevent and treat Bombyx mori nuclear polyhedrosis virus. 2+ ) can also increase the weight of silkworm cocoons themselves.

[0004] The technical solution of the present invention is as follows:

[0005] In the first aspect of the present invention, the present invention provides the use of a manganese-containing compound in the preparation of a drug for preventing and treating Bombyx mori nuclear polyhedrosis virus, wherein the manganese-containing compound is applied to silkworms, thereby enabling the silkworms to obtain disease resistance against Bombyx mori nuclear polyhedrosis virus (BmNPV). The application is to allow the silkworms to ingest the manganese-containing compound by oral feeding.

[0006] In one embodiment, the administering comprises adding the manganese-containing compound to the silkworm's food and feeding the silkworm with the food.

[0007] In one embodiment, the applying is spraying the manganese-containing compound onto mulberry leaves, which are food for the silkworms, and using the mulberry leaves as food for the silkworms.

[0008] In the present invention, the manganese-containing compound is not particularly limited, and any manganese-containing compound that does not poison and affect the growth of silkworms can be used. Preferably, the manganese-containing compound is a divalent manganese-containing compound. Examples of the manganese-containing compound include any one of manganese chloride, manganese bromide, manganese iodide, manganese sulfate, manganese nitrate, manganese perchlorate, manganese acetate, manganese carbonate, manganese borate, manganese phosphate, manganese hydrobromide, manganese tartrate, manganese fumarate, manganese maleate, manganese lactate, manganese benzenesulfonate, manganese pantothenate, manganese ascorbate, manganese phosphate salt, manganese carbonate salt, and manganese hydroxide, and any combination thereof.

[0009] In the present invention, when the manganese-containing compound is used for silkworms, the Mn 2+ concentration is 50 - 100 uM.

[0010] In the present invention, the administration time of the manganese-containing compound can be before infection with BmNPV or within 12 h after infection with BmNPV.

[0011] In the second aspect of the present invention, the present invention provides the use of a manganese-containing compound in the preparation of a drug for increasing the self-weight of silkworm cocoons. The manganese-containing compound includes manganese chloride and manganese sulfate.

[0012] In the present invention, in addition to the manganese-containing compound, the drug further includes a pharmaceutically acceptable excipient. The selection and dosage of the excipient are based on the principle of not having a negative impact or toxicity on the growth of silkworms.

[0013] In the present invention, the dosage forms of the drug include: powder, solid granule, and liquid preparation.

[0014] Beneficial effects

[0015] (1) The present invention discovers that the Mn 2+ solution can effectively inhibit the replication of Bombyx mori nucleopolyhedrovirus (BmNPV) in cells and silkworms, providing a new idea for the prevention and treatment of BmNPV infection in silkworms, and also laying a foundation for the prevention and treatment of virus infection in sericulture production.

[0016] (2) The present invention discovers that when the Mn 2+ solution acts on silkworms, it can not only enhance the immune ability of silkworms against BmNPV, but also increase the self-weight of silkworms and the amount of cocoons.

[0017] (3) The manganese-containing compound provided by the present invention has a low drug cost, is simple and convenient to use, has more environmental value than pesticides, is energy-saving and environmentally friendly, has a low price, and has an obvious prevention and treatment effect. Description of the drawings

[0018] Figure 1, After poisoning and then feeding mulberry leaves, the effects of different treatments on the virus in silkworms and the morphology of cocoons. Among them, A is the replication quantity of viral gDNA in the midgut; B is the replication quantity of viral gDNA in the hemocyte; C is the replication quantity of viral gDNA in the fatbody, and D is the morphology of cocoons in different groups.

[0019] Figure 2 , After poisoning for 12 h and then feeding mulberry leaves, the concentration of the MnCl2 group sprayed on the mulberry leaves is 100 μM, and the effects of different treatments on silkworms and cocoons are analyzed. Among them, A is the comparison of the weights of larvae, B is the comparison of the weights of silkworm pupae, C is the comparison of the morbidity rate, D is the comparison of the mortality rate, E is the comparison of the cocooning rate, F is the comparison of the weights of cocoons, G is the comparison of the cocoon layer rate, H is the comparison of the weights of cocoon shells; I is the replication quantity of viral gDNA in the midgut; J is the replication quantity of viral gDNA in the hemocyte; K is the replication quantity of viral gDNA in the fatbody.

[0020] Figure 3 , After poisoning for 24 h and then feeding mulberry leaves, the concentration of the MnCl2 group sprayed on the mulberry leaves is 100 μM, and the effects of different treatments on silkworms and cocoons are analyzed. Among them, A is the comparison of the weights of larvae, B is the comparison of the weights of silkworm pupae, C is the comparison of the morbidity rate, D is the comparison of the mortality rate, E is the comparison of the weights of cocoons, F is the comparison of the cocooning rate, G is the comparison of the cocoon layer rate, H is the comparison of the weights of cocoon shells. I is the quantity of viral gDNA in the midgut; J is the quantity of viral gDNA in the hemocyte; K is the quantity of viral gDNA in the fatbody.

[0021] Figure 4 , The inhibitory effects (C, D, E) of different treatments on viral VP39 mRNA in different parts (midgut, hemocyte, fatbody) of silkworms, and the amounts of gDNA (F, G, H), as well as the effects (A, B) of different treatments on the weight of silkworms themselves and the consumption of mulberry leaves.

[0022] Figure 5 , After poisoning and then feeding mulberry leaves, the effects of different treatments and different concentrations of MnSO4 on silkworms. Among them, A is the comparison of the weights of larvae, B is the comparison of the morbidity rate, C is the comparison of the mortality rate, D is the comparison of the cocooning rate, F is the comparison of the weights of cocoon shells, G is the comparison of the weights of pupae, H is the comparison of the cocoon layer rate, I is the comparison of the weights of cocoons.

[0023] Figure 6, After virus inoculation and feeding mulberry leaves, the effects of different treatments and different concentrations of MnSO4 on the morphology of silkworms and cocoons. Among them, A is the amount of viral gDNA in the midgut; B is the amount of viral gDNA in the hemocyte; C is the amount of viral gDNA in the fat body; D is the morphology of cocoons in different groups.

[0024] Figure 7 , After virus inoculation and feeding mulberry leaves, the effects of different groups on the virus in silkworms. Among them, A is the amount of viral gDNA in the midgut; B is the amount of viral gDNA in the hemocyte; C is the amount of viral gDNA in the fat body.

[0025] Figure 8 , After virus inoculation and feeding mulberry leaves, the effects of different treatments on the morphology of silkworms and cocoons. Among them, A is the comparison of the weight of larvae; B is the comparison of the weight of pupae; C is the comparison of the morbidity rate; D is the comparison of the mortality rate; E is the comparison of the cocooning rate; F is the comparison of the weight of cocoons; G is the comparison of the weight of cocoon shells; H is the comparison of the cocoon layer rate; I is the morphology of cocoons in different treatments. Detailed implementation mode

[0026] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] Example 1:

[0028] 1.1 Reagent: MnCl2, molecular weight 197.90. The dissolution and storage concentration is 200 mM. Weigh 3.9858 g and dissolve it in 100 ml of water. When used, it is diluted to three gradients of 50 μM, 75 μM, and 100 μM for experimental research.

[0029] 1.2 Material: Medicated mulberry leaves: Pick fresh mulberry leaves, weigh a fixed weight, and spray a certain amount of 50 μM, 75 μM, and 100 μM Mn2+ on the mulberry leaves for feeding silkworms.

[0030] 1.3 Antiviral test of different concentrations of Mn2+

[0031] (1) In vivo experiment on silkworms: Select well-growing fifth-instar silkworms and divide them into 5 groups, namely the control group (Control), the polyhedra virus group (Polyhedra), the PM1 group (Polyhedra + 50 μM MnCl2), the PM2 group (Polyhedra + 75 μM MnCl2), and the PM3 group (Polyhedra + 100 μM MnCl2). During feeding, the control group was orally fed 5 μl of PBS; the polyhedra virus group was fed 5 μl of BmNPV (concentration of 10 7 ODV), and the PM1, PM2, and PM3 groups were respectively orally fed 5 μl of BmNPV (concentration of 10 7 ODV). After virus inoculation, different concentrations of Mn 2+ reagent (50 μM, 75 μM, 100 μM MnCl2) were sprayed on the surface of mulberry leaves in a fixed amount to feed the silkworms. The silkworms continued to feed on mulberry leaves of different concentrations. After continuing to feed for 24, 48, and 72 h, the larval blood, fat body, and midgut were taken for genomic DNA extraction, and the replication of the viral gene VP39 in the silkworm body was detected by fluorescence quantitative PCR. At the same time, the cocoon morphology of different groups was analyzed.

[0032] Results: It can be seen from Figure 1 that the use of different concentrations of Mn 2+ can inhibit the proliferation of multiple viruses in the bodies of silkworm larvae at 24 h, 48 h, and 72 h, and shows a concentration-dependent relationship. Regarding the virus inhibition effect of different concentrations, 100 μM has the best inhibitory effect on BmNPV in silkworms. From the perspective of the silkworm morphology, the addition of the virus can cause the silkworms to grow slowly, reduce the cocoon formation rate, and decrease the cocoon weight. However, the morphology of the group with the addition of high-concentration Mn 2+ is better than that of other concentration groups and the virus group.

[0033] (2) Experiment on feeding silkworms with medicated mulberry leaves: Select well-growing fifth-instar silkworms and divide them into 5 groups, namely the control group (Control), the polyhedra virus group (Polyhedra), and the PM group (Polyhedra + 100 μM MnCl2). During feeding, the control group was orally fed 5 μl of PBS; the polyhedra virus group and the PM group were respectively orally fed 5 μl of BmNPV (concentration of 10 7 ODV). After virus inoculation, the silkworms were normally fed. At 12 and 24 h after virus infection, medicated mulberry leaves were fed to prevent the outbreak of BmNPV virus. After continuing to feed for 24, 48, and 72 h, the larvae were taken for mRNA and genomic DNA extraction, and the replication of the viral gene VP39 in the silkworm body was detected by fluorescence quantitative PCR. At the same time, during the feeding process of the silkworms, the effects of different treatments on the silkworms and cocoons were observed and statistically analyzed.

[0034] Results: SeeFigure 2 and Figure 3 It can be seen that using 100 μM Mn within 12 h after the virus outbreak 2+ can effectively inhibit virus proliferation, and is superior to the control group in terms of larval weight, pupa weight, cocoon weight, cocoon layer rate, and cocoon shell weight, and is significantly superior to the virus-infected group, with a substantial reduction in the incidence and mortality rate. However, the control effect is poor when the drug is administered 24 h after the virus outbreak. At the same time, the results of this example show that adding Mn 2+ in the present invention can not only play an antiviral role, but also improve the weight and quality of silkworm cocoons.

[0035] Example 2

[0036] Take well-growing fifth-instar silkworms and divide them into 4 groups, namely the control group (Control), MnCl2 group, polyhedra virus group (Polyhedra), and Polyhedra + MnCl2 100 μM group. During feeding, the control group was orally fed 5 μl of PBS; for the polyhedra virus group, the other groups were orally fed 5 μl of BmNPV (concentration of 10 7 ODV). After virus inoculation, the silkworms were fed with medicated mulberry leaves. After continuing to feed for 24, 48, and 72 h, larvae were taken for RNA extraction. After reverse transcription, the expression of VP39 was detected by fluorescence quantitative PCR; at the same time, the changes in the body weights of silkworms in different groups were also evaluated.

[0037] Results: See Figure 4 It shows that adding Mn 2+ in the present invention can inhibit the transcription of the VP39 gene in the virus, and at the same time, using Mn 2+ increases the body weight of silkworms.

[0038] Example 3

[0039] Take well-growing fifth-instar silkworms and divide them into 4 groups, namely the control group (Control), polyhedra virus group (Polyhedra), PS1 group (Polyhedra + MnSO4 50 μM), and PS2 group (Polyhedra + MnSO4 100 μM). During feeding, the control group was orally fed 5 μl of PBS; for the polyhedra virus group, PS1 and PS2 were orally fed 5 μl of BmNPV (concentration of 10 7 ODV), and Mn 2+Silkworms were continuously fed with mulberry leaves containing different concentrations (50 μM, 100 μM) of the reagent. After 24, 48, and 72 hours of continuous feeding, the genomic gDNA was extracted from the larval blood, fat body, and midgut, and the replication of the viral gene VP39 in the silkworm body was detected by fluorescence quantitative PCR. At the same time, during the feeding process of the silkworms, the effects of different treatments on the silkworms and cocoons were observed and counted.

[0040] Results: Refer to Figure 5 , Figure 6 The results show that the use of 50 μM and 100 μM of MnSO4 can also play a role in inhibiting BmNPV, and it is superior to the control group in terms of larval weight, pupal weight, cocoon weight, cocoon layer rate, and cocoon shell weight, and is significantly superior to the virus-infected group, with a significant reduction in the incidence and mortality rate.

[0041] Example 4 confirmed that it is Mn 2+ , rather than Cl2, that plays a role

[0042] Take well-growing fifth-instar silkworms and divide them into 4 groups, namely the control group (Control), the polyhedra virus group (Polyhedra), the PMS group (Polyhedra + 50 μM MnCl2), and the PN group (Polyhedra + 200 μM NaCl). During the feeding, the control group was orally fed 5 μl of PBS; the polyhedra virus group, the PMS group, and the PN group were orally fed 5 μl of BmNPV (concentration of 10 7 ODV), and they were continuously fed with mulberry leaves sprayed with PBS, 50 μM MnCl2, or 200 μM NaCl. After 24, 48, and 72 hours of continuous feeding, the mRNA and genomic DNA were extracted from the larval blood, fat body, and midgut, and the replication of the viral gene VP39 in the silkworm body was detected by fluorescence quantitative PCR. At the same time, during the feeding process of the silkworms, the effects of different treatments on the silkworms and cocoons were observed and counted.

[0043] Results: Refer to Figures 7-8 It shows that the use of 50 μM MnCl2 can significantly inhibit the proliferation of the virus in the silkworm body, while 200 μM NaCl is close to the normal control group. The same pattern is observed in terms of larval weight, pupal weight, cocoon weight, cocoon layer rate, and cocoon shell weight, that is, it is confirmed that it is Mn 2+ in the reagent that plays a role, rather than Cl2.

[0044] In summary, through the detection of silkworms, the present invention found that a concentration of 50 - 100 μM Mn 2+ can well inhibit the BmNPV virus. It not only has a good inhibitory effect on the virus, but also has a promoting effect on the silkworms themselves, increasing Mn 2+It promotes the weight of the silkworm itself, and the cocoon weight correspondingly increases, and the food intake also greatly increases. By dissecting the blood, fat body, and midgut to extract DNA and RNA samples for detection, the fluorescence quantitative detection shows a good inhibitory effect on the virus expression level, which is consistent with the phenotypic performance of the silkworm. It can effectively inhibit the replication of Bombyx mori nucleopolyhedrovirus (BmNPV) in cells and the silkworm body.

[0045] The above content further elaborates on the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as falling within the protection scope determined by the claims submitted for the present invention.

Claims

1. Use of a manganese-containing compound in the preparation of a drug for preventing and treating Bombyx mori nuclear polyhedrosis virus, characterized in that, Applying a manganese-containing compound to silkworms enables the silkworms to acquire disease resistance against Bombyx mori nucleopolyhedrovirus (BmNPV), and the manganese-containing compound is manganese chloride or manganese sulfate.

2. The application according to claim 1, characterized in that, The application is by orally adding the manganese-containing compound to the silkworms.

3. The application according to claim 1, characterized in that The application is by adding the manganese-containing compound to the food of the silkworms and feeding the food to the silkworms.

4. The application according to claim 3, characterized in that The application is by spraying the manganese-containing compound onto the mulberry leaves, which are the food of the silkworms, and using the mulberry leaves as the food for the silkworms.

5. The application according to claim 1, characterized in that When the manganese-containing compound is used, the Mn 2+ concentration is 50 - 100 uM.

6. The application according to claim 1, characterized in that, The administration time of the manganese-containing compound is before infection with BmNPV or within 12 h after infection with BmNPV.

7. Use of a manganese-containing compound in the preparation of a drug for increasing the body weight of silkworm cocoons, characterized in that, The manganese-containing compound is manganese chloride or manganese sulfate.

8. The application according to claim 1 or claim 7, characterized in that The drug also contains a pharmaceutically acceptable excipient.

9. The application according to claim 1 or claim 7, characterized in that, The dosage form of the drug is powder, solid granule or liquid preparation.

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