Use of EGCG in preparation of medicine for preventing and treating bombyx mori nuclear polyhedrosis virus

Abstract

The application belongs to the field of agricultural biotechnology, and particularly relates to application of EGCG in preparation of a medicine for preventing and treating Bombyx mori nuclear polyhedrosis virus (BmNPV). The application finds through extensive research that EGCG can effectively inhibit replication and transmission of BmNPV, reduce damage of the virus to a host, and improve economic benefits of a silkworm mulberry industry, thereby providing a new strategy for prevention and treatment of BmNPV of Bombyx mori.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology. Specifically, this invention relates to the application of EGCG in the preparation of drugs for the prevention and treatment of silkworm nucleopolyhedrovirus (BmNPV). Background Technology

[0002] Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly pathogenic baculovirus belonging to the Baculoviridae family. The virus particle is rod-shaped, approximately 400 nanometers long and 90 nanometers in diameter, consisting of a capsid enclosing a double-stranded DNA core and an outer envelope. BmNPV is a key limiting factor in the sericulture industry; infection can cause swelling of the silkworm's body surface, systemic suppuration, and even death, severely hindering the development of sericulture. The virus invades midgut epithelial cells, replicates in the cell nucleus, and then spreads to tissues such as fat bodies and hemocytes, ultimately leading to host death. The resulting loss in cocoon production accounts for more than 60% of total silkworm disease losses.

[0003] Currently, the main measures for controlling this disease include breeding resistant varieties, improving management techniques, using chemical prevention, and researching chemical control. Some conventional control methods include physical isolation, chemical disinfectants, and traditional antiviral drugs, but these methods have problems such as incomplete blocking of virus transmission, environmental pollution, the development of drug resistance, and drug residues. Therefore, exploring safe, efficient, and environmentally friendly methods to combat BmNPV has become an urgent research need.

[0004] EGCG, a classic green tea polyphenol extract, has been found to possess strong antiviral activity in recent years. For example, studies have shown its inhibitory effects on viruses such as influenza A virus, human immunodeficiency virus (HIV), herpesvirus, and hepatitis C virus (HCV). Its mechanism of action involves multiple levels, including directly blocking the binding of viruses to host cells, inhibiting viral replication enzyme activity, and regulating the host cell's immune response. However, the activity and efficacy of EGCG against silkworm virus have not yet been reported. Summary of the Invention

[0005] Developing highly effective and low-toxicity agents against silkworm nucleopolyhedrovirus (BmNPV) is a crucial problem urgently needing to be solved in the sericulture industry. This invention, through systematic screening and in-depth research, is the first to discover that epigallocatechin gallate (EGCG) can effectively inhibit the replication and spread of BmNPV in silkworms, thus providing a novel solution for the prevention and control of BmNPV infection in silkworms. This invention is based on this finding.

[0006] In one aspect, the present invention discloses the use of EGCG in the preparation of a medicament for the prevention and / or treatment of silkworm nucleopolyhedrovirus (BmNPV) infection.

[0007] In one embodiment, the drug further includes pharmaceutically acceptable excipients.

[0008] In this invention, the dosage form of the drug includes, but is not limited to, solution, wettable powder or granules.

[0009] Another aspect of the present invention discloses a method for preventing and controlling silkworm nucleopolyhedrovirus, the method comprising administering an effective dose of an EGCG-containing preparation to silkworms.

[0010] In one embodiment, the preparation may be administered before the silkworm is infected with BmNPV.

[0011] In one embodiment, the preparation may be administered within 12 hours after the silkworm is infected with BmNPV.

[0012] In a preferred embodiment, the preparation is applied by mixing it into mulberry leaves for silkworms to consume.

[0013] Beneficial effects

[0014] Green and safe: The EGCG of this invention, as a natural product, has good biodegradability, low residue in the environment, and low toxicity to non-target organisms, which meets the requirements of green and sustainable development of sericulture.

[0015] Highly effective antiviral: The EGCG of this invention can effectively block the replication and intercellular spread of BmNPV. Especially when intervened in the early stage of viral infection (within 12 hours), it can significantly inhibit viral proliferation and has a significantly improved antiviral ability compared with conventional antioxidants, providing a precise and effective intervention window for the prevention and treatment of BmNPV. Attached Figure Description

[0016] Figure 1 This study demonstrates the effects of different compound treatments on the activity of BmNPV-infected cells.

[0017] Figure 2 This study shows the effect of different compound treatments on the relative expression level of the BmNPV virus GP64 gene mRNA.

[0018] Figure 3 The results of the toxicity test of EGCG on silkworm BmN cells showed that EGCG had no effect on cell growth.

[0019] Figure 4 Edu staining was used to detect cell apoptosis after EGCG treatment.

[0020] Figure 5 Dead cell staining method to detect the viability of cells.

[0021] Figure 6 Effects of different concentrations of EGCG on viral gene (VP39) expression and replication in cells.

[0022] Figure 7 Western blot analysis was used to detect the effect of EGCG on the expression level of viral GP64 protein.

[0023] Figure 8 The inhibitory effect of EGCG on the replication of BmNPV in the hemolymph of silkworm.

[0024] Figure 9 The inhibitory effect of EGCG on the replication of BmNPV in the fat body of silkworm.

[0025] Figure 10 The inhibitory effect of EGCG on the replication of BmNPV in the midgut of silkworm. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0027] Example 1: Screening of compounds against BmNPV

[0028] Assay Methods: To screen for effective anti-BmNPV compounds, silkworm BmN cells were treated with an equivalent concentration (10 µM) of EGCG and various reducing agents (including β-mercaptoethanol / BME, dithiothreitol / DTT, tris(2-carboxyethyl)phosphine / TCEP, glutathione / GSH, and N-acetylcysteine / NAC). Cell viability was assessed by detecting OD490 values ​​at different time points (24, 48, and 72 hpi). A DMSO solvent control group was also included for comparison.

[0029] Test results: The results are as follows Figure 1As shown, at an initial screening concentration of 10 µM, the OD490 value of the EGCG-treated group remained the highest in the late infection stage (72 hpi) compared to the DMSO control group, indicating that it could most effectively maintain cell viability. Further concentration gradient experiments confirmed that EGCG at a concentration of 10 mM achieved the best inhibitory effect on BmNPV, significantly superior to other reducing agents tested.

[0030] Example 2: The significant advantage of EGCG in inhibiting BmNPV GP64 gene transcription

[0031] Methods: To evaluate and compare the inhibitory effects of EGCG and various known reducing agents / antioxidants on key BmNPV genes at the transcriptional level, we used RT-qPCR to detect the relative mRNA expression levels of the viral GP64 gene in different treatment groups (including β-mercaptoethanol / BME, dithiothreitol / DTT, tris(2-carboxyethyl)phosphine / TCEP, glutathione / GSH, N-acetylcysteine / NAC, and EGCG). All compounds and the solvent control group (DMSO) were treated under the same conditions.

[0032] Test results: The results are as follows Figure 2 As shown, compared with the DMSO control group, all tested reducing agents / antioxidants significantly downregulated the mRNA expression level of the GP64 gene (**P<0.01, ***P<0.001). Among them, the EGCG treatment group had the lowest relative expression level of GP64 mRNA, and its inhibitory effect was significantly better than all other reducing agents. This indicates that EGCG has the most prominent inhibitory effect on the BmNPV GP64 gene at the transcriptional level.

[0033] Example 3: Detection of EGCG toxicity to BmN in silkworm cells

[0034] BmN cells from silkworms were seeded into 96-well plates (5 × 10³ cells / well) and cultured overnight. Then, EGCG solutions with concentrations of 20 μM and 40 μM were added, respectively. Blank control (culture medium) and negative control (cells + culture medium) were set up. Cell viability was detected by MTT assay at 0 h, 24 h, 48 h, 72 h and 96 h.

[0035] Test results: As shown in Figure 3, 20uM and 40uM EGCG were not toxic to BmN cells, indicating that they have good safety.

[0036] Example 4: Detection of the cellular efficacy of EGCG against silkworm nucleopolyhedrovirus (BmNPV)

[0037] 4.1 Cell death inhibition experiment

[0038] Test method: After BmN cells were infected with BmNPV, they were treated with 20uM and 40uM EGCG, and cell apoptosis was detected by Edu staining (green fluorescence indicates apoptotic cells, and blue indicates Edu nuclear staining).

[0039] Results: As shown in Figure 4, the number of apoptotic cells in the BmNPV-infected group was significantly higher than that in the uninfected group, while the number of apoptotic cells in the EGCG-treated group was significantly lower than that in the BmNPV-infected group, and the effect was better with increasing concentration, indicating that EGCG can alleviate virus-induced cell death.

[0040] 4.2 Protective effect against cell death

[0041] Test method: To evaluate the effect of EGCG on cell death induced by BmNPV infection, BmN cells were treated with 20 μM and 40 μM EGCG after viral infection, and the cell viability was detected by dead cell staining.

[0042] Results: The results are as follows Figure 5 As shown, under BmNPV infection conditions, the number of dead cells in the EGCG-treated group was significantly reduced compared to the DMSO solvent control group, and this protective effect was concentration-dependent, meaning that treatment with 40 μM EGCG was more effective than that with 20 μM. This indicates that EGCG can effectively inhibit cell death induced by BmNPV infection.

[0043] 4.3 Virus proliferation inhibition experiment

[0044] Test method: BmN cells were treated with EGCG (20 uM, 40 uM) for 24 h and then infected with BmNPV-EGFP. gDNA was extracted at 24 h, 48 h and 72 h, respectively, and the viral genomic DNA and the copy number of gene VP39 were detected by qPCR.

[0045] Test results: As shown in Figure 6, compared with the DMSO control group, 20 uM and 40 uM EGCG both showed good inhibitory effects on the virus and could significantly inhibit viral proliferation.

[0046] Example 5: Effect of EGCG on viral GP64 protein expression

[0047] Test method: Using β-Tubulin as an internal control, Western blot was used to detect the GP64 protein level in BmNPV-infected cells after 48h and 72h of treatment with 40 uMEGCG.

[0048] Results: As shown in Figure 7, the expression level of GP64 protein in the EGCG-treated group was significantly lower than that in the untreated group, indicating that it can inhibit viral envelope protein synthesis and block viral assembly and release.

[0049] Example 6: In vivo anti-BmNPV experiment using EGCG

[0050] Test method: Fifth instar silkworms were divided into three groups (control group, 20 uM, and 40 uM EGCG). After oral infection with BmNPV (within 12 hours), the EGCG-licking group and the control group were given the same volume of DMSO in their mouths. After 24, 48, and 72 hours of continued rearing, larvae were collected for genomic DNA extraction, and the replication of the key viral genes GP64 and VP39 in the silkworms was detected by real-time quantitative PCR.

[0051] Test results: As shown in Figure 8- Figure 10 As shown, mRNA and genomic DNA of the virus were extracted from hemolymph, fat body, and midgut at 24h, 48h, and 72h in the EGCG group. The replication of viral genes GP64 and VP39 in silkworms was detected by real-time PCR. The results showed that all concentrations inhibited the proliferation of BmNPV, and the inhibitory effect increased with time.

[0052] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. Use of EGCG in the preparation of drugs for the prevention and / or treatment of silkworm nucleopolyhedrovirus (BmNPV) infection.

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The dosage forms of the drug include solutions, wettable powders, or granules.

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