Use of vertepafen 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 verteporfin in preparation of a drug for preventing and treating Bombyx mori nuclear polyhedrosis virus (BmNPV). The application finds, through extensive research, that verteporfin can effectively inhibit replication and transmission of BmNPV, reduce damage of the virus to a host, and improve economic benefits of a silkworm and mulberry industry, thereby providing a new strategy for prevention and treatment of BmNPV of the silkworm.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to the application of verteporfen 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] Verteporfen, a benzoporphyrin derivative, is commonly used as a photosensitizer in photodynamic therapy and is a drug for treating abnormal vascular diseases of the eye. CN113546076A discloses that verteporfen has a therapeutic effect on COVID-19, and its antiviral mechanism may be related to reactive oxygen species generation, energy metabolism regulation, and autophagy induction. However, its effect on the prevention and treatment of BmNPV in silkworms is still unclear. Summary of the Invention

[0005] The research and exploration of highly effective and low-toxicity anti-BmNPV drugs is a common need in this field. This invention, through extensive research, has found that verteporfen can effectively inhibit the replication and spread of BmNPV while promoting the growth and development of silkworms, providing a new strategy for the prevention and control of BmNPV in silkworms, thus completing this invention.

[0006] In one aspect, the present invention discloses the use of verteporfen in the preparation of a drug for the prevention and treatment of silkworm nucleopolyhedrovirus (BmNPV).

[0007] In one embodiment, the medicament of the present invention further includes pharmaceutically acceptable excipients.

[0008] In this invention, the dosage form of the drug is a liquid preparation, a wettable powder, or granules.

[0009] In one aspect, the present invention also discloses a method for preventing and controlling silkworm nucleopolyhedrovirus, the method comprising administering a preparation containing verteporfen to silkworms.

[0010] In one embodiment, the formulation is administered before silkworm infection (BmNPV); in another embodiment, the formulation is administered within 12 hours after silkworm infection (BmNPV).

[0011] In one embodiment, the preparation is administered to silkworms by feeding. Beneficial effects

[0012] Highly effective antiviral: Verteporfin can block the replication and spread of BmNPV. When applied in the early stage of viral infection (within 12 hours), it can significantly inhibit viral proliferation, providing a precise intervention opportunity for the prevention and treatment of BmNPV.

[0013] Green and environmentally friendly: Verteporfen is biodegradable, avoiding the harm of traditional pesticides to the environment and non-target organisms, which meets the needs of green and sustainable development of the sericulture industry. Attached Figure Description

[0014] Figure 1: Results of viroteporin toxicity test on silkworm cell BmN. The results show that viroteporin has no effect on the growth of silkworm cells.

[0015] Figure 2: TUNEL staining to detect cell apoptosis after verteporfen treatment.

[0016] Figure 3: Effects of different concentrations of verteporfen on viral gene (VP39) expression and replication in cells.

[0017] Figure 4: Western blot analysis of the effect of verteporfen on the expression level of viral GP64 protein.

[0018] Figure 5: Flow cytometry quantitative analysis of the virus inhibition of BmNPV-infected cells after verteporfen treatment.

[0019] Figure 6: Inhibitory effect of verteporfen on BmNPV replication in silkworm (midgut, hemolymph, fat body). Detailed Implementation

[0020] 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.

[0021] Example 1: Detection of verteporfen's toxicity to BmN in silkworm cells

[0022] BmN cells from silkworms were seeded into 96-well plates (5 × 10³ cells / well) and cultured overnight. Verteporfin solutions at concentrations of 3 μM and 6 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.

[0023] Test results: As shown in Figure 1, 3 uM and 6 uM verteporfen showed no toxicity to BmN cells, indicating good safety.

[0024] Example 2: Detection of the cellular efficacy of verteporfen against silkworm nucleopolyhedrovirus (BmNPV)

[0025] 2.1 Apoptosis Inhibition Experiment

[0026] Test method: After BmN cells were infected with BmNPV, they were treated with 3 uM and 6 uM verteporfen, and apoptosis was detected by TUNEL staining (green fluorescence indicates apoptotic cells, and blue indicates DAPI nuclear staining).

[0027] Results: As shown in Figure 2, 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 verteporfen treatment group was significantly lower than that in the BmNPV-infected group, and the effect was better with increasing concentration, indicating that verteporfen can alleviate virus-induced apoptosis.

[0028] 2.2 Virus proliferation inhibition experiment

[0029] Test method: BmN cells were treated with verteporfen (3 uM, 6 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.

[0030] Test results: As shown in Figure 3, compared with the DMSO control group, 3uM and 6uM verteporfen both showed good inhibitory effects on the virus and could significantly inhibit viral proliferation.

[0031] Example 3: Effect of verteporfen on viral GP64 protein expression

[0032] Test method: Using β-Tubulin as an internal control, Western blot was used to detect the GP64 protein level in BmNPV-infected cells after treatment with 6 uM vertepofen for 48 h and 72 h.

[0033] Results: As shown in Figure 4, the expression level of GP64 protein in the verteporfen treatment group was lower than that in the untreated group, indicating that it can inhibit viral envelope protein synthesis and block viral assembly and release.

[0034] Example 4: Flow cytometry analysis of virus inhibition efficiency

[0035] Test method: After infecting BmN cells with BmNPV-EGFP, and treating them with verteporfen (3 uM, 6 uM), the proportion of EGFP-positive cells was detected by flow cytometry at 48h and 72h.

[0036] Test results: As shown in Figure 5, after 72 hours, the proportion of EGFP-positive cells in the verteporfen treatment group decreased compared with the control group, and the inhibition efficiency increased over time.

[0037] Example 5: In vivo anti-BmNPV experiment with verteporfen

[0038] Test method: Fifth instar silkworms were divided into three groups (control group, 3 uM, and 6 uM verteporfen). After oral infection with BmNPV (within 12 hours), the verteporfen-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 viral gene VP39 in the silkworms was detected by real-time PCR.

[0039] Test results: As shown in Figure 6, mRNA and genomic DNA of the virus were extracted from the midgut, hemolymph, and fat body of the verteporfen group at 24h, 48h, and 72h. The replication of the viral gene 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.

[0040] 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. Application of verteporfen in the preparation of drugs for the prevention and treatment of silkworm nucleopolyhedrovirus (BmNPV).

2. The application of verteporfen according to claim 1 in the preparation of a drug for the prevention and treatment of silkworm nucleopolyhedrovirus (BmNPV), characterized in that, The drug also includes pharmaceutically acceptable excipients.

3. The application of verteporfen according to claim 1 in the preparation of a drug for the prevention and treatment of silkworm nucleopolyhedrovirus (BmNPV), characterized in that, The dosage form of the drug is a liquid preparation, a wettable powder, or granules.

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