Application of endoplasmic reticulum protein antioxidant in prevention and treatment of bombyx mori nuclear polyhedrosis virus

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to application of an endoplasmic reticulum protein antioxidant (reducing agent) in prevention and treatment of bombyx mori nuclear polyhedrosis virus. A screening test finds that the related endoplasmic reticulum protein antioxidants beta-mercaptoethanol (BME), dithiothreitol (DTT) and / or tris (2-carboxyethyl) phosphine (TCEP) have the effect of inhibiting the bombyx mori nuclear polyhedrosis virus (BmNPV), and the antioxidants interfere the replication and proliferation of the virus by destroying disulfide bonds in key proteins of the BmNPV, so that the bombyx mori nuclear polyhedrosis virus can be inhibited. The stability and activity of virions are directly influenced, so that the infection of the viruses on the silkworms is inhibited, a brand new thought is provided for the development of antiviral drugs of the silkworms, and the application value is good.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, and in particular, it relates to the application of endoplasmic reticulum protein antioxidants in the prevention and control of silkworm nucleopolyhedrovirus. This case is a divisional application of CN202510151313.5, which was filed on February 11, 2025. Background Technology

[0002] The silkworm, also known as the mulberry silkworm, is an insect belonging to the genus *Bombyx mori* in the family Bombycidae. It undergoes holometabolous metamorphosis, with its development divided into four stages: egg, larva, pupa, and adult. Silkworm farming plays a vital role in my country's agricultural economy and silk industry development. Bombyx mori nucleopolyhedrovirus (BmNPV) is the most damaging of the three major viral diseases in sericulture, frequently causing outbreaks in sericulture countries worldwide. It is highly contagious, difficult to control, and often results in significant economic losses. Primary infection of NPV occurs in the midgut epithelial tissue, with primary target cells including columnar cells and regenerating cells. Once NPV enters the cell, it undergoes a series of processes including viral replication, packaging, and cell death, utilizing the host cell for self-replication. While the silkworm holds an irreplaceable position in the silk industry, the high frequency of BmNPV-induced disease remains a bottleneck restricting the development of sericulture. Existing control methods, such as chemical agents and traditional disease management methods, have limitations in effectiveness and can cause environmental pollution. Currently, there are no reported effective drugs for silkworm nucleopolyhedrovirus (BmNPV) infection. Therefore, there is an urgent need for a method that can rapidly and effectively inhibit BmNPV infection, laying a scientific foundation for studying the mechanism of silkworm resistance to BmNPV and virus control in silkworm production.

[0003] Studies have shown that viral infection can trigger the rapid synthesis of large amounts of viral proteins in host cells, leading to the accumulation of misfolded proteins, which in turn induces endoplasmic reticulum stress and cellular oxidative damage. Antioxidants are one of the means of preventing and controlling viral infection. However, the relationship between BmNPV virus infection prevention and control and antioxidants in silkworms needs further research. Summary of the Invention

[0004] To further improve the prevention and control of silkworm nucleopolyhedrovirus (BmNPV) and provide ideas for the prevention and control of this virus, this invention, through long-term research, discovered that antioxidants (reducing agents) targeting endoplasmic reticulum proteins can exert a good antiviral effect in silkworms, thus completing this invention.

[0005] In one aspect, the present invention discloses the application of endoplasmic reticulum protein antioxidants (reducing agents) in the preparation of agents for the prevention and treatment of silkworm nucleopolyhedrovirus (BmNPV).

[0006] In one embodiment, the antioxidant (reducing agent) is β-mercaptoethanol (BME), dithiothreitol (DTT), and / or tris(2-carboxyethyl)phosphine (TCEP); more preferably, the antioxidant (reducing agent) is β-mercaptoethanol (BME).

[0007] In one aspect, the present invention discloses a formulation for preventing and controlling silkworm nucleopolyhedrovirus (BmNPV), the formulation containing β-mercaptoethanol (BME), dithiothreitol (DTT) and / or tris(2-carboxyethyl)phosphine (TCEP).

[0008] In one embodiment, the formulation is a powder, granule, or liquid formulation; preferably, the formulation is a liquid formulation.

[0009] In one embodiment, the preparation is applied to silkworms via feeding to prevent infection with silkworm nucleopolyhedrovirus (BmNPV). Preferably, the preparation is a liquid preparation, which is sprayed onto the mulberry leaves that the silkworms eat before feeding.

[0010] In one embodiment, the concentration of the preparation used is 2 mM for silkworm bodies and 2 mM for cells.

[0011] In one embodiment, the preparation is administered before or within 12 hours of silkworm infection with BmNPV.

[0012] In one embodiment, the formulation includes pharmaceutically acceptable excipients that enhance the stability and palatability of the drug.

[0013] One method of the present invention discloses a method for preventing and controlling silkworm nucleopolyhedrovirus (BmNPV) by applying an endoplasmic reticulum protein antioxidant (reducing agent) to silkworms; preferably, the antioxidant is β-mercaptoethanol (BME), dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), glutathione (GSH), N-acetylcysteine ​​(NAC), and sodium sulfide (Na2S); more preferably, the antioxidant (reducing agent) is β-mercaptoethanol (BME), dithiothreitol (DTT), and / or tris(2-carboxyethyl)phosphine (TCEP). Beneficial effects

[0014] (1) Through research and testing, this invention found that applying endoplasmic reticulum protein antioxidants (reducing agents) to silkworms can interfere with virus replication and proliferation by breaking the disulfide bonds in the key protein of BmNPV, thereby directly affecting the stability and activity of virus particles and inhibiting virus infection of silkworms. This provides a new idea for the development of antiviral drugs for silkworms and has good application value.

[0015] (2) Reduce the burden of viral infection on host cells and improve the health and productivity of silkworms. This invention demonstrates that the application of BME not only effectively inhibits the proliferation of BmNPV, but also protects the functional stability of host cells by indirectly alleviating the cellular stress response caused by viral infection. Further research found that while reducing viral proteins, BME helps maintain protein homeostasis within silkworm cells, optimizes cellular metabolic processes, and promotes healthy growth and improved productivity of silkworms.

[0016] (3) Excellent antiviral effect and application prospects contribute to technological innovation in sericulture. Compared with traditional antiviral methods, this invention uses redox reagents as the core antiviral strategy, which is precise and efficient. By controlling the dosage and application method (such as feeding or spraying mulberry leaves), BME can play a significant inhibitory role in the early stage of viral infection and early intervention. Combined with acceptable pharmaceutical excipients, the stability and palatability of the drug are further enhanced, providing technical support for the green and sustainable development of the sericulture industry and demonstrating extremely high development and application potential. Attached Figure Description

[0017] Figure 1 The fluorescence intensity of silkworm BmN cells infected with BmNPV-EGFP was shown at different time points after treatment with six redox drugs. A represents BME, B represents TCEP, C represents DTT, D represents GSH, E represents NAC, and F represents Na2S.

[0018] Figure 2 To quantitatively analyze the viral inhibition efficiency of BmNPV-infected cells after treatment with BME, DTT, and TCEP by flow cytometry.

[0019] Figure 3 The effect of BME treatment on BmNPV-infected cells was detected by TUNEL staining and MTT assay. Figure 3 Green fluorescence in A represents the presence of apoptotic DNA fragments in cells, while blue represents DAPI-stained nucleic acids. Cell viability at different time points (24 h, 48 h, 72 h) was assessed using the MTT assay (B), measured by OD... 490 The value represents cell activity.

[0020] Figure 4 To detect the virus that inhibits BmNPV proliferation after BME drug treatment. GP64 Gene expression level analysis.

[0021] Figure 5 To detect the virus that inhibits BmNPV proliferation after BME drug treatment. GP64 The effect of gene gDNA copy number.

[0022] Figure 6 The study aimed to detect the effect of BME drug treatment on BmNPV proliferation in cells by measuring the intensity of the GP64 viral protein band. This was compared with the blank control and the virus infection control, and the intensity of the GP64 band after BME treatment was measured.

[0023] Figure 7 To detect the effect of BME treatment on the proliferation of BmNPV in silkworms. GP64 Gene expression status. In the figure, A represents the viral gDNA copy number in the midgut after drug administration, B represents the viral gDNA copy number in hemocytes, C represents the viral gDNA copy number in the fat body, D represents the viral GP64 gene expression level in the midgut after drug administration, E represents the viral GP64 gene expression level in hemocytes, and F represents a comparison of viral GP64 gene expression levels in the fat body. Detailed Implementation

[0024] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Example 1: Screening of antioxidants (reducing agents)

[0025] One day in advance, silkworm BmN cells were seeded in 12-well plates, and the cell density reached 80%-90% the following day. Six different reducing agents—β-mercaptoethanol (BME), dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), glutathione (GSH), N-acetylcysteine ​​(NAC), and sodium sulfide (Na2S)—were added to the cell culture plates at equal concentrations (1 mM, 2 mM, and 4 mM). Twelve hours after drug addition, BmNPV virus was added, and fluorescence intensity of EGFP-BmNPV in the cells was observed at different time points (24 h, 48 h, and 72 h). See also... Figure 1Fluorescence microscopy revealed that, compared to the control group, the fluorescence intensity of BmNPV-tagged BmNPV viruses decreased after treatment with equal concentrations of BME, TCEP, and DTT for 24, 48, and 72 hours, indicating that BME, TCEP, and DTT had a good inhibitory effect on viral proliferation. Similarly, compared to the control group, treatment with GSH, NAC, and Na2S did not significantly change the viral fluorescence intensity. Further tests with BME, TCEP, and DTT treatment were subsequently conducted. Example 2: Quantitative analysis of the virus inhibition efficiency of BmNPV-infected cells after treatment with BME, DTT, and TCEP by flow cytometry

[0026] Using flow cytometry with DMSO as a control, the viral inhibition effects of BME, DTT, and TCEP at a concentration of 2 mM on cells infected with BmNPV-EGFP were evaluated and quantitatively assessed 24 hours after drug administration.

[0027] See Figure 2 Compared with the control group, the proportion of apoptotic cells in the BME-treated group was significantly lower than that in the DTT and TCEP-treated groups, indicating that BME has a strong effect in inhibiting viral infection and the resulting apoptosis. The apoptosis rate in the BME group remained at a low level, indicating that it can effectively inhibit the proliferation of BmNPV and reduce cell damage caused by viral infection.

[0028] In contrast, the proportion of apoptotic cells was higher in the DTT and TCEP treatment groups, especially in the TCEP group, where the number of apoptotic cells did not decrease significantly, indicating a weaker inhibitory effect on the virus. The DTT treatment group also failed to significantly reduce the proportion of apoptotic cells after 24 hours, possibly due to the limitations of its antiviral effect. Therefore, BME demonstrated a significant antiviral effect in this experiment, effectively inhibiting apoptosis induced by BmNPV infection, while the effects of DTT and TCEP were relatively weaker. Example 3: Effect of BME drug treatment on BmNPV-infected cells

[0029] Apoptosis of cells treated with BME was detected by TUNEL staining. Green fluorescence represents the presence of apoptotic DNA fragments in the cells, while blue represents DAPI-stained nucleic acids.

[0030] See Figure 3TUNEL staining results showed that in the control group (BmNPV-) uninfected with BmNPV, no green fluorescence was observed, indicating that no significant apoptosis occurred. In the control group (BmNPV+), i.e., the BmNPV-infected group (untreated with BME), green fluorescence was clearly visible, indicating that BmNPV infection induced apoptosis. In the BME-treated infected group (BME(BmNPV+)), green fluorescence was significantly reduced, and the number of apoptotic cells was significantly decreased compared to the untreated group. This indicates that BME can alleviate apoptosis induced by BmNPV infection.

[0031] Cell viability was further assessed using the MTT assay at different time points (24 h, 48 h, and 72 h), with OD490 values ​​representing cell viability. MTT results showed that at 24 hpi post-infection, there was no significant difference in OD490 values ​​between the BME-treated and untreated groups, indicating that BME had minimal impact on cell viability in the early stages of viral infection. At 48 hpi and 72 hpi, the OD490 values ​​of the BME-treated group were significantly higher than those of the untreated group (Control, BmNPV+). This indicates that BME treatment can effectively improve cell viability in the later stages of BmNPV infection and reduce viral damage to cells.

[0032] In summary, TUNEL staining results showed that BME treatment significantly inhibited apoptosis induced by BmNPV infection; simultaneously, MTT assay indicated that BME significantly improved the survival rate of BmNPV-infected cells. These results suggest that BME has antiviral proliferation and cell viability protection effects. Example 3: Effect of BME drug treatment on BmNPV proliferation

[0033] Well-grown BmN cells were infected with BmNPV and then treated, divided into a control group (DMSO) and an experimental group (BME) treated with different concentration gradients of 1, 2, and 4 mM for 24 h, 48 h, and 72 h. The virus was then extracted. GP64 mRNA was reverse transcribed into cDNA, and viral gene expression was detected using RT-qPCR. See also Figure 4 The results showed that, compared with the control group, BME effectively downregulated viral gene expression, and the inhibitory effect became more pronounced over time. Furthermore, there was no significant difference in the effect of BME concentrations of 2 mM and 4 mM on viral replication. Therefore, it can be concluded that, in in vitro experiments, a BME concentration of 2 mM showed the best inhibitory effect on silkworm BmNPV virus.

[0034] Furthermore, the changes in the GP64 gene gDNA copy number of BmNPV proliferation were detected after treatment with 2 mM BME for 24 h, 48 h, and 72 h. Using an equal volume of culture medium as a control, viral particles (BV) were detected in the supernatant separated from collected cells after centrifugation. GP64 For gene copy number information, see [link to gene copy number information]. Figure 5 Based on the exported RT-qPCR data, the results were analyzed after calculation using an EXCE1 worksheet and plotting with GraphPad Prism 9.3.1. The results showed that, compared with the control, BME effectively inhibited viral gDNA copying with the change of time gradient, further indicating that the reducing agent BME has a good inhibitory effect on the replication of silkworm BmNPV virus. Example 4: Detection of viral GP64 protein in virus-infected cells after BME drug treatment

[0035] The expression level of viral GP64 protein was detected by Western blot to assess the effect of BME treatment on viral GP64 protein levels in BmNPV-infected BmN cells. GP64 is the major envelope protein of BmNPV, and its expression level reflects viral replication and proliferation in host cells. β-Tubulin was used as an internal control to verify the consistency of loading levels across samples. See also Figure 6 The β-Tubulin band intensities were generally consistent across all experimental groups, indicating uniform sample loading and reliable experimental results. In the control group (Control +), the expression level of GP64 protein was significantly higher in the untreated BME-infected group (+ BmNPV, - BME), indicating active replication and proliferation of BmNPV in cells. In the BME-treated groups (Control +, + BME), the expression level of GP64 protein was significantly reduced, with a significantly weaker expression intensity compared to the untreated group, indicating that BME treatment can effectively inhibit BmNPV proliferation. In the uninfected group (Control -), no GP64 protein expression was detected in the uninfected BmNPV group (- BmNPV, ± BME), further demonstrating that GP64 protein is specifically associated with viral replication. Western blot results showed that BME treatment can significantly reduce the expression level of GP64 protein in BmNPV-infected cells, indicating that BME has a strong inhibitory effect on BmNPV replication and proliferation. Example 5: Detection of the GP64 gene of BmNPV virus in silkworms after BME treatment.

[0036] Fifth-instar healthy silkworms were used to test the effects of BME 1mM and 2mM on BmNPV virus replication. The copy number of BmNPV GP64 gDNA in the midgut, fat body, and hemolymph of silkworms was detected by gDNA template qPCR. (See [reference]) Figure 7 The study found that the copy number after knockdown was significantly lower than that in the control group, indicating that BME treatment inhibited viral replication. Subsequently, the gene expression levels of viral GP64 in the midgut, fat body, and hemolymph of p50 silkworms infected with BmNPV were detected. Compared with the control group, the mRNA expression level in the experimental group was significantly downregulated, and the treatment with 2mM BME was more effective, further validating the inhibitory effect of BME on viral replication.

[0037] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of endoplasmic reticulum protein antioxidants in the preparation of agents for the prevention and treatment of silkworm nucleopolyhedrovirus (BmNPV), characterized in that, The endoplasmic reticulum protein antioxidant is tris(2-carboxyethyl)phosphine (TCEP).

2. The application according to claim 1, characterized in that, The formulation is a powder, granule, or liquid formulation, preferably a liquid formulation.

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

4. The application according to claim 1, characterized in that, The preparation is applied to silkworms by feeding to prevent infection with silkworm nucleopolyhedrovirus (BmNPV); preferably, the preparation is a liquid preparation, which is sprayed onto the mulberry leaves that the silkworms eat before feeding.

5. The application according to claim 4, characterized in that, The concentration of the preparation used is 2 mM for silkworm bodies and 2 mM for cells.

6. The formulation according to claim 5, characterized in that, The preparation is administered before silkworms are infected with BmNPV, or within 12 hours after infection with BmNPV.

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