Application of endoplasmic reticulum protein antioxidants in the prevention and treatment of Bombyx mori nuclear polyhedrosis virus

Abstract

The present invention belongs to the field of agricultural biotechnology, and in particular, relates to the use of endoplasmic reticulum protein antioxidants (reducing agents) in the prevention and treatment of Bombyx mori nuclear polyhedrosis virus. Screening and testing have found that the endoplasmic reticulum protein antioxidants involved in the present invention, β-mercaptoethanol (BME), dithiothreitol (DTT), and / or tris(2-carboxyethyl)phosphine (TCEP), have the effect of inhibiting Bombyx mori nuclear polyhedrosis virus (BmNPV). These antioxidants disrupt the disulfide bonds in key BmNPV proteins, interfering with viral replication and proliferation, directly affecting the stability and activity of viral particles, thereby inhibiting viral infection of silkworms. This provides a new approach for the development of antiviral drugs for silkworms and has good application value.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and in particular, relates to the application of an endoplasmic reticulum protein antioxidant in preventing and treating silkworm nuclear polyhedrosis virus. Background Art

[0002] The silkworm, also known as the mulberry silkworm, is an insect of the genus Bombyx mori, family Bombycidae. It undergoes complete metamorphosis, with four developmental stages: egg, larva, pupa, and adult. Silkworm farming plays a vital role in my country's agricultural economy and silk industry. Bombyx mori nuclear polyhedrosis virus (BmNPV) is the most devastating of the three major viral diseases affecting sericulture. Outbreaks occur frequently in silkworm-raising countries worldwide. It is highly contagious and difficult to control, often causing significant economic losses. Primary infection with NPV occurs in the midgut epithelium, with primary target cells including columnar cells and regenerative cells. Once NPV enters the cell, a series of viral processes, including viral replication, packaging, and cell death, occur, allowing the virus to exploit the host cell for its own reproduction. The silkworm holds an irreplaceable position in the silk industry, but the high incidence of BmNPV remains a bottleneck hindering the industry's development. Existing control measures, such as chemical agents and traditional disease management methods, have limitations and pose environmental risks. Currently, there is no specific drug reported for Bombyx mori nuclear polyhedrosis virus (BmNPV) infection. Therefore, there is an urgent need for a method that can quickly and efficiently inhibit BmNPV infection to lay a scientific foundation for studying the mechanism of silkworm resistance to BmNPV and virus prevention and control in sericulture 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, further inducing endoplasmic reticulum stress and cellular oxidative damage. Antioxidation is one of the means of preventing and controlling viral infection. However, the relationship between BmNPV virus infection prevention and control in silkworms and antioxidants needs further study. Summary of the Invention

[0004] In order to further enhance the prevention and treatment effect of Bombyx mori nuclear polyhedrosis virus (BmNPV) and provide ideas for the prevention and treatment of the virus, the present invention has been completed through long-term research and found that antioxidants (reducing agents) targeting endoplasmic reticulum proteins can exert a good antiviral effect in silkworms.

[0005] In one aspect of the present invention, the present invention discloses the use of an endoplasmic reticulum protein antioxidant (reducing agent) in the preparation of a preparation for preventing and treating Bombyx mori nuclear polyhedrosis virus (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 of the present invention, a preparation for controlling Bombyx mori nuclear polyhedrosis virus (BmNPV) is disclosed, wherein the preparation contains β-mercaptoethanol (BME), dithiothreitol (DTT) and / or tris(2-carboxyethyl)phosphine (TCEP).

[0008] In one embodiment, the preparation is a powder, a granule preparation, or a liquid preparation. Preferably, the preparation is a liquid preparation.

[0009] In one embodiment, the preparation is administered to silkworms by feeding them to prevent and treat Bombyx mori nuclear polyhedrosis virus (BmNPV) infection. Preferably, the preparation is a liquid preparation, which is sprayed onto mulberry leaves, the food of the silkworms, for feeding.

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

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

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

[0013] A method of the present invention discloses a method for preventing and controlling Bombyx mori nuclear polyhedrosis virus (BmNPV), comprising applying an endoplasmic reticulum protein antioxidant (reducing agent) to the silkworm; 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, the present invention found that applying endoplasmic reticulum protein antioxidants (reducing agents) to silkworms can disrupt the disulfide bonds in BmNPV key proteins, interfere with the replication and proliferation of the virus, and directly affect the stability and activity of virus particles, thereby inhibiting the virus from infecting 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 silkworm health and productivity. The present study showed that the application of BME not only effectively inhibits the proliferation of BmNPV, but also protects the functional stability of host cells by indirectly reducing the cellular stress response caused by viral infection. Further studies have found that BME helps maintain protein homeostasis in silkworm cells while reducing viral proteins, optimizing cellular metabolic processes, and promoting the healthy growth and production performance of silkworms.

[0016] (3) Excellent antiviral effect and application prospects, helping sericulture technology innovation. Compared with traditional antiviral methods, the present 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 mulberry leaf spraying), BME can exert a significant inhibitory effect 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, showing extremely high development and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 , are the fluorescence intensities in silkworm BmN cells infected with BmNPV-EGFP at different time periods after treatment with six redox drugs, among which A is BME, B is TCEP, C is DTT, D is GSH, E is NAC, and F is Na2S.

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

[0019] Figure 3 , combined with TUNEL staining and MTT to detect the effect of BME drug treatment on BmNPV infected cells, among which, Figure 3 In A, green fluorescence represents the presence of apoptotic DNA fragments in cells, and blue represents DAPI-stained nucleic acids. The MTT assay (B) was used to assess cell survival at different time points (24 h, 48 h, and 72 h). 490 Values ​​indicate cell viability.

[0020] Figure 4 , to detect the virus that proliferates BmNPV after BME drug treatment GP64 Gene expression analysis.

[0021] Figure 5 , to detect the virus that proliferates BmNPV after BME drug treatment GP64 Effect of gene gDNA copy number.

[0022] Figure 6 2. Detection of viral GP64 protein to investigate the effect of BME drug treatment on BmNPV proliferation in cells. The brightness of the target protein GP64 band in the BME-treated group was detected compared with the blank control and virus-infected control.

[0023] Figure 7 , to detect the effect of BME treatment on the proliferation of BmNPV in silkworms GP64 Gene expression. In the figure, A shows the viral gDNA copy number in the midgut after drug addition, B shows the viral gDNA copy number in the hemolymph, C shows the viral gDNA copy number in the fat body, D shows the viral GP64 gene expression level in the midgut after drug addition, E shows the viral GP64 gene expression level in the hemolymph, and F shows the comparison of viral GP64 gene expression levels in the fat body. DETAILED DESCRIPTION

[0024] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0025] Example 1: Screening of antioxidants (reducing agents)

[0026] Bombyx mori BmN cells were plated in 12-well plates one day in advance, and the cell density reached 80%-90% the next day. Six different reducing agents, including 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 (1mM, 2mM, and 4mM). BmNPV was added 12 hours after drug addition, and fluorescence images were taken at different time points (24h, 48h, and 72h) to observe the intensity of the EGFP-BmNPV fluorescence signal in the cells after drug addition. Figure 1Fluorescence microscopy revealed that, compared with the control group, the fluorescence intensity of GFP-tagged BmNPV virus was reduced after 24, 48, and 72 hours of treatment with equivalent concentrations of BME, TCEP, and DTT. This indicates that BME, TCEP, and DTT effectively inhibit viral proliferation. Similarly, treatment with GSH, NAC, and Na₂S did not significantly alter viral fluorescence intensity compared with the control group. Further testing with BME, TCEP, and DTT will be conducted.

[0027] Example 2: Flow cytometry quantitative analysis of viral inhibition efficiency of BmNPV-infected cells after treatment with BME, DTT, and TCEP

[0028] By flow cytometry with DMSO as the control, the three reagents BME, DTT and TCEP were detected at a concentration of 2mM to treat cells infected with BmNPV-EGFP, and quantitative analysis was performed to evaluate the viral inhibitory effect of the relevant drugs 24 hours after addition.

[0029] See also 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 is highly effective in inhibiting viral infection and its induced apoptosis. The apoptosis rate in the BME-treated group remained low, indicating that it effectively inhibited the proliferation of BmNPV and reduced cellular damage caused by viral infection.

[0030] In contrast, the DTT and TCEP treatment groups showed a higher proportion of apoptotic cells, particularly in the TCEP group, where the number of apoptotic cells did not significantly decrease, indicating a weaker inhibitory effect on the virus. DTT treatment also failed to significantly reduce the proportion of apoptotic cells after 24 hours, likely due to the limitations of its antiviral effect. Therefore, BME demonstrated significant antiviral activity in this experiment, effectively inhibiting cell apoptosis induced by BmNPV infection, while the effects of DTT and TCEP were relatively weak.

[0031] Example 3 Effects of BME Drug Treatment on BmNPV-Infected Cells

[0032] Cell apoptosis after BME treatment was detected by TUNEL staining, where green fluorescence represents the presence of apoptotic DNA fragments in the cells, and blue represents DAPI-stained nucleic acids.

[0033] See also Figure 3TUNEL staining results showed that: In the control (BmNPV-) group, which was not infected with BmNPV, no green fluorescence was observed through TUNEL staining, indicating that the cells did not undergo significant apoptosis. In the control (BmNPV+) group, which was the BmNPV-infected group (untreated BME), green fluorescence was clearly visible, indicating that BmNPV infection caused cell apoptosis. In the BME-treated group (BME(BmNPV+)), green fluorescence was significantly reduced, and the number of apoptotic cells was significantly lower than that in the untreated group. This indicates that BME can alleviate cell apoptosis caused by BmNPV infection.

[0034] Cell viability was further assessed using the MTT assay at different time points (24, 48, and 72 hours), with OD490 values ​​used to indicate cell viability. MTT assay results showed no significant difference in OD490 values ​​between the BME-treated and untreated groups at 24 hours post-infection (hpi), indicating that BME has minimal effect on cell viability during the early stages of viral infection. At 48 hours (48 hpi) and 72 hours (72 hpi), OD490 values ​​in the BME-treated group were significantly higher than those in the untreated group (Control, BmNPV+). This suggests that BME treatment effectively enhances cell viability during the late stages of BmNPV infection and mitigates viral damage.

[0035] In summary, TUNEL staining results showed that BME treatment significantly inhibited cell apoptosis caused by BmNPV infection, while MTT assays showed that BME significantly increased the survival rate of BmNPV-infected cells. These results suggest that BME has antiviral proliferation and cell-protective effects.

[0036] Example 4: Effects of BME Drug Treatment on BmNPV Proliferation

[0037] Well-grown BmN cells were selected and infected with BmNPV and then treated. They were divided into DMSO control group and BME experimental group, and treated with different concentration gradients of 1, 2, and 4 mM for 24 h, 48 h, and 72 h. GP64 mRNA, reverse transcribe to synthesize cDNA and use RT-qPCR to detect viral gene expression. Figure 4 The results showed that compared with the control group, BME effectively downregulated viral gene expression, with the inhibitory effect becoming more pronounced over time. Furthermore, there was no significant difference in the effect of BME on viral replication between 2mM and 4mM concentrations. Therefore, it can be concluded that 2mM BME has the best inhibitory effect on BmNPV in vitro.

[0038] Furthermore, the changes in the gDNA copy number of the GP64 gene of BmNPV proliferation were detected after 24h, 48h, and 72h of treatment with 2mM BME. Using an equal volume of culture medium as a control, the virus particles BV in the supernatant separated by centrifugation were detected. GP64 For gene copy number, see Figure 5 Based on the exported RT-qPCR data, the results were calculated through EXCEl worksheet and analyzed after drawing in GraphPad Prism 9.3.1. The results showed that compared with the Control, BME effectively inhibited the viral gDNA copy as the time gradient changed, further indicating that the reducing agent BME has a good effect in inhibiting the replication of the silkworm BmNPV virus.

[0039] Example 5 Detection of viral GP64 protein in virus-infected cells after BME drug treatment

[0040] Western blot was used to detect the expression level of viral GP64 protein in BmNPV-infected BmN cells to evaluate the effect of BME drug treatment on viral GP64 protein. GP64 is the main envelope protein of BmNPV, and its expression level can reflect the replication and proliferation of the virus in host cells. β-Tubulin was used as an internal control to verify the consistency of loading amount among samples. Figure 6 The β-Tubulin band intensity was essentially consistent across all experimental groups, indicating uniform sample loading and reliable experimental results. In the control group (Control+), GP64 protein expression levels were significantly higher in the untreated BmNPV-infected group (+ BmNPV, - BME), indicating active BmNPV replication and proliferation in the cells. In the BME-treated groups (Control+, + BME), GP64 protein expression levels were significantly reduced in the BME-treated infected group (+ BmNPV, + BME), with expression intensity significantly weakened compared to the untreated group, demonstrating that BME treatment effectively inhibits BmNPV proliferation. In the uninfected group (Control-), GP64 protein expression was undetectable in the uninfected group (- BmNPV, ± BME), further demonstrating that GP64 protein is specifically associated with viral replication. Western blot results showed that BME treatment significantly reduced GP64 protein expression in BmNPV-infected cells, indicating that BME has a strong inhibitory effect on BmNPV replication and proliferation.

[0041] Example 6 Detection of viral GP64 gene in silkworms infected with BmNPV by BME treatment

[0042] The effects of BME 1mM and 2mM on BmNPV replication were examined in 5th instar silkworms with good growth. The gDNA copy number of BmNPV GP64 in the midgut, fat body and hemolymph of silkworms was detected by qPCR using gDNA templates. Figure 7 , and 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 of viral GP64 in the midgut, fat body, and hemolymph of the p50 strain of silkworms infected with BmNPV was detected. Compared with the control group, the mRNA expression level in the experimental group was significantly downregulated, and the treatment effect of 2mM BME was better, which further verified the inhibitory effect of BME on viral replication.

[0043] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Application of an endoplasmic reticulum protein antioxidant in the preparation of a preparation for preventing and treating Bombyx mori nuclear polyhedrosis virus (BmNPV), characterized in that: The preparation is used for silkworms, and the antioxidant is beta-mercaptoethanol (BME).

2. The use according to claim 1, characterized in that The preparation is a powder, a granule preparation or a liquid preparation.

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

Images