Application of 4 mu 8C in preparation of preparation for preventing and treating bombyx mori nuclear polyhedrosis virus

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to application of 4 mu 8C in preparation of a medicine for preventing and treating bombyx mori nuclear polyhedrosis virus. Bioinformatics analysis, gene knockout, RT-PCR detection and other modes find that BmIRE1alpha is used as an important molecular target for regulating and controlling bombyx mori nuclear polyhedrosis virus (BmNPV) replication, an inhibitor 4mu8C not only blocks virus replication, but also regulates and controls a bombyx mori endoplasmic reticulum stress pathway and the expression level of key genes, and the BmIRE1alpha can be used for regulating and controlling bombyx mori nuclear polyhedrosis virus (BmNPV) replication. And the cell stress burden caused by virus infection is relieved. Researches show that 4 mu 8C has an accurate and efficient antiviral effect, has more potential compared with traditional pesticides, avoids damage to the environment and non-target organisms, provides new technical support for green and sustainable development of sericulture, and has great development and application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology. Specifically, the present invention relates to the application of 4μ8C in the preparation of drugs for preventing and treating Bombyx mori nuclear polyhedrosis virus. Background Art

[0002] The silkworm (Bombyx mori) is the world's most important silk-producing insect, belonging to the family Bombycidae of the order Lepidoptera. Silkworms have a high reproduction rate and adaptability during breeding, making them the foundation of the global silk industry. Bombyx mori nuclear polyhedrosisvirus (BmNPV) is one of the common pathogenic viruses in silkworm breeding. It is a double-stranded DNA virus classified in the family Baculoviridae. Large-scale infection can lead to severe mortality in the silkworm population, causing huge losses to the sericulture industry. BmNPV not only affects the growth and development of silkworms, but also suppresses the silkworm's immune system. The virus evades the host's immune response through a variety of mechanisms, including regulating the host's cell apoptosis and endoplasmic reticulum stress response. The endoplasmic reticulum stress response of host cells is a key point in the process of viral infection, because endoplasmic reticulum stress can help the virus successfully replicate and spread in the cell. So far, there is no effective method to prevent and control BmNPV. Therefore, it is of great significance to study the inhibitory effects of Bombyx mori nuclear polyhedrosis virus.

[0003] 4μ8C (IRE1 Inhibitor III) is a small molecule chemical inhibitor. Studies have shown that it can specifically inhibit the activity of IRE1, a key enzyme in the endoplasmic reticulum stress response. IRE1 plays an important role in the unfolded protein response (UPR) of cells and regulates the ability of cells to cope with endoplasmic reticulum stress. The endoplasmic reticulum stress response plays an important role in the process of viral infection. Many viruses activate the endoplasmic reticulum stress response to help them replicate and escape the attack of the host immune system. However, the relationship between IRE1 and BmNPV infection in silkworms has not been further studied and explored, which will provide new ideas for the prevention and treatment of BmNPV. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an application of 4μ8C (IRE1 Inhibitor III) for preparing drugs for treating or preventing Bombyx mori nuclear polyhedrosis virus. Through in-depth research and exploration, the present invention found that when BmNPV infects silkworm cells, it activates the IRE1-mediated endoplasmic reticulum stress pathway, promoting the replication and proliferation of the virus. Due to the key role of IRE1 in the viral replication process, inhibiting its activity is expected to become a new antiviral strategy. As an IRE1 inhibitor, 4μ8C can reduce the endoplasmic reticulum stress response by blocking the RNase activity of IRE1, thereby improving the survival rate of host cells, providing new ideas for the development of antiviral drugs for silkworms.

[0005] In one aspect of the present invention, the present invention provides a method for preventing and treating Bombyx mori nuclear polyhedrosis virus, the method comprising applying a preparation containing 4μ8C to the silkworm. The preparation can inhibit the activity of the silkworm endoplasmic reticulum key enzyme IRE1, thereby inhibiting the infection of BmNPV to the silkworm. The application comprises adding the preparation containing 4μ8C to the food of the silkworm for feeding, or spraying it on mulberry leaves for feeding the silkworm. The concentration of the 4μ8C used is 4μM for silkworm bodies and 3μM for cells. The administration time of the 4μ8C is before the silkworm is infected with BmNPV, or within 12 hours after infection with BmNPV.

[0006] In one aspect of the present invention, the present invention provides the use of the inhibitor 4μ8C in the preparation of a preparation for preventing and controlling Bombyx mori nuclear polyhedrosis virus.

[0007] In one embodiment, the formulation is administered to the silkworms by feeding.

[0008] In one embodiment, the concentration of 4μ8C in the preparation is 4 μM for silkworm bodies and 3 μM for cells.

[0009] In one embodiment, the administration time of the 4μ8C-containing formulation is before the silkworm is infected with BmNPV, or within 12 hours after infection with BmNPV.

[0010] In one embodiment, the formulation includes a pharmaceutically acceptable excipient that can enhance drug stability and palatability.

[0011] In one embodiment, the formulation is in the form of a powder, solid granules or a liquid formulation. Those skilled in the art can select the desired formulation according to the feeding method of the silkworm. Preferably, the formulation is a liquid formulation, which is convenient for silkworm feeding and absorption.

[0012] The present invention discloses the use of 4μ8C in the preparation of drugs for inhibiting BmNPV, and clarifies its mechanism of action in inhibiting BmNPV replication. By rationally mixing 4μ8C in the food of silkworms, it can be used for post-infection treatment; or by preventively adding 4μ8C 24 hours before virus infection, it can effectively prevent the proliferation of the virus and play a role in preventing infection. Beneficial Effects

[0013] (1) The present invention found that 4μ8C can effectively inhibit the proliferation of Bombyx mori nuclear polyhedrosis virus (BmNPV) in cells and silkworms, revealing that BmIRE1α is an important molecular target for regulating viral replication, providing a new strategy for precise virus prevention and control, and laying a scientific foundation for virus prevention and control in the silkworm industry. 4μ8C not only blocks viral replication, but also regulates the expression levels of the silkworm endoplasmic reticulum stress pathway and key genes, reducing the cellular stress burden caused by viral infection, and is beneficial to the healthy growth and production performance of silkworms.

[0014] (2) The present invention combines high-throughput RNA-seq and bioinformatics analysis to systematically reveal that the differentially expressed genes in cells after 4μ8C treatment are concentrated on antiviral immune pathways, endoplasmic reticulum protein homeostasis regulation and cellular metabolic balance. Precise targeting provides more core targets for the development of anti-BmNPV viral drugs.

[0015] (3) The present invention is that 4μ8C has a precise and efficient antiviral effect, which is more promising than traditional pesticides. It avoids damage to the environment and non-target organisms, provides new technical support for the green and sustainable development of the silkworm industry, and has great development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 , detect the expression analysis of BmIRE1α in different developmental stages of silkworm and after virus infection. A is the expression level of BmIRE1α in different developmental stages of silkworm, B is the expression in different tissues of silkworm (such as hemolymph, midgut and epidermis, etc.), and C is the gene expression analysis of BmIRE1α in cells at different time periods after infection with BmNPV-EGFP.

[0017] Figure 2 , knock down BmIRE1α by siRNA transfection technology to study its effect on the replication of the virus BmNPV. Among them: A is the expression level of the VP39 gene during BmNPV virus infection at different time periods, B is the gene detection efficiency of BmIRE1α in cells at different time periods, and C is the intensity analysis of the EGFP-BmNPV fluorescence signal at different time periods after siBmIRE1α treatment detected by fluorescence microscopy.

[0018] Figure 3, and knock down BmIRE1α to detect its effect on BmNPV virus replication. Among them: AI is the replication of BmNPV virus VP39 gene and BmIRE1α gene expression in silkworms after siBmIRE1α. The gDNA copy number of BmNPV VP39 in the hemolymph (A), fat body (D) and midgut (G) of silkworms was detected by gDNA template qPCR; the gene expression of viral VP39 in hemolymph (B), fat body (E), midgut (H) after infection with BmNPV virus and the efficiency of BmIRE1α knockdown in hemolymph (C), fat body (F), and midgut (I).

[0019] Figure 4 IXA4 treatment can rescue cells that inhibit BmNPV proliferation. A detected siUFBP1+IXA4 and used siNC+DMSO and siUFBP1+DMSO as controls to detect the proliferation of BmNPV after infection. VP39 Gene expression, B detects siUFBP1+IXA4. Using siNC+DMSO and siUFBP1+DMSO as controls, detects GP64 gene expression after virus infection. C detects siUFBP1+IXA4. Using siNC+DMSO and siUFBP1+DMSO as controls, detects GP64 gene expression after virus infection. BmBIP Gene expression. As above, D detects BmGRP94, E detects BmCANX, and F detects BmXBP1 gene expression. G detects siUFBP1+IXA4. Using siNC+DMSO and siUFBP1+DMSO as controls, the expression of VP39 gene gDNA after virus infection was detected. H Fluorescence microscopy was used to detect the intensity of the green fluorescence signal of BmNPV-EGFP in cells.

[0020] Figure 5 , to rescue cells that were stimulated by BmUFBP1 to promote BmNPV proliferation by using 4μ8C (IRE1α inhibitor). Among them, cells were transfected with overexpressed BmUFBP1 for 12 h, treated with 4μ8C (at a concentration of 3μM), and infected with the virus BmNPV-EGFP. The control group (Vector and BmUFBP1) and the 4μ8C treatment group (BmUFBP1+4μ8C) were tested respectively. VP39 (A), GP64(B), BmBIP (C), BmXBP1 (D) Gene expression level. E is the intensity of green fluorescence signal of BmNPV-EGFP in cells detected by fluorescence microscopy in Vector+DMSO (control group), BmUFBP1+DMSO (virus promotion group), and BmUFBP1+4μ8C (4μ8C treatment group).

[0021] Figure 6, is the RNA-seq analysis of silkworm BmN cells treated with 4μ8C. A is DEGs GO enrichment analysis, B is pathway enrichment analysis, C is gene annotation, and D is GSEA enrichment analysis to analyze the effects of 4μ8C on the endoplasmic reticulum stress pathway (IRE1α) and its downstream transcriptional and metabolic pathways.

[0022] Figure 7 , In order to use 4μ8C treatment to affect the proliferation of BmNPV in cells, the key genes of viral GP64 and endoplasmic reticulum stress were detected. Among them, the control group DMSO and the experimental group 4μ8C were treated with the same concentration of 3μM and infected with the virus BmNPV-EGFP. The detection time periods were 24h, 48h and 72h respectively GP64 (A), BmXBP1 (B), BmBIP (C), BmCANX (D), BmGRP94(E) Gene expression level. F is the intensity of green fluorescence signal of BmNPV-EGFP in cells detected by fluorescence microscopy with DMSO (control group) and 4μ8C (experimental group). DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The equipment and reagents used in the embodiments and test examples can be obtained from commercial sources unless otherwise specified. 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 intended to limit the present invention. Embodiment 1: 1.1 Bioinformatics analysis of BmIRE1

[0024] The protein sequence of BmIRE1α from Bombyx mori (NCBI accession number XP_037872628.1) was aligned with homologous sequences from other species. Specifically, BmIRE1α (XP_037872628.1) and IRE1 from A. gambiaeα (XP_562694.5), D. plexippus (XP_061377098.1), ps uthus (XP_013169478.1), D. rerio (XP_001919350.3), ms musculus (XP_037293644.1), ae aegypti (XP_021710632.1), and the residues in the sequence are colored according to the scores assigned to the columns. The phylogenetic tree of BmIRE1α and other homologous sequences was constructed using the neighbor-joining method using MEGA11 software. It was found that the BmIRE1α domain was highly conserved and was most closely related to the IRE1 protein of cotton bollworm.

[0025] 1.2 BmIRE1α plays an important regulatory role in silkworm resistance to BmNPV virus replication The 5th instar silkworms with good growth were divided into 8 groups and 10 groups according to different experimental objectives to detect the expression of BmIRE1α in different developmental stages and tissues of silkworms. Figure 1 By extracting mRNA, reverse transcription and synthesizing cDNA, and RT-qPCR analysis, it was found that BmIRE1α was highly expressed in the 4th instar larvae and in stages and tissues closely related to development, such as hemolymph, Malpighian tubules and epidermis, indicating that it plays an important regulatory role in the silkworm. Next, the expression of the BmIRE1α gene in BmN cells infected with EGFP-tagged BmNPV virus for 24h, 48h, and 72h was detected. It was found that compared with the control group, during the process of virus infection, at a certain stage, BmIRE1α would inhibit the continued replication of the virus and the expression level would increase. This suggests that BmIRE1α may play an important role in inhibiting viral proliferation. Effect of α-treatment on BmNPV proliferation in cells and detection of viral VP39 gene expression

[0026] See also Figure 2 By detecting cells transfected with BmIRE1α siRNA 24h, 48h and 72h after BmNPV virus infection, it was found that the expression of viral VP39 was significantly downregulated after knocking down BmIRE1α. The same result was obtained by fluorescence microscopy. After BmIRE1α was knocked down, the fluorescence intensity of BmNPV virus with EGFP label was weakened, indicating that the presence of BmIRE1α promoted the proliferation of the virus. By knocking down BmIRE1α, the expression of genes related to the endoplasmic reticulum stress response was weakened, which may lead to the obstruction of viral replication, which is reflected in the decrease of fluorescence intensity.

[0027] Furthermore, we selected 5th instar silkworms with good growth to detect the effect of knocking down BmIRE1α on BmNPV virus replication. Figure 3 , mainly detected the silkworm hemolymph (see Figure 3 AC), fat body (see Figure 3 mid DF) and midgut (see Figure 3The gene copy number and expression level of VP39 and the expression level of BmIRE1α in the midgut (GI) of silkworms were first detected by gDNA template qPCR. It was found that the copy number of BmNPV VP39 in the hemolymph (A), fat body (D) and midgut (G) of silkworms was significantly lower than that in the control group, indicating that knocking down BmIRE1α inhibited the replication of the virus. Subsequently, the gene expression level of viral VP39 in hemolymph (B), fat body (E) and midgut (H) after infection with BmNPV virus and the efficiency of BmIRE1α knockdown in hemolymph (C), fat body (F) and midgut (I) were detected. It was found that the mRNA expression level of the knockdown group was significantly downregulated and the expression level of BmIRE1α in the siBmIRE1α-1 and siBmIRE1α-2 groups was significantly decreased, which further verified the inhibitory effect of knocking down BmIRE1α on viral replication. It can reverse the knockdown of BmUFBP1 and reactivate IRE1α in the endoplasmic reticulum stress pathway, thereby promoting the replication of BmNPV virus

[0028] First, the synthetic siUFBP1 was designed and transfected into BmN cells for 12 h, and then the cells were treated with the BmIRE1α activator IXA4 (3 μM) and infected with BmNPV-EGFP. Figure 4 , qRT-PCR detection of BmNPV virus VP39 and GP64 ( Figure 4 AB), key genes related to endoplasmic reticulum stress BmBIP, BmGRP94, BmCANX, BmXBP1 ( Figure 4 CF) mRNA level; Figure 4 G) Using gDNA as template, BmGAPDH As an internal reference, qPCR detection of BmNPV virus VP39 The gDNA copy number of the gene was then measured by ( Figure 4 Middle H) Fluorescence microscopy was used to detect the expression level of BmNPV-EGFP in BmN cells.

[0029] Results: BmIRE1α activator IXA4 activated IRE1α protein in the endoplasmic reticulum stress pathway and reversed the inhibition of viral replication ( Figure 4 AB), mainly by activating key genes in the downstream stress response pathways such as BmBIP, BmGRP94, BmCANX, BmXBP1 ( Figure 4 CF) and other expressions, and the same results were obtained by fluorescence microscopy ( Figure 4H). BmUFBP1 is a protein involved in the endoplasmic reticulum stress response, and its knockdown usually affects viral replication. IXA4 is a BmIRE1α agonist that can activate the IRE1α protein in the endoplasmic reticulum stress pathway, thereby activating downstream stress response pathways (such as XBP1, etc.). When the endoplasmic reticulum stress pathway is activated, it usually helps the replication of the virus because the virus may rely on this pathway to improve its survival and proliferation environment. Inhibitor treatment can rescue cells affected by BmNPV proliferation caused by BmUFBP1 overexpression

[0030] The cells were transfected with the BmUFBP1 overexpression plasmid for 12 h, then treated with the BmIRE1α inhibitor 4μ8C (at a concentration of 3μM) and infected with BmNPV-EGFP. VP39, GP64, BmBIP, BmXBP1 Gene expression level, and fluorescence microscopy was used to detect the expression of BmNPV-EGFP in BmN cells using Vector+DMSO (control group), BmUFBP1+DMSO (virus promotion group), and BmUFBP1+4μ8C (4μ8C treatment group).

[0031] The results show that: Figure 5 Middle (AB), under the condition of overexpression of BmUFBP1, viral gene VP39 and GP64 The expression of BmUFBP1 was significantly upregulated, indicating that BmUFBP1 promoted viral replication. After adding the endoplasmic reticulum stress inhibitor 4μ8C, the expression of viral genes decreased significantly, indicating that endoplasmic reticulum stress may play an important role in BmUFBP1 promoting viral proliferation. Figure 5 In the middle (CD), the expression of ER stress-related genes was detected. After overexpression of BmUFBP1, the mRNA level of ER stress marker gene BmBIP was significantly increased, indicating that BmUFBP1 can activate ER stress. After adding 4μ8C, the expression of BmBIP decreased significantly, further proving that 4μ8C inhibited the ER stress signaling pathway. BmXBP1 BmUFBP1 also activated its expression, but the activation gradually weakened after 48h and 72h, indicating that endoplasmic reticulum stress is more active in the early stage of viral infection. Figure 5In Figure E, fluorescence microscopy revealed that compared with the control group (Vector+DMSO), the fluorescence intensity of the BmUFBP1 overexpression group (BmUFBP1+DMSO) was significantly enhanced, and after the addition of the 4μ8C inhibitor (BmUFBP1+4μ8C), the fluorescence intensity was significantly weakened, indicating that the endoplasmic reticulum stress inhibitor can significantly reduce the replication ability of the virus. This further verifies the intervention effect of 4μ8C on viral proliferation in inhibiting the BmIRE1α-related pathway. 1.6 RNA-seq analysis of silkworm BmN cells treated with 4μ8c

[0032] The silkworm BmN cells treated with the BmIRE1α inhibitor 4μ8c were used for RNA-seq to screen out differentially expressed genes (DEGs). The effects of 4μ8C on the endoplasmic reticulum stress pathway (IRE1α) and its downstream transcriptional and metabolic pathways were analyzed by GO enrichment analysis, pathway enrichment analysis, gene annotation and GSEA enrichment analysis of DEGs.

[0033] See also Figure 6 , the results showed that: GO analysis showed that 4μ8C treatment had a significant effect on genes related to endoplasmic reticulum stress, transport process and cell structure. The main biological processes (BP) involved included cell proliferation, response to stress, and transport process; the cell components (CC) involved were significantly enriched in the Golgi apparatus membrane and extracellular matrix; the molecular functions (MF) involved were receptor binding and enzymeregulator activity. These results support the mechanism that 4μ8C regulates endoplasmic reticulum stress pathways and cell metabolic signals, thereby intervening in viral infection.

[0034] Pathway enrichment analysis of DEGs showed that 4μ8C treatment may inhibit viral replication by affecting Golgi apparatus-related function and lipid transport and metabolism, suggesting its potential molecular mechanism of action.

[0035] The results of differential gene annotation showed that genes related to lipid transport and metabolism, signal transduction mechanisms, chromatin structure and dynamics were widely annotated, indicating that 4μ8C may interfere with the synthesis or metabolism of cell membrane components and indirectly inhibit virus packaging and transmission. The enrichment of signal transduction mechanisms may be related to virus-induced stress signals and immune responses.

[0036] GSEA analysis showed that folate biosynthesis, pyrimidine metabolism, and ribosome signaling pathways indicated that 4μ8C may inhibit viral proliferation by regulating nucleic acid synthesis and metabolic processes. The activity of ribosome-related pathways suggested that it may regulate the synthesis of viral proteins by affecting protein translation efficiency.

[0037] In summary, 4μ8C significantly affects the endoplasmic reticulum stress pathway (IRE1α) and its downstream transcriptional and metabolic pathways. GO and pathway analysis showed that 4μ8C regulates lipid metabolism, Golgi function, and nucleic acid metabolism, and may directly or indirectly interfere with viral replication and transmission. 1.7 Detection of viral GP64 and endoplasmic reticulum stress key genes affecting BmNPV proliferation in cells using 4μ8c treatment

[0038] The control group was treated with DMSO, the experimental group was treated with 4μ8C, and 3μM was added at the same concentration, and the virus BmNPV-EGFP was infected. The time periods of 24h, 48h and 72h were tested respectively GP64 , BmXBP1 , BmBIP , BmCANX , BmGRP94 Gene expression level. Fluorescence microscopy was used to detect the intensity of green fluorescence signals of BmNPV-EGFP in cells treated with DMSO (control group) and 4μ8C (experimental group).

[0039] See also Figure 7 The experimental results showed that compared with the control group, 3μM 4μ8C could effectively inhibit the gene expression of viral GP64 ( Figure 7 (A) Meanwhile, as time goes by, 4μ8C will significantly inhibit key genes related to endoplasmic reticulum pathway stress. BmXBP1 (B) BmBIP (C) BmCANX (D) BmGRP94(E). Also, see Figure 7 Middle F, fluorescence microscopy revealed that the green fluorescence intensity of EGFP-BmNPV treated with 4μ8C was weakened compared with the control group.

[0040] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope determined by the claims submitted for the present invention.

Claims

1. A method for preventing and treating Bombyx mori nuclear polyhedrosis virus, characterized in that: The method comprises administering a preparation containing 4μ8C to silkworms.

2. Application of inhibitor 4μ8C in the preparation of preparations for the prevention and treatment of Bombyx mori nuclear polyhedrosis virus.

3. The use according to claim 2, characterized in that: The formulation contains the inhibitor 4μ8C.

4. The use according to claim 3, characterized in that: The preparation further contains a pharmaceutically acceptable excipient; preferably, the excipient is a substance that enhances the stability of 4μ8C or enhances the palatability of the preparation.

5. The use according to claim 2, characterized in that: The dosage form of the preparation is powder, solid granules or liquid preparation.

6. The use according to claim 2, characterized in that: The concentration of 4μ8C used in the preparation is 4μM for silkworm bodies and 3μM for cells.

7. The use according to claim 2, characterized in that: The preparation is used before the silkworm is infected with BmNPV, or within 12 hours after being infected with BmNPV.

8. The use according to claim 2, characterized in that: The formulation is sprayed on mulberry leaves and then administered to silkworms through feeding.

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