Application of 4μ8C in the preparation of a preparation for preventing and treating Bombyx mori nuclear polyhedrosis virus

Abstract

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 nucleopolyhedrovirus. Through bioinformatics analysis, gene knockout, RT-PCR detection and other methods, the present invention discovers that BmIRE1α is an important molecular target for regulating the replication of Bombyx mori nucleopolyhedrovirus (BmNPV). The inhibitor 4μ8C not only blocks virus replication, but also regulates the endoplasmic reticulum stress pathway and the expression levels of key genes in Bombyx mori, reducing the cellular stress burden caused by virus infection. The research of the present invention shows that 4μ8C has precise and efficient antiviral effects, is more potential than traditional pesticides, avoids damage to the environment and non-target organisms, provides new technical support for the green and sustainable development of the sericulture industry, 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 most important silk-producing insect globally and belongs to the family Bombycidae of the order Lepidoptera. The silkworm has a high reproductive rate and adaptability during the breeding process, thus becoming the basis of the global silk industry. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the common pathogenic viruses during the silkworm breeding process. It belongs to double-stranded DNA viruses and is classified in the family Baculoviridae. Large-scale infections can lead to severe deaths 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 immune system of silkworms. The virus evades the host immune response through multiple mechanisms, including regulating host cell apoptosis, endoplasmic reticulum stress response, etc. The endoplasmic reticulum stress response of host cells is a key point during the virus infection process because endoplasmic reticulum stress can help the virus successfully replicate and spread within the cell. So far, there is no relatively effective method for preventing and treating the BmNPV virus. Therefore, it is of great significance to study drugs that inhibit the Bombyx mori nuclear polyhedrosis virus.

[0003] 4μ8C (IRE1 Inhibitor III) is a small molecule chemical inhibitor. Research shows 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 cell's ability to respond to endoplasmic reticulum stress. The endoplasmic reticulum stress response plays an important role during the virus infection process. Many viruses activate the endoplasmic reticulum stress response to help their replication and evade the attack of the host immune system. However, the relationship between IRE1 in silkworms and the infection of BmNPV 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 object of the present invention is to provide an application of preparing a drug for treating or preventing Bombyx mori nucleopolyhedrovirus based on 4μ8C (IRE1 Inhibitor III). Through in-depth research and exploration, the present invention discovers that when BmNPV infects Bombyx mori cells, it will activate the IRE1-mediated endoplasmic reticulum stress pathway, promoting the replication and proliferation of the virus. Due to the key role of IRE1 in the virus replication process, inhibiting its activity is expected to become a new antiviral strategy. As an IRE1 inhibitor, 4μ8C can weaken the endoplasmic reticulum stress response by blocking the RNase activity of IRE1, thereby increasing the survival rate of host cells, providing new ideas for the development of antiviral drugs for Bombyx mori.

[0005] In one aspect of the present invention, the present invention provides a method for preventing and treating Bombyx mori nucleopolyhedrovirus, and the method is to apply a preparation containing 4μ8C to Bombyx mori. The preparation can inhibit the activity of the endoplasmic reticulum enzyme IRE1 of Bombyx mori, thereby inhibiting the infection of BmNPV to Bombyx mori. The application is to add the preparation containing 4μ8C to the food of Bombyx mori for feeding, or spray it on mulberry leaves for feeding Bombyx mori. The usage concentration of 4μ8C is 4μM for silkworm bodies and 3μM for cells. The administration time of 4μ8C is before Bombyx mori is infected with BmNPV, or within 12 hours after being infected with BmNPV.

[0006] In one aspect of the present invention, the present invention provides the application of the inhibitor 4μ8C in preparing a preparation for preventing and treating Bombyx mori nucleopolyhedrovirus.

[0007] In one embodiment, the preparation is applied to Bombyx mori by feeding.

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

[0009] In one embodiment, the administration time of the preparation containing 4μ8C is before Bombyx mori is infected with BmNPV, or within 12 hours after being infected with BmNPV.

[0010] In one embodiment, the preparation includes pharmaceutically acceptable excipients, and the excipients can enhance the drug stability and palatability.

[0011] In one embodiment, the dosage form of the preparation is powder, solid granule or liquid preparation. Those skilled in the art can select the required dosage form according to the feeding method of Bombyx mori. Preferably, the dosage form is liquid preparation, which is convenient for the feeding and absorption of Bombyx mori.

[0012] The application of 4μ8C in the preparation of drugs for inhibiting BmNPV is disclosed in the present invention, and its mechanism of action in inhibiting BmNPV replication is clarified. By reasonably mixing 4μ8C in the food of silkworms, it can be used for the treatment after infection; or 4μ8C is prophylactically added 24 hours before virus infection to effectively prevent the proliferation of the virus and play a role in preventing infection. Beneficial effects

[0013] (1)The present invention discovers that 4μ8C can effectively inhibit the proliferation of Bombyx mori nucleopolyhedrovirus (BmNPV) in cells and silkworms, reveals that BmIRE1α is an important molecular target for regulating virus replication, provides a new strategy for the precise prevention and control of the virus, and lays a scientific foundation for the prevention and control of sericulture viruses. 4μ8C not only blocks virus replication, but also regulates the endoplasmic reticulum stress pathway and the expression levels of key genes in silkworms, reduces the cellular stress burden caused by virus infection, and is beneficial to the healthy growth and improvement of 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 the antiviral immune pathway, endoplasmic reticulum protein homeostasis regulation, and cellular metabolic balance, and the precise targeting provides more core targets for the development of anti-BmNPV virus drugs.

[0015] (3)The present invention lies in the fact that 4μ8C has precise and efficient antiviral effects, has more potential than traditional pesticides, avoids damage to the environment and non-target organisms, provides new technical support for the green and sustainable development of sericulture, and has great development and application prospects. Description of the drawings

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

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

[0018] Figure 3, detect the effect of knocking down BmIRE1α on the replication of BmNPV virus. Among them: A-I are the analysis of the replication of BmNPV virus VP39 gene and the expression of BmIRE1α gene in silkworms after siBmIRE1α. Use gDNA template qPCR to detect the gDNA copy number of BmNPV VP39 in the hemolymph (A), fat body (D) and midgut (G) of silkworms; after infecting with BmNPV virus, the gene expression level of virus VP39 in the hemolymph (B), fat body (E), midgut (H) and the knockdown efficiency of BmIRE1α in the hemolymph (C), fat body (F), midgut (I).

[0019] Figure 4 , IXA4 treatment can rescue the cells in which siBmUFBP1 inhibits BmNPV proliferation. Among them, A detects siUFBP1+IXA4 with siNC+DMSO and siUFBP1+DMSO as controls, and detects the gene expression level after virus infection VP39 , B detects siUFBP1+IXA4 with siNC+DMSO and siUFBP1+DMSO as controls, and detects the gene expression level of GP64 after virus infection, C detects siUFBP1+IXA4 with siNC+DMSO and siUFBP1+DMSO as controls, and detects the gene expression level after virus infection BmBIP . Similarly, D detects the gene expression level of BmGRP94, E detects the gene expression level of BmCANX, and F detects the gene expression level of BmXBP1. G detects the gDNA expression level of VP39 gene after infecting with virus with siUFBP1+IXA4 with siNC+DMSO and siUFBP1+DMSO as controls. H uses fluorescence microscopy to detect the intensity of green fluorescence signal of BmNPV-EGFP in cells.

[0020] Figure 5 , to use 4μ8C (IRE1α inhibitor) treatment to rescue the cells in which BmUFBP1 promotes BmNPV proliferation. Among them, cells transfected with overexpressed BmUFBP1 are treated for 12 h with 4μ8C (concentration: 3 μM) and infected with virus BmNPV-EGFP. The control group (Vector and BmUFBP1) and the 4μ8C treatment group (BmUFBP1+4μ8C) are used to detect the gene expression levels respectively VP39 (A), GP64(B), BmBIP (C), BmXBP1 (D) . E uses fluorescence microscopy to detect the intensity of green fluorescence signal of BmNPV-EGFP in cells in Vector+DMSO (control group), BmUFBP1+DMSO (virus promotion group), BmUFBP1+4μ8C (4μ8C treatment group).

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

[0022] Figure 7 , To investigate the effect of 4μ8C treatment on the proliferation of BmNPV in cells, the virus GP64 and key genes of 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 gene expression levels were detected at 24h, 48h, and 72h time periods respectively. GP64 (A), BmXBP1(B), BmBIP (C), BmCANX (D), BmGRP94(E) F is to detect the intensity of the green fluorescence signal of BmNPV-EGFP in cells by using a fluorescence microscope for DMSO (control group) and 4μ8C (experimental group). Detailed implementation methods

[0023] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. 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 used to limit the present invention.

[0024] Example 1:

[0025] 1.1 Bioinformatics analysis of BmIRE1

[0026] The protein sequence of BmIRE1α (NCBI accession number XP_037872628.1) of the silkworm was subjected to homologous multiple sequence alignment with other species. Specifically, BmIRE1α (XP_037872628.1) was aligned with IRE1α from A. gambiae (XP_562694.5), D. plexippus (XP_061377098.1), P. satyrus (XP_013169478.1), D. rerio (XP_001919350.3), M. musculus (XP_037293644.1), A. aegypti (XP_021710632.1). The residues in the sequences were colored according to the scores assigned to the columns. Using MEGA11 software, a phylogenetic tree of BmIRE1α and other homologous sequences was constructed by the neighbor-joining method. It was found that the BmIRE1α domain was highly conserved and was most closely related to the IRE1 protein of Helicoverpa armigera.

[0027] 1.2 BmIRE1α plays an important regulatory role in the anti-BmNPV virus replication in the silkworm

[0028] Well-growing fifth-instar silkworms were taken and divided into 8 groups and 10 groups respectively according to different experimental objectives to detect the expression levels of BmIRE1α at different developmental stages and tissues of the silkworm. See Figure 1 , through mRNA extraction, reverse transcription to synthesize cDNA and RT-qPCR analysis, it was found that BmIRE1α was highly expressed in the fourth-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 body. Immediately afterwards, the expression levels of the BmIRE1α gene in BmN cells infected with BmNPV virus with an EGFP tag for 24 h, 48 h, and 72 h were detected. It was found that compared with the control group, during the process of virus infection, at a certain stage, BmIRE1α would inhibit the continuous replication of the virus and the expression level would increase. This implies that BmIRE1α may play an important role in inhibiting virus proliferation.

[0029] Effect of α treatment on the proliferation of BmNPV in cells and detection of virus VP39 gene expression

[0030] See Figure 2, By detecting the cells transfected with BmIRE1α siRNA and infected with BmNPV virus for 24 h, 48 h, and 72 h, it was found that after knocking down BmIRE1α, the expression level of viral VP39 was significantly downregulated. The same result was obtained by fluorescence microscopy. After BmIRE1α was knocked down, the fluorescence intensity of BmNPV virus with EGFP tag was weakened, indicating that the presence of BmIRE1α promoted virus proliferation. By knocking down BmIRE1α, the expression of genes related to endoplasmic reticulum stress response was weakened, which might lead to the inhibition of virus replication, reflected as the decrease in fluorescence intensity.

[0031] Furthermore, fifth-instar silkworms with good growth were selected to detect the effect of knocking down BmIRE1α on BmNPV virus replication. See Figure 3 , mainly detected the changes in the gene copy number and expression level of VP39 and the expression level of BmIRE1α in the hemolymph (see Figure 3 A-C in it), fat body (see Figure 3 D-F in it), and midgut (see Figure 3 G-I in it) of the silkworm body. First, qPCR was used with gDNA template to detect the gDNA copy number of BmNPV VP39 in the hemolymph (A), fat body (D), and midgut (G) of the silkworm body. It was found that the copy number in the knockdown group (siBmIRE1α-1 and siBmIRE1α-2) was significantly lower than that in the control group, indicating that knocking down BmIRE1α inhibited virus replication. Subsequently, the gene expression level of viral VP39 in the hemolymph (B), fat body (E), and midgut (H) and the knockdown efficiency of BmIRE1α in the hemolymph (C), fat body (F), and midgut (I) after infecting with BmNPV virus were detected. It was found that the mRNA expression level in 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 virus replication.

[0032] Knocking down BmUFBP1 can reverse the situation, reactivate IRE1α in the endoplasmic reticulum stress pathway, and thus promote the replication of BmNPV virus

[0033] First, siUFBP1 was designed and synthesized for transfection of BmN cells for 12 h, then the cells were treated with BmIRE1α activator IXA4 (3 μM) and infected with BmNPV-EGFP. See Figure 4 , qRT-PCR was used to detect the mRNA levels of BmNPV virus VP39 and GP64 ( Figure 4 A-B in it), key genes related to endoplasmic reticulum stress BmBIP, BmGRP94, BmCANX, BmXBP1 ( Figure 4 C-F in it); ( Figure 4 G in it) Using gDNA as a template, withBmGAPDH As an internal reference, the gDNA copy number of the BmNPV virus was detected by qPCR VP39 and the expression level of BmNPV-EGFP in BmN cells was subsequently detected by fluorescence microscopy ( Figure 4 in H).

[0034] Results: The BmIRE1α activator IXA4 can activate the IRE1α protein in the endoplasmic reticulum stress pathway to reverse the inhibition of virus replication ( Figure 4 in A-B), importantly by activating key genes in the downstream stress response pathway such as BmBIP, BmGRP94, BmCANX, BmXBP1 ( Figure 4 in C-F), etc. The same results were obtained by fluorescence microscopy detection ( Figure 4 in H). BmUFBP1 is a protein involved in the endoplasmic reticulum stress response, and its knockdown usually affects virus replication. IXA4 is a BmIRE1α agonist that can activate the IRE1α protein in the endoplasmic reticulum stress pathway, thereby activating the downstream stress response pathway (such as XBP1, etc.). When the endoplasmic reticulum stress pathway is activated, it usually helps virus replication because the virus may rely on this pathway to improve its survival and proliferation environment.

[0035] Treatment with an inhibitor can rescue the cells affected by BmNPV proliferation caused by overexpression of BmUFBP1

[0036] Construct a plasmid overexpressing BmUFBP1 and transfect cells for 12 h, then treat with the BmIRE1α inhibitor 4μ8C (concentration 3 μM), and infect with BmNPV-EGFP. Detect the gene expression level by qRT-PCR VP39, GP64, BmBIP, BmXBP1 and at the same time use fluorescence microscopy to detect the expression level of BmNPV-EGFP in BmN cells in Vector+DMSO (control group), BmUFBP1+DMSO (virus promotion group), and BmUFBP1+4μ8C (4μ8C treatment group).

[0037] Results showed: See Figure 5 in (A-B), in the case of overexpressing BmUFBP1, the expression levels of the virus genes VP39 and GP64 were significantly upregulated, indicating that BmUFBP1 promoted virus replication. After adding the endoplasmic reticulum stress inhibitor 4μ8C, the virus gene expression level decreased significantly, indicating that endoplasmic reticulum stress may play an important role in BmUFBP1 promoting virus proliferation. See Figure 5In (C-D), the expression of genes related to endoplasmic reticulum stress was detected. After overexpressing BmUFBP1, the mRNA level of the endoplasmic reticulum stress marker gene BmBIP increased significantly, indicating that BmUFBP1 can activate endoplasmic reticulum stress. After adding 4μ8C, the expression of BmBIP decreased significantly, further proving that 4μ8C inhibits the endoplasmic reticulum stress signaling pathway. For BmXBP1 genes, BmUFBP1 also activated their expression, but this activation gradually weakened after 48 h and 72 h, indicating that endoplasmic reticulum stress is more active in the early stage of virus infection. See Figure 5 In E, fluorescence microscopy detection found that: compared with the control group (Vector + DMSO), the fluorescence intensity of the BmUFBP1 overexpression group (BmUFBP1 + DMSO) was significantly enhanced. After adding the 4μ8C inhibitor (BmUFBP1 + 4μ8C), the fluorescence intensity decreased significantly, indicating that the endoplasmic reticulum stress inhibitor can significantly reduce the virus replication ability. This further verified the intervention effect of 4μ8C on virus proliferation in inhibiting the BmIRE1α-related pathway.

[0038] 1.6 RNA-seq analysis of silkworm BmN cells treated with 4μ8c

[0039] RNA-seq was performed on the differentially expressed genes (DEGs) screened from silkworm BmN cells treated with the BmIRE1α inhibitor 4μ8c. 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, respectively.

[0040] See Figure 6 , and the results showed that: GO analysis showed that the treatment with 4μ8C had significant effects on genes related to endoplasmic reticulum stress, transport processes, and cell structure. The main biological processes (BP) involved included cell proliferation, response to stress, and transport process; the cellular components (CC) involved were significantly enriched in the Golgi apparatus membrane and extracellular matrix; the molecular functions (MF) involved were receptor binding and enzyme regulator activity. These results support the mechanism by which 4μ8C regulates the endoplasmic reticulum stress pathway and cell metabolism signals to interfere with virus infection.

[0041] Pathway enrichment analysis of DEGs indicated that 4μ8C treatment might inhibit virus replication by affecting pathways such as Golgi apparatus-related functions and lipid transport and metabolism, suggesting its potential molecular mechanism of action.

[0042] The results of differential gene annotation showed that genes related to lipid transport and metabolism, signal transduction mechanisms, chromatin structure and dynamics, etc. were widely annotated, indicating that 4μ8C might interfere with the synthesis or metabolism of cell membrane components and indirectly inhibit virus packaging and transmission. The enrichment of signal transduction mechanisms might be related to virus-induced stress signals and immune responses.

[0043] GSEA analysis showed that folate biosynthesis, pyrimidine metabolism, and ribosomal signaling pathway indicated that 4μ8C might inhibit virus proliferation by regulating nucleic acid synthesis and metabolism processes. The activation of ribosome-related pathways suggested that it might regulate the synthesis of virus proteins by affecting protein translation efficiency.

[0044] In summary, 4μ8C had a significant impact on the endoplasmic reticulum stress pathway (IRE1α) and its downstream transcriptional and metabolic pathways. GO and pathway analysis showed that 4μ8C regulated lipid metabolism, Golgi function, and nucleic acid metabolism, and might directly or indirectly interfere with virus replication and transmission.

[0045] 1.7 Detection of viral GP64 and key genes of endoplasmic reticulum stress affecting the proliferation of BmNPV in cells treated with 4μ8c

[0046] Treat with the control group DMSO and the experimental group 4μ8C at the same concentration of 3μM and infect with the virus BmNPV-EGFP. Detect the gene expression levels at 24h, 48h, and 72h time periods respectively. GP64 、 BmXBP1 、 BmBIP 、 BmCANX 、 BmGRP94 Use a fluorescence microscope to detect the intensity of the green fluorescence signal of BmNPV-EGFP in cells with DMSO (control group) and 4μ8C (experimental group).

[0047] SeeFigure 7 , the experimental results showed that: compared with the control group, 3 μM of 4μ8C could effectively inhibit the gene expression of viral GP64 ( Figure 7 A in), and at the same time, as time increased, 4μ8C would significantly inhibit the key genes related to endoplasmic reticulum pathway stress BmXBP1 (B), BmBIP (C), BmCANX (D), BmGRP94 (E). At the same time, referring to Figure 7 F in, fluorescence microscopy detection found that compared with the control group, the green fluorescence intensity of EGFP-BmNPV treated with 4μ8C was weakened.

[0048] The above content further elaborates on the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as falling within the protection scope determined by the claims submitted for the present invention.

Claims

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

2. The use according to claim 1, characterized in that: The formulation contains the inhibitor 4μ8C.

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

4. The use according to claim 3, characterized in that: The auxiliary material is a substance that enhances the stability of 4μ8C or enhances the palatability of the preparation.

5. The use according to claim 1, characterized in that: The dosage form of the preparation is powder, solid granules or liquid preparation.

6. The use according to claim 1, characterized in that: The preparation is used before the silkworm is infected with BmNPV, or within 12 hours after being infected with BmNPV.

7. The use according to claim 1, characterized in that: The formulation is sprayed on mulberry leaves and then administered to silkworms through feeding.

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