Application of bortezomib in preparation of medicine for resisting grouper iridovirus

Abstract

The invention discloses an application of bortezomib in preparation of a medicine for resisting grouper iridovirus. Researches show that bortezomib has a remarkable inhibiting effect on grouper iridovirus, and is low in cytotoxicity and good in safety. The bortezomib can obviously reduce the fluorescence signal intensity of virus protein, inhibit the transcriptional level of main capsid protein MCP and envelope protein VP19 of the virus and reduce the copy number of DNA and mRNA of the virus; the bortezomib can effectively inhibit grouper iridovirus infection and effectively block synthesis of virus protein, so that efficient inhibition of grouper iridovirus infection is achieved, and along with prolonging of virus infection time, bortezomib can also remarkably inhibit virus gene transcription, protein synthesis and genome replication. Therefore, bortezomib not only can effectively prevent and treat grouper iridovirus infection, but also has the characteristics of high specificity, low toxicity, remarkable antiviral effect and the like, and also has important application value in prevention and control of iridovirus-related diseases in aquaculture industry.

Description

Technical Field

[0001] This invention relates to the field of antiviral drugs for aquaculture, specifically to the application of bortezomib in the preparation of drugs against grouper iridovirus. Background Technology

[0002] Grouper, an important economic fish belonging to the genus *Salmonella* in the family Serranidae of the order Perciformes, has become a significant species in aquaculture due to its delicious flesh and high nutritional value. In recent years, with advancements in aquaculture technology and increasing market demand, grouper farming has developed rapidly. However, increased stocking density and expanded industry scale have led to frequent outbreaks of viral diseases, severely hindering the industry's development. Among these, infections caused by viruses of the family Iridoviridae have become a key issue threatening the sustainable development of grouper farming.

[0003] Iridoviruses are a class of double-stranded DNA viruses with an icosahedral structure, whose genome size ranges from 140 to 303 kb, and whose virus particle diameter is approximately 125 to 380 nm. Among them, grouper iridovirus, also known as Singapore grouper iridovirus (SGIV), is a new species of the genus Ranavirus in the family Iridoviridae. It is a highly pathogenic iridovirus isolated and identified from diseased grouper farmed in Singapore. This virus is characterized by rapid transmission, high mortality (up to 80% or more), and significant harm, causing severe economic losses to the grouper aquaculture industry. Currently, the control of this virus faces technical challenges such as a lack of effective drugs and limited vaccine protection. Developing new, highly effective, and safe anti-SGIV drugs has become an urgent need in the field of aquatic disease control and is of great significance for ensuring the healthy development of the grouper aquaculture industry.

[0004] Bortezomib is a highly selective 26S proteasome inhibitor that primarily blocks the ubiquitin-proteasome pathway by reversibly inhibiting chymotrypsin-like activity. As the first proteasome inhibitor approved for clinical use, its significant pharmacological activity has been well-established in the treatment of mammalian tumors. However, its application in the treatment of viruses in aquaculture has not been reported, particularly in the treatment of fish diseases caused by grouper iridovirus, which remains an unknown area. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an application of bortezomib in the preparation of drugs against iridovirus.

[0006] A second object of the present invention is to provide the use of bortezomib in the preparation of a medicament for the prevention or treatment of iridovirus disease in fish caused by iridovirus infection.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: Experimental studies of this invention demonstrate that bortezomib at low concentrations (0.02–50 μM) can effectively inhibit iridovirus infection in grouper cells and exhibits low biotoxicity to host cells. Further research shows that bortezomib can attenuate the cytopathic effect induced by iridovirus, inhibit the transcriptional levels of the major capsid protein MCP and envelope protein VP19 of SGIV virus, reduce the protein expression level of MCP, and decrease the copy number of viral DNA and mRNA. The number of fluorescent cells expressing SGIV MCP was significantly reduced in the bortezomib-treated group, and the fluorescence signal intensity of viral proteins decreased, effectively blocking viral protein synthesis and interfering with replication in grouper cells, thus achieving highly efficient inhibition of iridovirus infection. Furthermore, with prolonged viral infection time, bortezomib can also significantly inhibit viral gene transcription, protein synthesis, and genome replication. Therefore, bortezomib can effectively prevent and treat iridovirus infection, and its low toxicity and significant antiviral effect make it of significant application value in the prevention and control of iridovirus-related diseases in aquaculture.

[0008] Therefore, the present invention provides the use of bortezomib in the preparation of medicaments against iridovirus.

[0009] This invention also provides the use of bortezomib in the preparation of medicaments for the prevention or treatment of iridovirus disease in fish caused by iridovirus infection.

[0010] Furthermore, the fish in question is a grouper.

[0011] Furthermore, the iridovirus is a grouper iridovirus.

[0012] Furthermore, the drug achieves treatment by reducing cytopathic effects and inhibiting the expression of the iridovirus MCP and VP19 genes.

[0013] Furthermore, the concentration of bortezomib in the drug is 0.01–50 μM.

[0014] Furthermore, the concentration of bortezomib in the drug is 1 μM.

[0015] Furthermore, the drug also contains pharmaceutically acceptable excipients.

[0016] Furthermore, the excipients are pharmaceutically acceptable salts and derivatives, carriers, and excipients.

[0017] Furthermore, the drug is formulated as an injection, powder, capsule, or granule.

[0018] In summary, this invention demonstrates that bortezomib significantly inhibits SGIV infection in grouper and can be used as an antiviral drug. It significantly inhibits SGIV infection even at low concentrations, exhibits no significant toxic side effects on host cells, and demonstrates high safety, meeting the requirements for sustainable development in aquaculture. This drug provides a feasible virus control solution for grouper aquaculture and has broad market application prospects. This invention not only provides a novel drug option for the prevention and treatment of grouper iridovirus disease but also expands the application scope of bortezomib in aquaculture, possessing significant practical value for promoting the high-quality development of ecological grouper aquaculture.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of bortezomib in the preparation of drugs against iridovirus. The research of this invention shows that bortezomib has a significant inhibitory effect on grouper iridovirus, with low cytotoxicity and good safety. Bortezomib not only significantly reduces the fluorescence signal intensity of viral proteins, inhibits the transcription levels of the major capsid protein MCP and envelope protein VP19, and reduces the copy number of viral DNA and mRNA; it also effectively blocks the synthesis of viral proteins, thereby achieving highly efficient inhibition of grouper iridovirus infection. Furthermore, with the extension of viral infection time, bortezomib can also significantly inhibit viral gene transcription, protein synthesis, and genome replication. Therefore, bortezomib can not only effectively prevent and treat grouper iridovirus infection, possessing high specificity, low toxicity, and significant antiviral effects, but also has important application value in the prevention and control of iridovirus-related diseases in aquaculture. Attached Figure Description

[0020] Figure 1 The effects of different concentrations of bortezomib on the toxicity of grouper spleen cells.

[0021] Figure 2 The effects of different concentrations of bortezomib on the lesioning degree of iridovirus in grouper.

[0022] Figure 3 To investigate the inhibitory effect of different concentrations of bortezomib on genes related to grouper iridovirus.

[0023] Figure 4 The results show the inhibition of viral MCP protein fluorescence expression by bortezomib treatment.

[0024] Figure 5 Changes in gene and protein expression in bortezomib-treated cells after infection with SGIV at different time points.

[0025] Figure 6The effect of bortezomib-treated cells on SGIV infection at different time points on the replication of grouper iridovirus nucleic acid. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0027] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0028] (1) Bortezomib: purchased from MedChemExpress (catalog number: 179324-69-7), HPLC purity ≥99.97%, molecular formula C 19 H 25 BN4O4 has a molecular weight of 384.24 and its structural formula is as follows:

[0029] (2) The Grouper Spleen cell (GS) of the oblique grouper is stored in the applicant’s laboratory and is available to the public from the applicant for use only to repeat the experiments of this invention.

[0030] (3) The grouper iridovirus (Guangxi strain, SGIV) was isolated from diseased Epinephelus coioides and is stored in the applicant's laboratory. It is available to the public from the applicant and is used only for repeating experiments of this invention. In the following examples, the SGIV virus infection experiments were all conducted under the condition of multiplicity of infection (MOI) = 2.

[0031] (4) Primer sequence: SGIV major capsid protein (MCP) gene: Forward primer (qMCP-F): 5'-GCACGCTTCTCTCACCTTCA-3' (SEQ ID NO.1) Reverse primer (qMCP-R): 5'-AACGGCAACGGGAGCACTA-3' (SEQ ID NO.2) SGIV envelope protein (VP19) gene: Forward primer (qVP19-F): 5'-TCCAAGGGAGAAACTGTAAG-3' (SEQ ID NO.3) Reverse primer (qVP19-R): 5'-GGGGTAAGCGTGAAGACT-3' (SEQ ID NO.4) Primers for the internal reference gene β-actin: Forward primer (β-actin-F): 5'-TACGAGCTGCCTGACGGACA-3' (SEQ ID NO.5) Reverse primer (β-actin-R): 5'-GGCTGTGATCTCCTTCTGCA-3' (SEQ ID NO.6) (All primers were synthesized by Qingke Biotechnology Co., Ltd.) (5) The quantitative experimental data involved in the following examples are the mean ± standard error (Mean ± SEM) of three independent repeated experiments. Statistical analysis was performed using SPSS 20.0 software. Student's t-test was used to compare differences between groups, and the significance level was set at: * P <0.05 (significant),** P <0.01 (highly significant).

[0032] Example 1: Drug toxicity test of bortezomib I. Experimental Methods The toxic effect of bortezomib on GS cells was detected using the CCK-8 assay. Cell suspension (100 μL / well) was seeded in 96-well plates and cultured at 28°C for 18 h to allow cell adhesion. Experimental groups were prepared with L15 medium containing different concentrations of bortezomib (0.02 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, and 50 μM), with six replicates. A solvent control group containing 0.1% DMSO and a cell-free control group were also included. Cells were cultured at 28°C for another 24 h, and cell morphology was observed under an optical microscope. The supernatant was then removed, and the cells were washed three times with fresh medium. 100 μL of 10% CCK-8 solution was added to each well, and the cells were incubated at 28°C for 1–4 h. The absorbance of cells in each group at 450 nm was then measured using a microplate reader (Thermo Fisher Science, USA) to determine the effect of bortezomib on GS cell viability. Relative cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%. All results are expressed as the mean ± standard deviation of three independent experiments.

[0033] II. Experimental Results GS cells were treated with different concentrations of bortezomib (0.02-50 μM) for 24 h. Cell viability was detected by the CCK-8 assay, and the results are as follows: Figure 1As shown, there was no significant difference between the experimental group and the control group. This confirms that bortezomib at this concentration range has no significant toxic effect on GS cells.

[0034] Example 2: Detection of antiviral activity of bortezomib The antiviral effect of different concentrations of bortezomib against SGIV was evaluated using in vitro cell experiments. GS cells were first seeded into 24-well plates and cultured to a monolayer, then pretreated with bortezomib at concentration gradients of 0.02-50 μM for 2 hours each time; subsequently, they were inoculated with SGIV virus (MOI=2). Cytopathic effect (CPE) was observed under a microscope 24 hours after infection. Total RNA was extracted from each group of cells, and after passing nucleic acid concentration and purity tests, cDNA synthesis was performed according to the reverse transcription reaction system in Table 1 and the reaction procedure in Table 2. The expression levels of SGIV MCP and VP19 genes were then detected using SYBR Green quantitative PCR, with three replicates for each sample, using actin as an internal control gene. -ΔΔCt Relative expression levels were calculated using a method similar to that used in traditional Chinese medicine. Viral protein expression was analyzed using Western blot, with β-Tubulin used as an internal control for standardization. All experiments were independently repeated three times.

[0035] 1. Ce1l Total RNA Isolation Kit and ReverTraAce ® The operating procedure for the qPCR RT Kit is as follows: (1) Add 250 μL of Buffer cRL1 to the collected cell sample and mix thoroughly to lyse the cells; (2) Transfer the lysis buffer to a DNA-Cleaning Column, centrifuge at 12,000 rpm (13,400×g) for 2 minutes, and collect the filtrate; (3) Add 1.6 times the volume of Buffer cRL2 to the filtrate, mix gently, transfer to RNA-only column, centrifuge at 12,000 rpm for 1 minute, and discard the filtrate; (4) Add 500 μL Buffer RW1, centrifuge at 12,000 rpm for 1 minute, and discard the filtrate; (5) Add 700 μL Buffer RW1, centrifuge at 12,000 rpm for 1 minute, and discard the filtrate; (6) Repeat step (5) again.

[0036] (7) Centrifuge the empty column at 12,000 rpm for 2 minutes to remove residual washing liquid; (8) Transfer the purification column to a new EP tube, add 30 μL of RNase-Free ddH2O preheated at 65℃, let stand at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute to collect RNA; (9) Take 4 μL for agarose gel electrophoresis, and 1 μL to determine the concentration (A260 / A280). The remaining sample is placed in a water bath at 65℃ for 5 minutes and then placed in an ice bath for later use.

[0037] (10) Using ReverTraAce ® cDNA synthesis was performed using the qPCR RT Kit (purchased from Shanghai Xinrui Biotechnology Co., Ltd., product number FSQ-101B). A 10 μL reverse transcription reaction system was prepared according to Table 1; the reverse transcription program of the PCR instrument was set according to Table 2. (11) Use the product immediately or store it at 4°C for a short period of time, or at -20°C for a long period of time.

[0038] Table 1 Reverse transcription system

[0039] Table 2 Reverse Transcription Procedure

[0040] 2. The experimental procedures for real-time PCR are as follows: A 10 μL reaction system was prepared using 2×SYBR Green Real-time PCR Mix (Toyobo, Japan), with the amounts of each component added as shown in Table 3. Real-time quantitative PCR was performed on a QuantStudio 5 real-time PCR instrument (Thermo Fisher Scientific, USA) according to the corresponding reaction program (Table 4). Finally, 2... -ΔΔCT The method normalizes the expression levels of target genes.

[0041] Table 3. Fluorescence Quantitative System

[0042] Table 4. Quantitative Fluorescence Procedure

[0043] 3. The experimental procedure for Western blot is as follows: (1) Sample preparation: After collecting cell samples from 12-well plates, add 40 μL Pierce IP lysis buffer for lysis, then add 10 μL 5× protein loading buffer, boil in water for 5 minutes to denature the protein, centrifuge at 12000×g for 3 minutes, and take the supernatant for use or store at -20℃.

[0044] (2) Gel preparation: Prepare SDS-PAGE gels. The separating gel system (10 mL) contains 5.0 mL of 30% acrylamide-bisacrylamide mixture, 2.6 mL of 1.5 M Tris-HCl (pH 8.8), 0.1 mL of 10% SDS, 0.1 mL of 10% ammonium persulfate, and 0.01 mL of TEMED. The stacking gel system (4 mL) contains 0.6 mL of 30% acrylamide-bisacrylamide mixture, 0.5 mL of 1 M Tris-HCl (pH 6.8), 0.04 mL of 10% SDS, 0.04 mL of 10% ammonium persulfate, and 0.01 mL of TEMED.

[0045] (3) Electrophoresis: Load protein samples of medium weight into each well and add pre-stained protein markers. Electrophore for 30 minutes at a constant voltage of 70V (stacking gel), and then electrophore for 60 minutes at a constant voltage of 110V (separating gel) until bromophenol blue reaches the bottom of the gel.

[0046] (4) Transfer: Before the transfer, activate the PVDF membrane with methanol for 1-2 minutes, assemble the transfer "sandwich" structure in sequence (sponge → filter paper → gel → membrane → filter paper → sponge), and use the wet transfer method to transfer the membrane at a constant current of 100mA for 60 minutes.

[0047] (5) Sealing: Place the PVDF membrane in 5% skim milk prepared with PBST and seal it at room temperature for 2-3 hours or overnight at 4°C.

[0048] (6) Primary antibody incubation: Place the PVDF membrane into the primary antibody solution diluted with the blocking solution and incubate at room temperature for 2-3 hours or at 4°C overnight.

[0049] (7) Secondary antibody incubation: Wash the membrane twice with PBST buffer, 5 minutes each time. Place the membrane in HRP-labeled secondary antibody solution diluted with blocking buffer and incubate at room temperature for 45 minutes.

[0050] (8) Color development and result analysis: Wash the membrane three times with PBST buffer, 10 minutes each time. Use the HRP-DAB substrate color development kit (TIANGEN) for color development, acquire images using a chemiluminescence imaging system, and perform grayscale analysis using ImageJ software.

[0051] II. Experimental Results The cytopathic effect was observed under a microscope, and the results were as follows: Figure 2 As shown, the untreated control group exhibited obvious cell shrinkage and rounding, as well as intercellular voids, while the cytopathic effect in the bortezomib-treated group was significantly reduced.

[0052] To evaluate the antiviral efficacy of bortezomib, the effects of different concentrations of the drug on SGIV virus gene expression were investigated. The qRT-PCR results are as follows: Figure 3As shown, the mRNA transcription levels of viral MCP and VP19 genes in the drug-treated group were significantly lower than those in the control group; Western blot analysis further confirmed that the synthesis of viral structural proteins was significantly inhibited. These results indicate that bortezomib has significant anti-SGIV activity.

[0053] Example 3: Fluorescence detection analysis of bortezomib's antiviral activity I. Experimental Methods To clarify the inhibitory effect of bortezomib on SGIV virus protein expression, immunofluorescence was used for analysis. The specific experimental method is as follows: GS cells were seeded in 24-well plates and cultured at 28°C for 18 hours to form a monolayer. The experimental group was pretreated for 2 hours with L-15 medium containing 1 μM bortezomib (dissolved in 0.1% DMSO), while a control group was prepared using 0.1% DMSO solvent. Subsequently, SGIV virus solution was seeded at MOI=2 and cultured at 28°C for another 24 hours. Cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and then blocked with 0.2% BSA (Sigma) for 30 minutes. Anti-SGIV MCP mouse monoclonal primary antibody (1:500 dilution) was added and incubated at room temperature for 2 hours. Then, anti-mouse IgG secondary antibody labeled with Alexa Fluor 555 (1:200 dilution) was added and incubated in the dark for 1 hour. Finally, the nuclei were stained with DAPI for 5 minutes.

[0054] II. Experimental Results Observation results of laser confocal microscopy (CLSM) are as follows Figure 4 As shown, compared with the DMSO control group, the number of MCP protein-positive cells in the 1 μM bortezomib treatment group was significantly reduced, and the fluorescence signal intensity was significantly weakened. These results indicate that the drug can effectively inhibit the expression of the SGIV structural protein MCP.

[0055] Example 4: Analysis of antiviral activity of bortezomib at different time points I. Experimental Methods To verify the inhibitory effect of 1 μM bortezomib on SGIV replication at different infection time points, a time-series analysis experiment was conducted. The specific steps were as follows: GS cells were seeded in 24-well plates and cultured at 28°C for 18 hours until a monolayer was formed. The experimental group was pretreated for 2 hours with L-15 medium containing 1 μM bortezomib (0.1% DMSO solvent), while a control group was set up with 0.1% DMSO solvent. Subsequently, SGIV virus suspension was inoculated, and samples were collected at 12, 24, and 36 hours post-infection. Total RNA was extracted using the Ce1l Total RNA Isolation Kit for qRT-PCR detection of viral MCP and VP19 gene transcription levels; DNA samples were extracted using the TlANamp Genomic DNA Kit for quantitative PCR analysis of viral genome replication; total protein was extracted using Pierce IP lysis buffer, and viral structural protein expression was detected by Western blot.

[0056] The operating procedure for the TlANamp Genomic DNA Kit is as follows: (1) Collect adherent cells.

[0057] (2) Add 200 μL of buffer GA and shake thoroughly to resuspend the cell pellet; (3) Add 20 μL of Proteinase K solution and vortex to mix; (4) Add 200 μL of buffer GB, mix thoroughly by inverting, place at 70°C for 10 minutes until the solution becomes clear, and briefly centrifuge to remove droplets from the inner wall of the tube cap; (5) Add 200 μL of anhydrous ethanol, shake well for 15 seconds, and centrifuge briefly to remove water droplets from the inner wall of the tube cap. (6) Transfer the mixture (containing any possible flocculent precipitate) to a CB3 adsorption column, centrifuge at 12,000 rpm (13,400×g) for 30 seconds, and discard the waste liquid; (7) Add 500 μL of buffer GD, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid; (8) Add 600 μL of PW rinsing solution, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid; (9) Repeat step 7; (10) Centrifuge the empty column at 12,000 rpm for 2 minutes to completely remove residual ethanol, and let it stand at room temperature for 2 minutes; (11) Transfer the adsorption column to a new centrifuge tube, add 50-200 μL of TE buffer to the center of the membrane, let it stand at room temperature for 2-5 minutes, then centrifuge at 12,000 rpm for 2 minutes to collect the DNA. Store the DNA product at -20℃.

[0058] II. Experimental Results Experimental results are as follows Figure 5 As shown, the 1 μM bortezomib treatment group exhibited significant antiviral activity at all time points. qPCR quantitative analysis revealed that, compared with the virus control group, the transcriptional levels of SGIV MCP and VP19 genes were significantly downregulated in the treatment group. Protein detection results further confirmed that viral structural protein synthesis was inhibited.

[0059] Results of viral nucleic acid quantitative analysis as follows Figure 6 As shown, both viral genomic DNA and mRNA copy numbers were significantly reduced.

[0060] The above data collectively demonstrate that bortezomib can inhibit the SGIV replication cycle through multiple pathways, including inhibiting viral gene transcription, blocking genome replication, and interfering with structural protein synthesis. This invention is the first to systematically elucidate the concentration- and temporal inhibitory effects of bortezomib on SGIV replication at the molecular level, providing an important theoretical basis for the application of this drug in the prevention and control of aquatic viruses.

Claims

1. Application of bortezomib in the preparation of drugs against iridovirus.

2. Application of bortezomib in the preparation of drugs for the prevention or treatment of iridovirus disease in fish caused by iridovirus infection.

3. The application according to claim 2, characterized in that, The fish in question is a grouper.

4. The application according to claim 1 or 2, characterized in that, The iris virus mentioned is the grouper iris virus.

5. The application according to claim 1 or 2, characterized in that, The drug achieves its therapeutic effect by reducing cytopathic effects and inhibiting the expression of the MCP and VP19 genes of iridovirus.

6. The application according to claim 1 or 2, characterized in that, The concentration of bortezomib in the drug is 0.01–50 μM.

7. The application according to claim 6, characterized in that, The concentration of bortezomib in the drug is 1 μM.

8. The application according to claim 1 or 2, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The excipients are pharmaceutically acceptable salts and derivatives, carriers, and excipients.

10. The application according to claim 1 or 2, characterized in that, The drug is prepared in the form of injection, powder, capsule, or granule.

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