Fish nervous necrosis virus resisting medicine and screening method thereof

Abstract

The invention discloses a fish nervous necrosis virus resisting medicine which is characterized by comprising baicalin. The invention also discloses a screening method of the drug for resisting the nervous necrosis virus of the fish, which comprises the following steps: constructing a recombinant prokaryotic expression plasmid with the HSC70 gene, transferring the plasmid into a BL21 (DE3) cell, inducing protein expression, and purifying to obtain the HSC70 protein; preparing to-be-detected compound solutions with different concentrations, and then dropwise adding the to-be-detected compound solutions into the solution containing the HSC70 protein for incubation; under the excitation wavelength of 285 nm, the fluorescence signal of the HSC70 protein in the range of 310-350 nm is detected, the compound for targeted inhibition of the HSC70 protein is determined according to the change of the fluorescence signal, and the fish nervous necrosis virus resisting medicine is screened out.

Description

Technical Field

[0001] This invention relates to the technical field of antiviral drugs for fish, specifically to an antiviral drug for fish nerve necrosis virus and its screening method. Background Technology

[0002] Nervous necrosis virus (NNV), belonging to the genus Betanodavirus in the family Nodaviridae, is a pathogen causing viral encephalopathy and retinopathy. NNV is a highly contagious virus affecting over 100 fish species, including many important economic fish such as European bass, Asian bass, and tongue sole. Once infected, juvenile fish experience rapid onset and progression of the disease, leading to 100% mortality. Despite control measures, NNV continues to infect globally, causing irreversible damage to aquaculture. Although two vaccines against NNV have been approved for marketing, the continuous evolution of NNV may weaken the effectiveness of existing vaccines, and the widespread adoption of fish vaccines in actual aquaculture remains a global challenge. Therefore, finding safe, effective, and practical new drugs against NNV is of great importance.

[0003] Natural products offer numerous advantages in antiviral applications, such as low susceptibility to inducing drug resistance mutations, low cost, and high eco-friendliness. Their use in aquaculture antiviral applications can contribute to the sustainable development of aquaculture. Furthermore, natural products typically have larger molecular weights, more sp3 carbon and oxygen atoms, and fewer nitrogen and halogen atoms, resulting in higher molecular rigidity and a greater abundance of hydrogen bond acceptors and donors. These structural characteristics allow for more precise binding to specific targets on viruses or host cells, making them particularly suitable for targeting complex biological processes such as protein-protein interactions. However, there are currently few reports on the development of anti-NNV drugs using natural products.

[0004] Currently, the screening of anti-NNV drugs mostly employs cell line and in vivo screening methods. However, these methods are costly, time-consuming, and cannot clearly identify the drug targets, making it difficult to elucidate their antiviral mechanisms. Furthermore, drug screening strategies that directly target the virus are prone to leading to drug resistance. Therefore, developing a novel, efficient, low-cost screening method with clearly defined targets is crucial. Summary of the Invention

[0005] One of the objectives of this invention is to provide a drug for treating fish nerve necrosis virus.

[0006] The second objective of this invention is to provide a method for screening drugs against fish nerve necrosis virus.

[0007] To achieve the first objective mentioned above, the present invention adopts the following technical solution:

[0008] An antiviral drug for fish nerve necrosis, characterized in that it includes baicalin.

[0009] The baicalin can be added to water to soak fish, at a dosage of 25 mg / L of water.

[0010] This invention also provides the application of baicalin in the preparation of drugs against fish nerve necrosis virus.

[0011] To achieve the second objective mentioned above, the present invention adopts the following technical solution: a method for screening drugs against fish nerve necrosis virus, comprising the following steps:

[0012] A recombinant prokaryotic expression plasmid carrying the HSC70 gene was constructed, and the plasmid was transformed into BL21(DE3) cells to induce protein expression. The HSC70 protein was then purified.

[0013] Different concentrations of test compound solutions were prepared, and then the test compound solutions were added dropwise to a solution containing HSC70 protein for incubation. The fluorescence signal of HSC70 protein in the range of 310-350 nm was detected at an excitation wavelength of 285 nm. Based on the changes in fluorescence signal, compounds that target and inhibit HSC70 protein were identified, and anti-fish nerve necrosis virus drugs were screened.

[0014] HSC70 protein, or Heat Shock Cognate Protein 70, is a member of the heat shock protein family and a molecular chaperone protein widely expressed in most cells. Studies have reported that HSC70 protein is one of the key receptors for NNV infection of host cells. Therefore, this invention establishes a screening method for anti-fish nerve necrosis virus drugs based on the intrinsic fluorescence properties of the HSC70 protein. Specifically, it screens for anti-fish nerve necrosis virus drugs based on changes in the fluorescence signal of the HSC70 protein. This screening method can rapidly screen for anti-fish nerve necrosis virus drugs, is low-cost, and clearly identifies the drug targets.

[0015] The anti-fish nerve necrosis virus drug screened in this invention uses the HSC70 protein as an antiviral target to inhibit viral infection, which is a promising antiviral approach.

[0016] Preferably, after identifying compounds that target and inhibit the HSC70 protein, the present invention screens out anti-fish nerve necrosis virus drugs through cell-level viral lesion effect inhibition experiments and in vivo experiments using live fish models infected with the virus.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] Experiments have confirmed that the baicalin screened in this invention can significantly inhibit NNV replication, reduce virus-induced cytopathic effects, and upregulate the expression of antiviral genes. In in vivo experiments, it can significantly reduce NNV expression levels in fish, increasing the survival rate of NNV-infected sea bass by 29.63%. Furthermore, baicalin has the natural advantage of being derived from traditional Chinese medicine, making it environmentally friendly, low-cost, and potentially a safe and effective anti-NNV drug with broad application prospects.

[0019] This invention establishes for the first time a high-throughput screening method based on HSC70 intrinsic fluorescence quenching, overcoming the shortcomings of existing drug screening technologies such as long cycle, high cost, and unclear target. Attached Figure Description

[0020] Figure 1 This is an SDS-PAGE image of purified recombinant HSC70 protein stained with Coomassie brilliant blue; lane 1 is the protein marker, and lane 2 is the purified HSC70 protein.

[0021] Figure 2 This graph shows the quenching results of 30 candidate compounds on the intrinsic fluorescence of the HSC70 protein; the horizontal axis represents wavelength (nm), and the vertical axis represents fluorescence intensity (au).

[0022] Figure 3 The results of screening compounds targeting HSC70 are shown in Figure A; A shows the inhibition rate of HSC70 protein at a compound concentration of 50 μM, with those exceeding 90% shown in red; B shows the structural formula of compounds with an inhibition rate exceeding 90%.

[0023] Figure 4 The graph shows the safe concentration results of the screened compounds; where a is pungent tannin, b is naringenin-7-O-glucoside, c is berberine II, and d is baicalin.

[0024] Figure 5 This diagram shows the docking results of baicalin with the HSC70 protein molecule. AD represent different domains of HSC70 targeted by baicalin.

[0025] Figure 6 The figure shows the results of the in vitro antiviral experiment of baicalin; A represents the cytopathic effect, and B represents the CP expression level of NNV.

[0026] Figure 7 The figure shows the effect of baicalin on HSC70 gene expression.

[0027] Figure 8 Baicalin IC 50 Resulting image;

[0028] Figure 9The figure shows the effect of baicalin on antiviral gene expression; A represents IFN gene expression; B represents Mx gene expression; C represents ISG15 gene expression; and D represents Viperin gene expression.

[0029] Figure 10 Figure 1 shows the results of the in vivo antiviral experiment of baicalin; A shows the effect of baicalin on the survival rate of sea bass infected with NNV; B shows the effect of baicalin on the viral expression level in the brain and eyes of sea bass infected with NNV. Detailed Implementation

[0030] The present invention will be further described in detail below through specific implementation examples, so that those skilled in the art can better understand and implement the technical solution of the present invention.

[0031] The following examples use sea bass fin rays ( Lateolabrax japonicus Experiments were conducted using the LJF (Large Bovine Fibrillation) cell line and Nervous necrosis virus (NNV; GeneBank: SBN147). The LJF cell line was established according to the techniques and conditions described in the literature [Le, Y., Li, YL, Jin, YL, et al., Establishment and characterization of a brain cell line from sea perch, Lateolabrax japonicus. In Vitro Cellular & Developmental Biology Animal. 2017, 53, 834-840.]. The cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibico) at 28°C. NNV was isolated from diseased sea bass tissue. The isolation method for NNV was performed according to the techniques or conditions described in the literature [Xing, J., Zhang, ZQ, Sheng, XZ et al., Identification and characterization of a new strain of nervous necrosis virus isolated from pearl gentian grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) in China. Aquaculture, 2020, 529, 735663.].

[0032] Example 1: Purification of the fusion expression protein pET-N-CST-HSC70

[0033] First, using sea bass tissue cDNA as a template, the HSC70 gene ORF (NCBI accession number: PV915707) was amplified by PCR using primers with homologous arms (F: 5'-tccaagcttctgcaggaattcATGTCTAAGGGACCAGCAGTTGG-3'; R: 5'-tctgtcgacgatatcgaattcTTAGTCAACCTCCTCAATGGTTGG-3'). After digestion with EcoRI, the plasmid was inserted into the expression vector pET-N-GST using homologous recombinase to obtain the recombinant plasmid pET-N-GST-HSC70. The recombinant plasmid pET-N-GST-HSC70 was transformed into DH5α, plated on LB agar plates containing antibiotics, and cultured until single colonies formed. A single colony was picked using a sterile inoculation loop and inoculated into liquid culture medium. Subsequent plasmid extraction and sequencing confirmed successful colony formation.

[0034] The recombinant plasmid was retransformed into *E. coli* BL21(DE3) cells and induced at 16°C for 14 h. Then, the protein was purified using the Beyotime GST protein purification kit according to the manufacturer's instructions. The purified protein was dissolved in elution buffer (50 mM Tris + 150 mM NaCl, pH 8.0). Subsequent staining with Coomassie Brilliant Blue was performed, and the staining results are shown below. Figure 1 As shown, the purified HSC70 protein band is single and bright, meeting the requirements for subsequent experiments.

[0035] Example 2: Steady-state and transient fluorescence spectroscopy for detecting the interaction between candidate compounds and recombinant proteins.

[0036] The candidate compounds included the following 30 natural compounds: (-)-α-Pinene, Alisol F, Aloperine, Arteannuin B, Artemisinin, Baicalin, Bergenin, Betulinic acid, Catalpol, Dehydroandrographolide succinat, Diallyl Trisulfide, Dihydromyricetin, Fangchinoline, Gomisin G, Guanosine, Hinokitiol, Hyperoside, Oxyresveratrol, Paederoside, Picroside II, Prunin, Pseudolaric acid B, Punicalin, Sophocarpine (monohydrate), Thiamine (hydrochloride), Tricin, Verbenalin, Zingibroside R1, α-lipoic acid, and α-Vitamin E. These were purchased from MCE and dissolved in DMSO to obtain 10 mM solutions, which were then stored at -80°C for an extended period.

[0037] Add 2 mL of HSC70 protein dissolved in Tris buffer to a dedicated cuvette. Using an Edinburgh Instruments (FLS1000) steady-state transient fluorescence spectrometer, fix the excitation wavelength at 285 nm, the emission wavelength at 290-500 nm, and the emission slit at 2 nm. Add the compound dropwise to the cuvette, setting compound concentrations of 0, 10, 20, 30, 40, and 50 μM. Scan the co-incubation system of different compound concentrations and HSC70 protein at 25 °C sequentially. The results are shown below. Figure 2 As shown. Figure 2 The results showed that the fluorescence intensity of the HSC70 protein changed when different concentrations of the compound were applied.

[0038] Based on the fluorescence intensity values ​​of each group, the inhibition rate of the compound on the protein was quantified using the formula QE(%)=(1-F / F0)×100 (F0: initial fluorescence intensity; F: intensity after addition of the compound). The results are as follows: Figure 3 As shown. In the candidate compound screening system, compounds with an inhibition rate of HSC70 greater than 90% at 50 μM were selected. Figure 3 The results showed that four compounds exhibited inhibition rates greater than 90% at 50 μM: pungent tannin, naringenin-7-O-glucoside, berberine II, and baicalin.

[0039] Example 3: Determination of the safe concentration of the screened compounds

[0040] LJF cells were seeded in 96-well plates and cultured overnight until a cell monolayer formed (culture conditions: 28°C, DMEM medium containing 10% fetal bovine serum). Then, different concentrations of pungent tannin, naringenin-7-O-glucoside, berberine II, and baicalin (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μM) were added, with three replicates per group; a control group was also included, which received the same concentration of DMSO. After incubation at 28°C for 48 h, 10 μL of CCK-8 reagent was added to each well and gently mixed. Incubation continued for 2 h to allow CCK-8 to react with live cells. The absorbance (OD value) of each well was then measured at 450 nm using a multi-plate reader. Based on the absorbance values ​​and the following formula: Cell viability = (Absorbance of experimental wells - Absorbance of blank wells) / (Absorbance of control wells - Absorbance of blank wells) × 100%, cell viability was calculated to assess the cytotoxicity of the drug. Experimental results are as follows... Figure 4 As shown in the figure. The results indicate that, within the experimental concentration range, pomegranate tannin, naringenin-7-O-glucoside, berberine II, and baicalin exhibit relatively weak cytotoxicity, with minimal impact on cell viability at low to medium concentrations (≤ 60 μM).

[0041] Example 4: Interaction between baicalin and HSC70 virtual molecules

[0042] Homology modeling of the target protein HSC70 was performed using the SWISS-MODEL online platform. The three-dimensional structure of baicalin was obtained from the PubChem database. Water molecules were removed and polar hydrogen was added to the protein structure using AutoDock Tools 1.5.7, and the structure was saved in PDBQT format. Docking analysis was performed using AutoDock Vina 1.2.3, with docking boxes set based on the functional domains of HSC70, and the obtained binding energies were compared and analyzed. The baicalin-HSC70 docking conformation was visualized using PyMol 2.5.2, and key interactions were analyzed. The molecular docking simulation results are shown below. Figure 5 As shown.

[0043] Figure 5 The results showed that baicalin could specifically bind to multiple domains of HSC70, and the binding energies were all below -7 kcal / mol, which met the energy threshold standard for strong interactions.

[0044] Example 5: In vitro antiviral infection experiment

[0045] LJF cells were pre-seeded into 6-well plates and cultured in an incubator. After cell monolayers formed, 2 mL of 10 μM pungent tannin, naringenin-7-O-glucoside, berberine II, and baicalin diluted with culture medium were added, followed by co-incubation with NNV at MOI=2. The virus group (NNV) was treated with the same volume of culture medium and DMSO concentration. 24 hours after infection, cell morphology was observed, the culture medium was discarded, and the cells were washed three times with PBS. Total RNA was extracted using the FastPure Cell / TissueTotal RNA Isolation Kit (Vazyme, China). Using 1 μg of RNA as a template, cDNA was synthesized via reverse transcription using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China) with random hexamer primers and Oligo(dT)18 primers. Amplification reactions were performed using Hieff® qPCR SYBR Green Master Mix (Yeasen, China) on a LightCycler® 480 real-time quantitative PCR system (Roche, Switzerland). Specific primers (F: 5'-GTCGGCTGATACTCCTGTGTG-3', R: 5'-CTCCAGTTCCAAGGCTGTAGT-3') were used to detect the expression level of viral capsid protein. The reaction program was: 95℃ pre-denaturation for 5 min; 95℃ for 10 s, 59℃ for 20 s, 72℃ for 20 s, for 40 cycles. Experimental results are shown below. Figure 6 As shown.

[0046] Figure 6 The results showed that baicalin could reduce cell shrinkage and rounding caused by NNV, and could significantly reduce the intracellular expression of NNV (*). P <0.05).

[0047] Example 6 Effect of baicalin on HSC70 expression

[0048] LJF cells were pre-seeded in 6-well plates, with NNV and NNV+10μM baicalin groups established. Virus stock solution was inoculated at 2 MOI, and a control group with an equal concentration of DMSO was set up. After incubation at 28℃ for 48 h, cells were washed three times with PBS, cells were collected, RNA was extracted, and cDNA was reverse transcribed. The expression of the HSC70 gene was detected using specific primers (F: 5'-CAGGGCAACAGGACCACAC-3'; R: 5'-TTGTCAGCACCATAGACGAGAT-3'). Experimental results are as follows: Figure 7 As shown.

[0049] Figure 7 The results showed that NNV significantly increased the expression level of HSC70, but baicalin could reduce the high expression of HSC70 induced by NNV (*). P <0.05).

[0050] Example 7: Half-maximal effective concentration (IC50) of baicalin 50 Determination of )

[0051] Digested LJF cells were seeded into 12-well plates. When a monolayer was formed, 1 ml of baicalin diluted with culture medium was added to achieve final concentrations of 3, 5, 10, and 15 μM. DMSO treatment was used as a negative control. After 1 h of treatment, 2 MOINNV was added, and the cells were incubated at 28°C for 48 h. Cells were collected, and total RNA was extracted using a column chromatography kit. Using the total RNA as a template, cDNA was synthesized using reverse transcriptase. -ΔΔCT The relative expression level of the NNV Capsid Protein (CP) gene was calculated using a method similar to that used in Origin 2024. Dose-response curves were plotted, and IC50 was calculated using a logistic regression curve. 50 Value. Experimental results are as follows: Figure 8 As shown.

[0052] Depend on Figure 8 It can be seen that baicalin inhibits viral load in a dose-dependent manner, with an IC50 value of 100%. 50 The value is 11.56 μM.

[0053] Example 8: Effects of baicalin on antiviral gene expression

[0054] Digested LJF cells were seeded into 6-well plates. Once a monolayer was formed, 2 ml of 10 μM baicalin diluted with culture medium was added. A DMSO group was used as a control. Cells were incubated at 28°C for 48 h. After incubation, cell samples were collected from each group for subsequent detection of antiviral gene (IFN, Mx, ISG15, and Viperin) expression levels. The primer sequences for quantitative real-time PCR are shown in Table 1. Experimental results are as follows: Figure 9 As shown.

[0055] Depend on Figure 9 It can be seen that 10 μM baicalin significantly enhances the expression of the host cell's intrinsic antiviral gene IFN-Mx / ISG15 / Viperin by activating the innate immune signaling pathway (*). P <0.05).

[0056] Table 1 Primers for antiviral gene detection

[0057] Primer names for real-time PCR Primer sequence IFN-F 5'-ggCACCgCCAgTgATgCTg-3' IFN-R 5'-TgAgAgCATgCAgTCgCTgC-3' ISG15-F 5'-CgAgACTgTgAgCAACTT-3' ISG15-R 5'-TTAACCAgAAgTgTCCTCAg-3' Mx-F 5'-CTAgAAgAgCAgATAgAggAgA-3' Mx-R 5'-TggCATCCTgAgTgAATgCTg-3' Viperin-F 5'-CgTTgACAACCTCTACAAgATC-3' Viperin-R 5'-CgATCAgCAgACACTggAACA-3'

[0058] Example 9: Testing the in vivo antiviral activity of baicalin

[0059] Healthy, disease-free sea bass aged one month were selected and divided into a blank control group, a virus infection control group, and a baicalin treatment group. The baicalin treatment group was prepared by dissolving an appropriate amount of baicalin in a small amount of DMSO, then diluting the stock solution with experimental water to the required concentration, ensuring that the final concentration of baicalin added to 20L of water was 25mg / L, and the DMSO concentration was controlled below 0.1%. The blank control group and the virus infection group used water containing the same amount of DMSO. The fish were transferred to aquaculture tanks containing the corresponding drug solution, with 27 sea bass placed in each group. After pre-soaking for 6 hours, the baicalin treatment group and the virus infection control group were intraperitoneally injected with NNV virus solution (60μL / fish, 3×10⁻⁶). 7 TCID 50 / mL), and the blank control group was injected with an equal volume of PBS.

[0060] The baicalin treatment group had half its volume of the solution replaced daily while maintaining a constant concentration, while the other groups had the same volume of water replaced. The water temperature was maintained at 25℃. The number of sea bass deaths in each group was recorded daily. The results at the end of the experiment are as follows: Figure 10 As shown. On day 10 post-infection, three sea bass were harvested from each group, and their brain and eye tissues were collected. Specific primers were used to detect the expression level of NNV CP in the tissues.

[0061] Depend on Figure 10 It was found that soaking in baicalin could enhance the sea bass's resistance to NNV. Compared with the virus-infected group, the baicalin-treated group increased the survival rate of NNV-infected sea bass by 29.63% and significantly reduced the virus expression level in the fish (*). P <0.05).

Claims

1. A drug for treating fish nerve necrosis virus, characterized in that, Including baicalin.

2. The anti-fish nerve necrosis virus drug according to claim 1, characterized in that, The dosage of baicalin is 25 mg / L in water.

3. A method for screening anti-fish nerve necrosis virus drugs as described in claim 1, characterized in that, Includes the following steps: A recombinant prokaryotic expression plasmid carrying the HSC70 gene was constructed, and the plasmid was transformed into BL21(DE3) cells to induce protein expression. The HSC70 protein was then purified. Different concentrations of test compound solutions were prepared, and then the test compound solutions were added dropwise to a solution containing HSC70 protein for incubation. The fluorescence signal of HSC70 protein in the range of 310-350 nm was detected at an excitation wavelength of 285 nm. Based on the changes in fluorescence signal, compounds that target and inhibit HSC70 protein were identified, and anti-fish nerve necrosis virus drugs were screened.

4. The screening method for anti-fish nerve necrosis virus drugs according to claim 3, characterized in that, After identifying compounds that target and inhibit the HSC70 protein, drugs against fish nerve necrosis virus were finally screened through cell-level viral lesion inhibition experiments and in vivo experiments using live fish models infected with the virus.

5. Application of baicalin in the preparation of drugs against fish nerve necrosis virus.

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