Application of microcystin LR and / or microcystin RR in preparation of product for inhibiting mandarin fish rhabdovirus

Abstract

The invention relates to novel application of microcystic toxins LR and RR in resisting mandarin fish rhabdovirus. The microcystic toxins LR and RR are found to have a remarkable mandarin fish rhabdovirus resisting effect, can obviously reduce the copy number of the mandarin fish rhabdovirus and reduce the virus titer of the mandarin fish rhabdovirus, have concentration dependence, can destroy the structure of the mandarin fish rhabdovirus and effectively improve the survival rate of fishes infected with the mandarin fish rhabdovirus, and are good in biological safety. Therefore, the microcystic toxins LR and RR can be used for preparing products such as drugs for resisting the mandarin fish rhabdovirus, fish feed or fish feed additives and the like, and can become an effective prevention and control means for preventing and controlling the mandarin fish rhabdovirus.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and aquatic virus control, specifically to the application of microcystin LR and / or microcystin RR in the preparation of products that inhibit mandarin fish rhabdovirus. Background Technology

[0002] Mandarin fish ( Siniperca chuatsi Mandarin fish (Siniperca chuatsi) is a unique and valuable freshwater economic fish species in my country, prized for its delicious flesh and high aquaculture and economic value. However, with the continuous expansion of intensive aquaculture, disease problems have become increasingly prominent, among which mandarin fish rhabdovirus (MRV) is a significant concern. Siniperca chuatsi rhabdovirus Viral diseases caused by SCRV have become one of the most serious threats to the healthy development of mandarin fish farming.

[0003] SCRV, belonging to the Rhabdoviridae family, is a major pathogen that seriously harms mandarin fish farming. The virus particles are typically bullet-shaped or rod-shaped, enveloped, and have a single-stranded negative-sense RNA genome. This virus is highly contagious and has a high pathogenicity rate. Infection can cause mandarin fish to experience decreased appetite, abnormal swimming, and darkening of body color. It can also lead to severe necrosis of major organs such as the liver, spleen, and kidneys, causing devastating damage to both fry and adult fish, with a mortality rate exceeding 90%, resulting in significant economic losses for fish farmers.

[0004] Currently, control measures against SCRV remain very limited, mainly facing the following challenges: First, there is a lack of commercially available vaccines. Although some exploration has been conducted on inactivated vaccines, live attenuated vaccines, and subunit vaccines, no safe and effective commercial vaccines have yet been launched due to high costs of in vitro virus culture, short immune protection periods, and potentially poor cross-protection between different strains. Second, there is a lack of specific antiviral drugs. Faced with outbreaks of viral diseases, fish farmers often lack direct and effective treatment methods. Existing measures mainly involve water disinfection, oral administration of traditional Chinese medicine, or immune enhancers. While these methods can improve the non-specific immunity of fish to some extent, their specific inhibitory effect on SCRV is unclear, and they suffer from unstable effects and unclear mechanisms of action. In addition, the application of chemical drugs is also limited. Some broad-spectrum antiviral chemical drugs (such as ribavirin) may theoretically be effective, but they pose risks of drug residues, environmental pollution, drug resistance, and potential impacts on the quality of aquatic products and consumer health, thus their application in aquaculture is strictly limited.

[0005] In conclusion, developing a highly efficient, safe, and specific anti-SCRV drug or inhibitor has become an urgent technological need for the mandarin fish farming industry. Summary of the Invention

[0006] The purpose of this invention is to provide the application of microcystin LR and / or RR in anti-SCRV, specifically involving their novel use in the preparation of anti-SCRV products, in order to solve the problem of the lack of efficient and specific SCRV inhibitors in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of microcystin LR and / or microcystin RR, or substances with microcystin LR and / or microcystin RR as the main components, in the preparation of products that inhibit mandarin fish rhabdovirus.

[0008] Furthermore, the two-dimensional structural formula of the microcystin LR is as follows: .

[0009] Furthermore, the two-dimensional structural formula of the microcystin RR is as follows: .

[0010] Furthermore, the safe concentrations for both microcystin LR and microcystin RR in CPB cells are 100 nM and below.

[0011] Furthermore, the microcystin LR and microcystin RR are used to reduce the copy number of mandarin fish bullet virus, reduce the viral titer of mandarin fish bullet virus, and disrupt the viral structure of mandarin fish bullet virus.

[0012] Furthermore, the product includes drugs and virus inhibitors. It can be added to fish feed or used as a fish feed additive to control mandarin fish rhabdovirus.

[0013] Secondly, based on the application of the first aspect, the present invention further provides the application of microcystin LR and / or microcystin RR, or substances with microcystin LR and / or microcystin RR as the main component, in the preparation of products for treating or preventing diseases caused by mandarin fish rhabdovirus infection.

[0014] Thirdly, the present invention also provides the use of microcystin LR and / or microcystin RR, or substances with microcystin LR and / or microcystin RR as the main component, in the preparation of products for improving clinical symptoms caused by mandarin fish rhabdovirus infection.

[0015] In the above applications, the "product" is preferably a pharmaceutical product or a virus inhibitor. The "mandarin fish rhabdovirus" specifically refers to *Siniperca chuatsi rhabdovirus* (SCRV).

[0016] Fourthly, the present invention provides an inhibitor of mandarin fish bullet virus. The active ingredient of the inhibitor comprises microcystin LR and / or microcystin RR, or a substance with microcystin LR and / or microcystin RR as the main component.

[0017] Furthermore, the SCRV inhibitor of the present invention may also contain pharmaceutically or aquaculture-acceptable carriers or excipients. This inhibitor can be formulated into various dosage forms suitable for aquatic administration, including but not limited to: oral formulations (such as powders, granules, and microcapsules for mixing with feed), bath preparations, and injections. The carriers or excipients may include diluents (such as starch and glucose), binders, disintegrants, lubricants, stabilizers, antioxidants, pH adjusters, etc.

[0018] The scientific basis and experimental conclusions of this invention are as follows: (1) Safety verification: The CCK-8 cell activity assay confirmed that microcystin LR and RR had no significant effect on the survival rate of mandarin fish host cells (CPB cells) within the set effective concentration range (survival rate >90%), indicating that they have good cell biocompatibility at the effective antiviral dose.

[0019] (2) Viral titer inhibition: using half-maximum tissue culture infectious dose (TCID) 50 The assay was performed using the method described above. The results showed that, compared to the virus control group, the SCRV virus titer in the cell culture supernatant of the experimental groups treated with microcystin LR or RR was significantly reduced. p The concentration-dependent inhibitory effect was <0.01%. The entire inhibition period was within 48 hours, and the virus killing time was controlled within 2 hours, which can quickly kill the virus and inhibit its reproduction for a long time, effectively reducing cell infection by the virus.

[0020] (3) Inhibition of viral genome replication: The DNA copy number of SCRV in extracellular fluid was detected by real-time quantitative PCR (qPCR). The data showed that after LR or RR treatment, the number of viral genome copies released into the culture supernatant was significantly lower than that in the control group (p<0.05), proving that it can effectively inhibit viral replication and / or release.

[0021] (4) Viral morphological observation: Transmission electron microscopy (TEM) showed that after short-term treatment with microcystin LR or RR, the typical bullet-shaped structure of SCRV virus particles was severely damaged, and the viral capsid became irregular, incomplete, and even disintegrated. This directly confirms morphologically that LR and RR have a direct destructive effect on SCRV virus particles.

[0022] In summary, the core contribution of this invention lies in the first-ever disclosure of novel applications of microcystin LR and RR against SCRV. This discovery, based on rigorous in vitro cell and virological experiments, clearly demonstrates their significant inhibitory effects on SCRV replication, infectivity, and viral particle structure at safe concentrations. This provides a novel lead compound and a clear technical direction for the development of specific antiviral drugs or aquatic virus inhibitors targeting SCRV, possessing significant theoretical and practical value. Attached Figure Description

[0023] One or more embodiments are illustrated by way of example with reference to the accompanying drawings. These illustrations do not constitute a limitation on the embodiments, and unless otherwise stated, the figures in the drawings are not to be limited by scale.

[0024] Figure 1 The diagram shows the two-dimensional structure of the microalgal toxin LR. Figure 2 This is a two-dimensional structural diagram of the microalgal toxin RR; Figure 3 The results of CCK8 assay were used to analyze the safety of microalgal toxins LR and RR on cpb cells. Figure 4 The effect of microalgal toxins LR and RR on SCRV virus titer; Figure 5 The trend of microalgal toxins LR and RR on SCRV virus titers; Figure 6 The results show the effects of microalgal toxins LR and RR on the DNA copy number of extracellular SCRV. Figure 7 The results show the trend of the effects of microalgal toxins LR and RR on the DNA copy number of extracellular SCRV; Figure 8 Image of SCRV particles under an electron microscope; Figure 9 This is an electron microscope image of SCRV particles after treatment with microalgal toxin LR; Figure 10 This is an electron microscope image of SCRV particles after treatment with microalgal toxin RR; Figure 11 The graph shows the therapeutic effects of microalgal toxins LR and RR on fish infected with SCRV virus. Detailed Implementation

[0025] Given the problems in the background technology, natural products have become an important source in the search for novel antiviral substances due to their structural diversity and novel mechanisms of action. Microcystins (MCs) are a class of monocyclic heptacapeptide hepatotoxicants produced by freshwater cyanobacteria (such as the genus *Microcystis*), among which microcystin LR (MC-LR) and microcystin RR (MC-RR) are the two most widely distributed and common subtypes in nature. For a long time, research has mainly focused on the strong hepatotoxicity of MCs and their harm to human and animal health. Their toxic mechanism mainly involves inhibiting the activity of protein phosphatases 1 (PP1) and 2A (PP2A), leading to excessive phosphorylation of intracellular proteins, thereby triggering cellular stress, cytoskeleton disruption, and programmed cell death. In recent years, research has begun to focus on the potential biological activities of MCs at non-toxic concentrations, including their potential value in anti-tumor activity and inducing apoptosis. However, to date, there have been no public reports or studies, either domestically or internationally, regarding whether microcystin LR and RR have direct antiviral functions, especially against aquatic animal viruses such as SCRV. This remains a completely unexplored area.

[0026] Based on the aforementioned industry status and technological gaps, the inventors have for the first time discovered and verified that microcystin LR and RR can significantly inhibit SCRV replication and infection at non-cytotoxic concentrations. This discovery not only provides novel candidate substances for the prevention and control of SCRV, but also opens up an unprecedented new avenue for the resource-based and high-value utilization of microcystin.

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The described embodiments are only a part of this invention, and not all of it. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0028] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0029] The Chinese perch brain cell line (CPB) and the Chinese perch rhabdovirus used in the following examples were obtained from the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences. They were cultured in a 28°C constant temperature incubator using Leibovitz's L-15 medium containing 10% fetal bovine serum (FBS).

[0030] The marine microalgal toxins in the following examples were identified as national Class II standard substances and purchased from the National Marine Environmental Monitoring Centre. The two-dimensional structural formulas of the two marine microalgal toxins are shown below. Figure 1 and Figure 2 The relevant information is shown in Table 1.

[0031] Table 1. Information on marine microalgal toxins

[0032] Note: a. Both CAS numbers and reference material numbers are used to identify chemical substances, but they differ in that: the CAS number (Chemical Abstracts Service Number) is a unique identifier assigned to each chemical substance by the Chemical Abstracts Service, regardless of the substance's source, purity, or extraction batch; the CAS number is uniform. The format and numbering method of reference material numbers vary depending on the standardization body.

[0033] The main reagents used in this experiment are shown in Table 2. The cell and virus experiments in this study were conducted in the Fish Disease Control Cell and Virus Laboratory of the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences.

[0034] Table 2 Main reagents used in the experiment

[0035] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0036] Example 1: Determination of the safe concentration range of microcystin LR and RR in CPB cells 1.1 Materials and Methods Cytotoxicity assay: CCK-8 assay was used. CPB cells were cultured at 1 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 h until adherence. The original culture medium was discarded, and fresh culture medium containing different concentrations of MC-LR or MC-RR (concentration gradient: 0, 0.05, 0.14, 1.23, 3.7, 11.11, 33.33, 100.00, 300.00 ng / mL) was added, with six replicates per concentration. A cell-free control was also included. After 48 h of further culture, 10 μL of CCK-8 solution (Dojindo) was added to each well, and after incubation for 4 h, the absorbance was measured at 450 nm using a BioTek Synergy H1 microplate reader.

[0037] Data processing: Cell viability (%) = (Experimental group OD) 450 - Blank Group OD 450 ) / (control group OD 450 - Blank Group OD 450 () × 100%. The half-maximal cytotoxicity concentration (CMC) was calculated using GraphPad Prism 9.0 software. 50 ).

[0038] 1.2 Results Safe concentrations of MC-LR and MC-RR are as follows Figure 3 As shown, a pharmacological excitatory effect was produced at low concentrations. Therefore, to ensure that subsequent antiviral experiments were conducted within a safe range, three concentrations of 100, 3.7, and 0.14 ng / mL were selected for subsequent experiments.

[0039] Example 2: Effects of LR and RR on SCRV virus titer Experimental grouping and viral titer determination (TCID) 50 Method): CPB cells were seeded into 96-well plates and cultured until a monolayer was formed. The culture medium was discarded, and the cells were washed with PBS and then treated according to the following groups.

[0040] Set up a control group: SCRV cells were discarded 2 h after inoculation and cultured in 2% FBS L-15 medium. To test the direct inactivation effects of LR and RR on SCRV, experimental group S1 was set up: SCRV was mixed with LR and RR in vitro for 2 h, the mixture was inoculated into cells for 2 h and then discarded, and 2% FBS L-15 medium was added for further culture. To test whether LR and RR have therapeutic effects on infected cells: Experimental group S2 was set up: SCRV was added to cells and discarded after 2 h, and LR and RR mixture diluted with 2% FBS L-15 medium was added and cultured for a longer time; To examine whether LR and RR enhanced CPB cell resistance to SCRV, experimental group S3 was set up: LR and RR cells were inoculated for 2 h, followed by SCRV inoculation for 2 h and then discarded. Cells were then cultured in 2% FBS L-15 medium. Each group consisted of 3 columns (24 wells) and cultured at 28℃. Supernatants were collected at 24 h, 48 h, and 72 h post-infection. The supernatants were serially diluted 10-fold, with each dilution seeded in 8 replicates. Cell pathogenesis effect (CPE) was recorded after 5-7 days. The Reed-Muench method was used to calculate the viral titer (log) at each time point. 10 TCID 50 / mL).

[0041] The results showed that both LR and RR could significantly reduce the viral titer of SCRV (see [link to study]). Figure 4 In the LR group, compared with the control group, the viral titers of S1-S3 decreased by 7.90E+07, 7.65E+07, and 8.45E+07, respectively, in a concentration-dependent manner (see [link to LR group]). Figure 5 As the concentration increases, the viral titer at a concentration of 100 ng / mL decreases by an average of 8.28E+07 compared to 0.14 ng / mL, which is extremely significant.

[0042] Example 3: Effects of LR and RR on SCRV DNA copy number The effects of LR and RR on SCRV DNA copy number were investigated by detecting SCRV DNA copy number in extracellular fluid using qPCR. The results showed that treatment with both LR and RR significantly reduced viral DNA copy number, indicating that they effectively inhibited viral replication and release (see [link to study]. Figure 6 ).

[0043] Direct inactivation pathway (S1 group: virus infected after extracellular pre-incubation with LR / RR): In this treatment group, even at lower concentrations, the extracellular viral DNA copy number showed a decreasing trend.

[0044] In the LR group, the viral load at low concentrations was reduced by an average of 1.03 copies / mg compared to the control group. As the LR concentration increased, the inhibitory effect continued to strengthen, with viral loads of 6.39, 5.44, and 4.81 copies / mg, respectively, compared to 6.83, 6.47, and 6.45 copies / mg in the control group. Meanwhile, the average viral loads in the RR group (S1-S3) were 5.64, 4.93, and 5.11 copies / mg, respectively, representing decreases of 0.98, 1.69, and 1.51 copies / mg compared to the control group.

[0045] In the RR group, although the inhibitory effect on viral particles was not significant compared to the LR group at low concentrations, it showed better inhibitory effects at medium and high concentrations, with an average reduction of 1.17 and 1.60 copies / mg, respectively. This indicates that LR / RR can act directly on SCRV viral particles without cell dependence, disrupting their infectivity or structural integrity, thereby blocking their ability to subsequently infect cells. This result provides crucial evidence for the direct viral inactivation effect of LR / RR.

[0046] Intracellular therapy route (S2 group: LR / RR administered after viral infection): A dose-dependent viral inhibition effect was also observed in the treatment with LR / RR following viral infection. Under LR treatment, the viral load in the S2 group decreased significantly with increasing concentration, reaching 5.20, 3.90, and 2.79 copies / mg, respectively, representing reductions of 1.62, 2.56, and 3.65 copies / mg compared to the viral control group.

[0047] The RR group also showed good antiviral effects at all three concentrations, exhibiting similar trends. The average viral load in the S2 group was 6.36, 5.53, and 5.01 copies / mg. Compared to the control group, the RR group showed a reduction of 1.07 copies / mg at low concentrations and an inhibitory effect of 2.22 copies / mg at high concentrations.

[0048] This indicates that LR / RR can still exert a significant therapeutic effect in the early stages of viral infection, possibly by interfering with the replication, assembly, or release of the virus within cells, effectively blocking the production of progeny viruses.

[0049] Cell pre-protection pathway (S3 group: cells are exposed to LR / RR before virus infection): Pre-treatment of cells with LR / RR before virus infection also showed a clear inhibitory effect.

[0050] In the LR treatment group, the extracellular viral loads were 6.18, 4.75, and 4.09 copies / mg at low, medium, and high pretreatment concentrations, respectively. Compared with the control group, viral release decreased by approximately 9.39%, 26.4%, and 36.4%, respectively, with the decrease increasing with increasing LR pretreatment concentration, showing a significant dose-response relationship.

[0051] A similar trend was observed in the RR treatment group. The extracellular viral load after pretreatment was 6.12, 5.16 and 4.02 copies / mg, respectively, which were approximately 7.7%, 28.9% and 38.3% lower than the control group. The inhibitory effect was particularly significant in the medium and high concentration groups.

[0052] All three treatment pathways confirmed that LR / RR possessed significant anti-SCRV activity, and its inhibitory effect increased with increasing concentration, demonstrating a clear dose-response relationship. Furthermore, the entire treatment cycle was controlled within 2 days, with a critical period of action within 2 hours, enabling rapid viral eradication and long-term control of viral infection. A cross-sectional comparison of the three treatment pathways revealed that pathway S1 demonstrated its direct viral inactivation capability; pathway S2 highlighted its post-infection therapeutic potential; and pathway S3 suggested its potential function in modulating the host cell's antiviral response. These results collectively indicate that LR / RR can exert its anti-SCRV effect through multiple pathways and mechanisms, providing a systematic and reliable experimental basis for its development into a viral inhibitor with a multimodal mechanism of action.

[0053] 3.1 Materials and Methods Cell infection and treatment: CPB cells were seeded in 12-well plates and cultured to 80% confluence. Grouping was the same as in Example 2. SCRV was infected with MOI=0.1, and after 1 h of adsorption, the medium was replaced with maintenance medium containing the corresponding toxin.

[0054] Sample collection: Cell supernatant and cell pellet were collected 24 h post-infection (after washing with PBS and scraping).

[0055] Viral nucleic acid extraction and qPCR: Supernatant (extracellular virus): Viral RNA was extracted using a viral RNA extraction kit (Qiagen). Total RNA was extracted using TRIzol reagent (Invitrogen), and genomic DNA contamination was removed by treatment with DNase I. Reverse transcription was performed using a PrimeScript RT kit (Takara). The copy number of the SCRV nucleoprotein (N) gene was detected using real-time quantitative PCR (qPCR).

[0056] Primer sequences: NF: 5'-CAGAGTCTCAACTCTGACATCCAGG-3'; NR: 5'-CAGTCTCCGTGAATACCCCAATCCAG-3'; Probe: 5'-FAM-CTCTGTCAACATTTGCTCATGTCGC-3'. A standard curve was plotted using in vitro transcribed N gene RNA as a standard to absolutely quantify viral RNA copy number.

[0057] 3.2 Results like Figure 6 As shown, at 24 h post-infection, the SCRV N gene copy number in the cell supernatant of both the LR and RR treatment groups was significantly lower than that of the virus control group (p<0.01), and this difference was concentration-dependent. Figure 7In both the LR and RR groups, the S2 pathway showed the highest concentration dependence and the best overall antiviral effect. In the LR group, compared with the control group, the average viral load of S1-S3 decreased by 1.03, 2.61, and 1.56 copies / mg, respectively; in the RR group, the average viral load of S1-S3 decreased by 0.98, 1.69, and 1.51 copies / mg, respectively.

[0058] Example 4: Electron microscopic observation of the destructive effects of LR and RR on SCRV particles Transmission electron microscopy observation of the destructive effects of LR and RR on SCRV particles. Transmission electron microscopy results show that after treatment with LR and RR, as... Figure 9 and Figure 10 The morphology of SCRV virus particles was significantly damaged, and the viral structure was destroyed, indicating that it has the function of directly destroying the viral capsid. 4.1 Materials and Methods Virus-toxin co-incubation: Take SCRV virus solution (~10) 11 100 μL of virus particles ( / mL) was mixed with an equal volume of PBS buffer (pH 7.4) containing 10 μM MC-LR or MC-RR. A PBS control group containing only the virus was set up. The mixture was incubated at 4°C for 12 h.

[0059] Negative staining and electron microscopy observation: 10 μL of the above-incubated mixture was dropped onto a Formvar-carbon coated copper grid and allowed to absorb for 2 min. Excess liquid was then blotted away with filter paper. The mixture was negatively stained with 2% (w / v) phosphotungstic acid (PTA, pH 6.8) for 1 min, blotted dry, and allowed to air dry at room temperature. Observation and imaging were performed using a JEM-1400Flash transmission electron microscope (JEOL, Japan) at an accelerating voltage of 80 kV.

[0060] 4.2 Results like Figure 8 As shown in the control group, untreated SCRV virus particles maintained their morphology intact, exhibiting typical characteristics of rhabdoviruses, with a length of approximately 180 nm and a diameter of approximately 70 nm, and a clearly defined capsid structure. Figure 9 (LR processing group) such as Figure 10 As shown in the (RR treatment group), after treatment with MC-LR or MC-RR, the structure of a large number of viral particles underwent significant changes: the capsid ruptured and deformed, contents leaked, and some even completely disintegrated into unstructured fragments. This result directly confirms that MC-LR and MC-RR can directly destroy the structural integrity of SCRV viral particles, which may be one of the important mechanisms by which they inhibit viral infectivity.

[0061] Example 5: Evaluation of the anti-SCRV effects of microcystin LR and RR in a mandarin fish model (animal experiment) This embodiment aims to verify the therapeutic or preventive effects of microcystin LR and RR on SCRV infection in vivo using an animal infection model.

[0062] 5.1 Materials and Methods Experimental animals: Healthy juvenile largemouth bass (Micropterus salmoides), measuring (10.0 ± 1.0) cm in length and (15.0 ± 3.0) g in weight. Before the experiment, they were acclimatized for two weeks in a recirculating aquaculture system (water temperature 25 ± 1℃, continuous aeration) to ensure no abnormalities or mortality. Largemouth bass are susceptible to SCRV and are a commonly used alternative animal model for evaluating anti-SCRV drugs.

[0063] Virus: SCRV virus solution (same as in Example 2), diluted with sterile phosphate-buffered saline (PBS) to a concentration of 1×10⁻⁶ before use. 5 TCID 50 / mL.

[0064] Toxin solution: Microcystin LR and RR standards were prepared into stock solutions with a concentration of 1 mg / mL using PBS containing 1% DMSO. The solutions were further diluted with PBS before use.

[0065] Experimental design: The experimental fish were randomly divided into 4 groups of 50 fish each, and placed in separate 100 L aquariums.

[0066] 1. Post-infection treatment group (Post-LR / RR): Each fish was first injected intraperitoneally with 100 μL of SCRV virus solution (1×10⁻⁶). 4 TCID 50 / tail). 12 hours after infection, inject 100 μL of PBS solution containing LR or RR at a concentration of 100 ng / mL into the peritoneum (dosage: 1 μg / g fish body weight).

[0067] 2. Virus Control: Each fish was intraperitoneally injected with 100 μL of SCRV virus solution (1×10⁻⁶). 4 TCID 50 / tail).

[0068] 3. Blank control group (Mock): Each fish was injected intraperitoneally with 100 μL of sterile PBS.

[0069] Note: LR and RR are set up as independent treatment groups.

[0070] Observation and Recording: Mortality monitoring: Observe and record the mortality of fish in each group daily for 10 consecutive days. Remove dead fish promptly.

[0071] Calculate the cumulative mortality rate (%) = (number of dead fish / initial number of fish in the group) × 100%.

[0072] Clinical symptom assessment: Observe and record the fish’s swimming, feeding, body color, and any abnormalities on the body surface and gills daily.

[0073] Data analysis: Statistical analysis was performed using GraphPad Prism software. Survival curves were compared using the Log-rank (Mantel-Cox) test. A p-value < 0.05 was considered statistically significant.

[0074] The results of this animal experiment indicate that ( Figure 11 In the control group, all 50 fish died from infection on day 7. However, the LR and RR groups, injected with 100 μL of 100 ng / mL drug, achieved survival rates of 64% and 74%, respectively. The surviving fish were uninfected and in good condition, demonstrating a faster and more effective approach compared to conventional control methods. Furthermore, compared to other treatments, LR and RR injections effectively curbed viral infection in the short term, reducing viral damage to the fish. High survival rates and rapid recovery were observed, with no reinfection observed within the following week. Microcystin toxins LR and RR showed clear protective effects against SCRV infection in mandarin fish (largemouth bass model), significantly reducing mortality, alleviating clinical symptoms, and mitigating histopathological damage. This provides crucial in vivo pharmacodynamic evidence for developing LR / RR into SCRV control products (such as injections, oral formulations, or bath solutions) for aquaculture, offering new insights into the development and application of microcystin toxins.

[0075] Microcystins are a class of cyclic heptapeptide toxins produced by cyanobacteria, with common subtypes including LR, RR, and YR. Previous studies have focused primarily on their toxicological effects, while their antiviral potential has not been fully explored. This invention is the first to discover that microcystins LR and RR have a significant inhibitory effect on SCRV, thus expanding their application scope.

[0076] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of microcystin LR and / or microcystin RR, or substances with microcystin LR and / or microcystin RR as the main components, in the preparation of products that inhibit mandarin fish rhabdovirus.

2. The application according to claim 1, characterized in that: The two-dimensional structural formula of the microcystin LR is as follows: .

3. The application according to claim 1, characterized in that: The two-dimensional structural formula of the microcystin RR is as follows: .

4. The application according to claim 1, characterized in that: The safe concentrations for both microcystin LR and microcystin RR in CPB cells are 100 nM and below.

5. The application according to claim 4, characterized in that: The microcystin LR and microcystin RR are used to reduce the copy number of mandarin fish bullet virus, reduce the viral titer of mandarin fish bullet virus, and disrupt the viral structure of mandarin fish bullet virus.

6. The application according to any one of claims 1 to 5, characterized in that: The products include drugs and virus inhibitors.

7. The use of microcystin LR and / or microcystin RR, or substances with microcystin LR and / or microcystin RR as the main component, in the preparation of products for the treatment or prevention of diseases caused by mandarin fish rhabdovirus infection.

8. The use of microcystin LR and / or microcystin RR, or substances with microcystin LR and / or microcystin RR as the main component, in the preparation of products that improve symptoms caused by mandarin fish rhabdovirus infection.

9. A mandarin fish rhabdovirus inhibitor, characterized in that: The active ingredient of the inhibitor includes microcystin LR and / or microcystin RR, or a substance with microcystin LR and / or microcystin RR as the main component.

10. The mandarin fish rhabdovirus inhibitor according to claim 9, characterized in that: The inhibitor also includes pharmaceutically or aquaculture-acceptable carriers or excipients; The inhibitors are formulated into various dosage forms suitable for aquatic administration, including: oral preparations, bath preparations, and injections; The carrier or excipients include diluents, binders, disintegrants, lubricants, stabilizers, antioxidants, and pH adjusters.

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