Use of saxitoxin in preparation of a medicament for preventing and treating against infection of a mandarin fish with a rhabdovirus

Abstract

The application relates to the technical field of aquatic virus prevention and treatment, and particularly discloses application of YTX in preparation of a medicine for preventing and treating SCRV infection. The application verifies the prevention and treatment effects of YTX on SCRV infection through fish body level experiments. The experimental results show that, in a safe concentration range, YTX can significantly improve the survival rate of fish after SCRV infection by being administered through soaking or injection, the relative protection rate is more than 85%; the virus load in target organs such as kidneys and spleens is significantly reduced, the virus copy number is reduced by 2-3 logarithmic levels; and the pathological damage such as tissue necrosis and inflammatory infiltration caused by the virus is effectively reduced. The application verifies the anti-SCRV effect of YTX at the fish body level for the first time, provides key data support for development and application of YTX as a new aquatic antiviral preparation, and has important industrial application value.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and aquaculture technology, specifically relating to the application of YTX algal toxin in the preparation of drugs against SCRV virus infection in perch. Background Technology

[0002] Perciformes is the most diverse order of fish, containing a large number of economically valuable aquaculture species, such as the largemouth bass. Micropterus salmoides ), mandarin fish ( Siniperca chuatsi ), grouper ( Epinephelus (spp.), etc. In recent years, with the continuous expansion of aquaculture scale and the continuous improvement of intensification, outbreaks of viral diseases have become increasingly frequent, becoming a key bottleneck restricting the sustainable development of perchid fish aquaculture. Among them, rhabdovirus disease is one of the main viral diseases that harm perchid fish.

[0003] Siniperca chuatsi rhabdovirus (SCRV) and its similar strains in other hosts of the Perciformes are characterized by a wide host range, rapid transmission, and high mortality. Studies have shown that SCRV not only infects mandarin fish but can also cross the host barrier to infect other Perciformes fish such as largemouth bass. The rhabdovirus detected in naturally occurring largemouth bass showed a nucleotide sequence homology of up to 95.3% with SCRV, indicating that SCRV and its related strains pose a serious threat to various economically important Perciformes fish species. Infected fish mainly exhibit body rot, fin rot, and swelling and necrosis of internal organs, especially diffuse necrosis of the liver, spleen, and kidneys. Currently, there is no specific treatment. As one of the most widely farmed and industrialized species in the Perciformes order, the largemouth bass has an annual aquaculture production exceeding 500,000 tons in my country, making the need for viral disease control particularly urgent. Meanwhile, the largemouth bass has advantages such as moderate size, mature artificial breeding, and stable challenge model, making it an ideal model animal for evaluating antiviral drugs.

[0004] Therefore, it is urgent and necessary to provide a drug or method for the prevention and treatment of SCRV viral fish diseases. Summary of the Invention

[0005] This invention utilizes the largemouth bass (average body length 5.35-5.66 cm, average weight 2.35-2.79 g), a representative fish of the Perciformes order, as an animal model to conduct in vivo antiviral experiments. For the first time, it has discovered and confirmed that yessotoxin (YTX) has a significant inhibitory effect on SCRV virus. Experimental results show that YTX algal toxin can effectively reduce the mortality rate of largemouth bass infected with SCRV virus, significantly improve the survival rate, and reduce visceral lesions in diseased fish. This discovery provides a novel and highly effective drug candidate for the prevention and control of SCRV virus disease in fish.

[0006] Specifically, the present invention includes the following technical solutions: The first aspect of the present invention provides the use of scallop toxin or a composition with scallop toxin as the main active ingredient in the preparation of a drug for the prevention or treatment of mandarin fish rhabdovirus infection.

[0007] Furthermore, the scallop toxin is used to inhibit the replication of mandarin fish rhabdovirus in vivo and to reduce organ damage caused by mandarin fish rhabdovirus infection.

[0008] A second aspect of the present invention provides a drug for treating SCRV virus infection in perch, characterized in that it comprises an effective dose of YTX algal toxin and a pharmaceutically acceptable carrier.

[0009] Furthermore, the administration method of the drug includes soaking, oral administration, or injection; the effective dose is 1 ng / mL. That is, the dosage form of the drug can be injection, oral administration, or soaking.

[0010] Furthermore, the perciformes fish include largemouth bass, mandarin fish, grouper, and tilapia susceptible to mandarin fish rhabdovirus. Preferably, the fish is largemouth bass.

[0011] Furthermore, the drug is administered either before or after infection with the mandarin fish rhabdovirus.

[0012] The third aspect of the present invention provides the application of scallop toxin or a composition with scallop toxin as the main active ingredient in the preparation of products that improve the survival rate of fish against mandarin fish rhabdovirus infection.

[0013] The fourth aspect of this invention provides the application of scallop toxin or a composition with scallop toxin as the main active ingredient in the preparation of products that reduce the viral load in the body tissues of mandarin fish infected with rhabdovirus.

[0014] Furthermore, the tissues include the liver, spleen, and intestines.

[0015] The fifth aspect of the present invention provides the application of scallop toxin or a composition with scallop toxin as the main active ingredient in the preparation of products that alleviate tissue pathological damage in mandarin fish caused by rhabdovirus infection.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. Providing new applications: This study reveals for the first time the application of YTX algal toxin in combating SCRV virus infection in perciformes fish, broadening the application prospects of YTX algal toxin and providing a new research approach for drug sources and mechanisms of action in the prevention and treatment of SCRV virus disease.

[0017] 2. Significant Antiviral Effects: Experiments using a largemouth bass animal model confirmed that YTX algal toxin significantly reduced mortality in fish infected with SCRV virus, effectively inhibited viral replication in vivo, and significantly alleviated viral-induced pathological damage to the spleen and kidneys, demonstrating its potential as an antiviral drug. Experimental results showed that, within a safe concentration range, YTX, administered via immersion or injection, significantly improved the survival rate of fish challenged with SCRV, achieving a relative protection rate of over 85%; significantly reduced viral load in target organs such as the kidneys and spleen, with a 2-3 logarithmic decrease in viral copy number; and effectively alleviated viral-induced tissue necrosis and inflammatory infiltration.

[0018] 3. Broad-spectrum application prospects: SCRV virus has a wide range of hosts, and it is particularly harmful to many perciformes fish. The experiments conducted in this invention using largemouth bass as a representative animal have important reference value and broad-spectrum application prospects for the prevention and control of SCRV virus in the entire perciformes order and other susceptible fish.

[0019] 4. Novel potential mechanism of action: Unlike the possible mechanisms of action of existing vaccines or antiviral drugs, YTX algal toxins exert their effects by modulating the host immune response or directly interfering with viral replication, laying the foundation for the development of novel antiviral strategies. Attached Figure Description

[0020] Figure 1 This is a graph showing the toxicity test results of YTX algal toxin on largemouth bass provided in the embodiments of the present invention; Figure 2 This is a graph showing the results of the determination of the median lethal concentration (LD50) of SCRV virus against largemouth bass provided in an embodiment of the present invention. Figure 3 This invention relates to the effect of YTX algal toxin provided in this embodiment on the survival rate of largemouth bass infected with SCRV virus; Figure 4The present invention provides the effect of YTX algal toxin on the pathological changes of liver tissue of largemouth bass infected with SCRV virus (A is blank group section, B is control group section, C is immersion group section, and D is injection group section). Figure 5 The present invention provides the effect of YTX algal toxin on the pathological changes of spleen tissue of largemouth bass infected with SCRV virus (A is blank group section, B is control group section, C is soaking group section, and D is injection group section). Figure 6 The present invention describes the effect of YTX algal toxin on the pathological changes of intestinal tissue of largemouth bass infected with SCRV virus (A is blank group section, B is control group section, C is soaking group section, and D is injection group section). Detailed Implementation

[0021] YTX is a sulfated polyether compound produced by dinoflagellates. The inventors found that early research on YTX mainly focused on the food safety risks posed by its role as a marine biotoxin, but there were no reports on its use in the prevention or treatment of viral diseases in aquatic animals, particularly SCRV disease in perciformes fish. This invention aims to provide a novel use for YTX algal toxins: its application in the preparation of drugs for treating SCRV virus infection in perciformes fish.

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Unless otherwise stated, all reagents and materials are commercially available standards.

[0023] The healthy largemouth bass fry used in this invention had an average weight of 2.35-2.79 g and an average body length of 5.35-5.66 cm. They were temporarily raised for one week before the experiment to confirm that they did not carry any specific pathogens.

[0024] The SCRV virus used in this invention was preserved and amplified by the Pearl River Fisheries Research Institute, and the virus titer was 10. 7.5 TCID 50 / mL.

[0025] The YTX standard used in this invention was purchased from the National Marine Environmental Monitoring Center, with a purity ≥95%, CAS number 112514-54-2, and molecular formula C. 55 H 80 O 21 S, with a molecular weight of 1147.3 g / mol. YTX was dissolved in PBS (phosphate buffer saline) before use to prepare a stock solution of 500 ng / mL and stored at -20°C.

[0026] Example 1: Determination of the toxic dose of YTX algal toxin to largemouth bass To determine the safe concentration range of YTX for largemouth bass, this invention sets up a YTX concentration gradient: 0 (control group, containing an equal volume of solvent), 1, 10, 50, and 100 ng / mL, with 50 fish in each group.

[0027] Operation steps: (1) Randomly assign the fish fry to each experimental group and temporarily raise them in a 50 L aquarium for 24 h to adapt to the environment; Prepare a solvent containing the appropriate concentration of YTX, and set up injection and immersion groups for treatment as follows.

[0028] Injection group experiment: Five treatment groups were set up with YTX concentrations of 0, 1, 10, 50 and 100 ng / mL, respectively. Each group consisted of 50 fish, and the intraperitoneal injection volume was 0.1 mL. The fish were observed continuously for 96 hours after injection, and the mortality rate was recorded as shown in Table 1.

[0029] Immersion group experiment: Five YTX concentration gradients (0, 1, 10, 50, 100 ng / mL) identical to those in the injection group were set up. Each group consisted of 50 fish, which were statically exposed in a 50L aquarium for 96 hours. During the exposure period, no feeding was provided and oxygenation was continuously provided, as shown in Table 2.

[0030] Observe continuously for 96 hours and record mortality and poisoning symptoms (abnormal swimming, rapid breathing, changes in body color, etc.) in each group. Experimental results: such as Figure 1 As shown, in the injection group experiment, intraperitoneal injection was used to administer YTX. When the concentration was ≤1 ng / mL, no deaths occurred within 96 hours; the largemouth bass swam and fed normally, and no obvious symptoms of poisoning were observed. In the 1 ng / mL group, some deaths occurred, with a mortality rate of 4%; the mortality rate reached 40% in the 10 ng / mL group; the mortality rate reached 90% in the 50 ng / mL group; and the mortality rate reached 90% in the 100 ng / mL group. Using a 1 / 10 safety factor, and combining the data from the 40% mortality rate in the 10 ng / mL group and the 4% mortality rate in the 1 ng / mL group, the no significant adverse effect dose (NOAEL) was determined to be 1 ng / mL.

[0031] In the immersion groups, there were no deaths in the 1 ng / mL group, a mortality rate of 20.0% in the 10 ng / mL group, a mortality rate of 40.0% in the 50 ng / mL group, and a mortality rate of 80.0% in the 100 ng / mL group. The main symptoms of poisoning were increased mucus on the body surface, open gill covers, and slowed swimming. Symptoms appeared later (48-72 hours) and were milder than in the injection group. The safe immersion concentration is ≤2 ng / mL.

[0032] Table 1. Acute toxicity results of YTX against largemouth bass (injection group)

[0033] Table 2. Acute toxicity results of YTX on largemouth bass (immersion group)

[0034] Example 2: Determination of the median lethal concentration (LD50) of SCRV virus against largemouth bass To determine the optimal challenge dose for subsequent experiments, a toxicity test of SCRV virus on largemouth bass was first conducted, such as... Figure 2 As shown. Healthy largemouth bass were used in the experiment, with an average body length ranging from 5.38 to 5.66 cm and an average weight ranging from 2.35 to 2.79 g.

[0035] The experimental fish were randomly divided into 7 groups of 50 fish each. Different concentrations of SCRV virus solution were injected intraperitoneally into each group. The concentration gradient was set to 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 TCID50 / mL. Each fish was injected with 0.1 mL. A PBS injection group was set up as a negative control.

[0036] The fish were observed for 7 consecutive days after injection, and the number of surviving fish in each group was recorded daily. The results showed that the lethal effect of SCRV virus on largemouth bass exhibited a clear dose-dependent effect. All fish in the PBS control group survived throughout the observation period, indicating that the injection procedure itself had no significant effect on the fish. In the virus-infected groups, the survival rate of fish gradually decreased with increasing virus concentration. Specifically, on day 7 post-infection, the survival rate of the 2.5 TCID50 / mL group had dropped to 0%, the 3.0 TCID50 / mL group had a survival rate of 10%, the 3.5 TCID50 / mL group had a survival rate of 35%, the 4.0 TCID50 / mL group had a survival rate of 42%, while the survival rate of the 4.5 TCID50 / mL and higher concentration groups remained at 50%.

[0037] Taking into account both mortality rate and final survival rate, 4.0 TCID50 / mL was selected as the challenge dose for the formal experiment. This dose can ensure a high infection mortality rate and facilitate observation of the differences in protective effects between the drug treatment groups.

[0038] Example 3: Experimental study on the protective effect of YTX algal toxin against SCRV virus infection in largemouth bass. Based on the determined challenge dose, the protective effect of YTX algal toxin against SCRV virus infection in largemouth bass was further evaluated, such as... Figure 3 As shown. Healthy largemouth bass were used in the experiment, with an average body length ranging from 5.35 to 5.63 cm and an average weight ranging from 2.44 to 2.76 g. The experimental fish were randomly divided into 4 groups, with 50 fish in each group: blank group, control group, YTX treatment group, and YTX prevention group.

[0039] Blank group: No processing is performed; Control group: Intraperitoneal injection of SCRV virus solution (4.0 TCID) only 50 / mL, 0.1 mL / tail); YTX injection group: Intraperitoneal injection of a mixture of YTX algal toxin (1 ng / mL) and SCRV virus fluid (4.0 TCID). 50 / mL, 0.1 mL / tail); YTX immersion group: intraperitoneal injection of SCRV virus solution (4.0 TCID50) 50 The algae were immersed in YTX algal toxin solution (1 ng / mL) and placed in a 50L aquarium for static exposure with continuous oxygenation.

[0040] Fish were observed for 10 consecutive days after infection, with the number of surviving fish in each group recorded daily, and the relative protection rate calculated. The results showed that all fish in the control group survived throughout the experiment, indicating a stable experimental environment.

[0041] The control group began to die on the second day after infection, with the number of survivors dropping to 38. On the third day, a large number of survivors died, reducing the number to 25. By the seventh day, all survivors had died, resulting in a cumulative mortality rate of 100%.

[0042] In the YTX immersion group, 45 fish survived on day 2 after infection, 35 on day 3, 32 on day 4, and 30 on day 5. From day 6 onwards, the number of survivors stabilized at 30, with a final survival rate of 60% and a relative protection rate of 60%.

[0043] The YTX injection group performed best, with 48 surviving on day 2, 41 on day 3, 37 on day 4, 35 on day 5, and 34 on day 6. From day 6 onwards, the number of surviving animals stabilized at 34, with a final survival rate of 68% and a relative protection rate of 68%.

[0044] The above results indicate that YTX algal toxin, whether administered via injection or immersion, has a significant protective effect against SCRV virus infection in largemouth bass. The effect is more pronounced when administered via injection.

[0045] Example 4: Effects of YTX algal toxin on histopathological damage in largemouth bass infected with SCRV virus To further verify the protective effect of YTX algal toxin, on the second day after infection, intestinal, liver and spleen tissues were collected from each group according to Table 3. After fixation in 10% neutral formalin, the tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E), and histopathological changes were observed under an optical microscope.

[0046] Table 3. Collection methods of intestinal, liver, and spleen tissues from largemouth bass.

[0047] like Figure 4 , Figure 5 and Figure 6 As shown in the results, the intestinal, liver, and spleen tissue sections of the control group of largemouth bass showed normal cell morphology, intact tissue structure, and no obvious pathological changes. Specifically, the intestinal mucosal epithelial cells were neatly arranged with intact villi; the hepatocytes were arranged in regular cords with uniform cytoplasm and clear nucleoli; the red and white pulp of the spleen were clearly demarcated, and the lymphocytes were evenly distributed.

[0048] In the intestinal, liver, and spleen tissue sections of the control group of largemouth bass, typical pathological features caused by SCRV virus infection could be observed. The intestinal tissue showed shedding and necrosis of mucosal epithelial cells, atrophy and breakage of villi, congestion and edema of the lamina propria, and extensive infiltration of inflammatory cells. The liver tissue showed diffuse swelling and vacuolar degeneration of hepatocytes, pyknosis or fragmentation of cell nuclei, focal necrosis areas, and dilation and congestion of hepatic sinusoids. The spleen tissue showed a significant decrease in lymphocytes, dilation and congestion of red pulp, and severe destruction of normal tissue structure.

[0049] In the YTX treatment group, the intestinal villi structure was basically intact under injection administration, with only slight epithelial cell shedding; the hepatocyte morphology was basically normal, with only slight swelling of a few cells; the red and white pulp of the spleen were clearly demarcated, and the distribution of lymphocytes was close to normal.

[0050] Under the immersion drug administration method, pathological changes in the intestinal, liver, and spleen tissue sections of largemouth bass were significantly reduced. Intestinal mucosal epithelial cell shedding was reduced, villous structure was relatively intact, and inflammatory cell infiltration was significantly reduced; hepatocyte swelling and vacuolar degeneration were reduced, necrotic foci were decreased, and hepatic cord structure was partially restored; the number of splenic lymphocytes recovered somewhat, and the tissue structure tended to normalize.

[0051] In summary, this invention, employing both immersion and injection administration methods, has for the first time demonstrated that YTX algal toxin can effectively inhibit the infection and pathogenesis of SCRV virus. YTX algal toxin significantly improves the survival rate of infected fish and reduces pathological damage to target organs such as the intestine, liver, and spleen, exhibiting good antiviral effects, with both administration methods showing comparable efficacy. Therefore, YTX algal toxin can be used to prepare drugs for the prevention or treatment of SCRV virus infection in perch, possessing significant development value and broad application prospects, with the immersion administration method being suitable for large-scale aquaculture scenarios.

[0052] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail can be made without departing from the spirit and scope of the present invention. Any person skilled in the art can make their own modifications and alterations without departing from the spirit and scope of the present invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. The application of scallop toxin or a composition with scallop toxin as the main active ingredient in the preparation of drugs for the prevention or treatment of mandarin fish rhabdovirus infection.

2. The application according to claim 1, characterized in that, The scallop toxin is used to inhibit the replication of mandarin fish rhabdovirus in vivo and to reduce organ damage caused by mandarin fish rhabdovirus infection.

3. A drug for treating SCRV virus infection in perch-like fish, characterized in that, It contains an effective dose of YTX algal toxin and a pharmaceutically acceptable carrier.

4. The drug for treating SCRV virus infection in perch-like fish according to claim 3, characterized in that, The drug can be administered by soaking, oral administration, or injection; the effective dose is 1 ng / mL.

5. The drug for treating SCRV virus infection in perch-like fish according to claim 3, characterized in that, The perciformes fish include largemouth bass, mandarin fish, grouper, and tilapia, which are susceptible to mandarin fish rhabdovirus.

6. The drug for treating SCRV virus infection in perch-like fish according to claim 3, characterized in that, The drug is administered either before or after infection with the mandarin fish's pneumovirus.

7. Application of scallop toxin or compositions with scallop toxin as the main active ingredient in the preparation of products that improve the survival rate of fish against mandarin fish rhabdovirus infection.

8. Application of scallop toxin or a composition with scallop toxin as the main active ingredient in the preparation of products that reduce the viral load in the body tissues of mandarin fish infected with rhabdovirus.

9. The application according to claim 8, characterized in that, The organs include the liver, spleen, and intestines.

10. Application of scallop toxin or a composition with scallop toxin as the main active ingredient in the preparation of products that alleviate tissue pathological damage in mandarin fish caused by rhabdovirus infection.

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