Use of trimetazidine in the preparation of a broad-spectrum antiviral drug for aquaculture

Abstract

The present application belongs to the field of disease prevention and treatment and aquaculture technology, and relates to application of trimetazidine in preparation of broad-spectrum antiviral drugs for aquaculture. The present application proves through tests that trimetazidine can inhibit WSSV and DIV-1 genomic mRNA transcription and replication of complete virions, improve the survival rate of shrimp and other aquatic animals after being infected with WSSV and DIV-1, and relieve the invasion of viruses on shrimp and other aquatic animals. Therefore, trimetazidine can endow shrimp and other aquatic animals with certain antiviral ability, so as to ensure the healthy development of shrimp and other aquatic animal breeding industry.

Description

Technical Field

[0001] This invention belongs to the field of disease prevention and aquaculture technology, and relates to the application of trimetazidine in the preparation of broad-spectrum antiviral drugs for aquaculture. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Decapod iridescent virus 1 (DIV-1) is one of the major pathogens prevalent in recent years. DIV-1 infection is most common in adult shrimp, with a mortality rate reaching 100%. Common clinical symptoms in shrimp infected with DIV-1 include lethargy and loss of appetite, and internal manifestations include a hollow stomach and intestines, and pale hepatopancreas. DIV-1 infection can cause large-scale outbreaks and deaths in farmed shrimp within a short period, resulting in huge economic losses. White Spot Syndrome Virus (WSSV) is a highly contagious and deadly pathogen that poses a significant threat to the global shrimp farming industry. WSSV can spread rapidly through both horizontal and vertical transmission, with cumulative mortality rates in shrimp reaching up to 100% within 3-7 days of infection. WSSV is highly destructive and spreads rapidly, already causing billions of dollars in losses to shrimp farming in China.

[0004] With the rapid development of the shrimp farming industry worldwide, the farming environment has become increasingly harsh. Infectious diseases affecting farmed shrimp are becoming more and more serious, spreading rapidly and widely, causing economic losses to the shrimp farming industry at a rate of nearly $1 billion annually, resulting in a significant impact. Therefore, the development of a new antiviral drug that can effectively inhibit WSSV and DIV-1 infections and treat white spot syndrome and decapod iridovirus disease would be of great significance for protecting the healthy development of the shrimp farming industry. Summary of the Invention

[0005] Trimetazidine is a drug that has attracted much attention in both human and animal studies. In human medicine, trimetazidine is mainly used to treat stable angina; while in animal studies, it is mainly used for the protection of organs such as the heart and liver. This invention demonstrates through experiments that trimetazidine can inhibit the transcription of WSSV and DIV-1 genomic mRNA and the replication of intact viral particles, improving the survival rate of shrimp and other aquatic animals infected with WSSV and DIV-1, and alleviating viral damage to shrimp and other aquatic animals. This indicates that trimetazidine can confer a certain antiviral ability to shrimp and other aquatic animals. Therefore, the anti-WSSV and DIV-1 effects discovered in this invention are different from the existing known medicinal uses of trimetazidine.

[0006] Based on the above research results, this invention provides the application of trimetazidine in the preparation of broad-spectrum antiviral drugs for aquaculture, which can effectively inhibit WSSV and DIV-1 infection, treat white spot syndrome and iridovirus disease in decapod shrimp, thereby ensuring the healthy development of shrimp and other aquatic animal farming.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The first aspect is the application of trimetazidine in the preparation of broad-spectrum antiviral drugs for aquaculture, wherein the virus is white spot syndrome virus or decapod iridovirus 1.

[0009] The trimetazidine described in this invention has the CAS number 5011-34-7, and its chemical structural formula is as follows:

[0010] .

[0011] In some implementation schemes, the antiviral drug is an anti-leukoderma syndrome drug or an anti-decapodiform iridovirus drug.

[0012] In some embodiments, the active ingredient of the drug contains trimetazidine or a pharmaceutically acceptable derivative thereof. The pharmaceutically acceptable derivatives described in this invention can be salts, esters, alcohols, ethers, glycosides, stereoisomers, hydrates, solvates, etc.

[0013] In some implementations, the drug is applied to aquatic animals. Specifically, the aquatic animals are crustaceans, such as shrimp, mud crabs, lobsters, barnacles, and krill. More specifically, the aquatic animals are shrimp.

[0014] In some embodiments, the drug is a composition. Specifically, the composition includes pharmaceutical excipients. These excipients may be binders, diluents, disintegrants, wetting agents, film-forming agents, antioxidants, palatability enhancers, penetration enhancers, preservatives, emulsifiers, dispersants, water, mineral oil, vegetable oil, etc.

[0015] In some embodiments, the dosage form of the drug includes, but is not limited to, wettable powder, water-dispersible granules, aqueous suspension, or dispersible oil suspension. The dosage form can be prepared by adding different pharmaceutical excipients.

[0016] In some implementations, the drug is added as a feed additive to the feed of aquatic animals. The drug is administered via liquid feed addition, including but not limited to premixed addition, direct addition, and coated addition, without specific limitations herein.

[0017] In some implementation schemes, the drug is administered via intramuscular injection.

[0018] In some implementation methods, the dosage of the drug is 0.1-0.65 mg / kg, that is, 0.1-0.65 mg per kilogram of aquatic animal body weight. The dosage can be 0.2-0.65 mg / kg, 0.3-0.65 mg / kg, 0.4-0.65 mg / kg, 0.45-0.65 mg / kg, 0.5-0.65 mg / kg, 0.1-0.63 mg / kg, 0.2-0.63 mg / kg, 0.3-0.63 mg / kg, 0.4-0.63 mg / kg, 0.45-0.63 mg / kg, 0.47-0.63 mg / kg, 0.5-0.63 mg / kg, etc.

[0019] Secondly, a method for preventing and controlling diseases in aquaculture involves applying a drug containing trimetazidine or a pharmaceutically acceptable derivative thereof to aquatic animals.

[0020] In some implementations, the aquatic animal is a crustacean, such as prawn, mud crab, lobster, barnacle, krill, etc. Specifically, the aquatic animal is prawn.

[0021] In some implementations, the dosage of the drug is 0.1-0.65 mg / kg.

[0022] The beneficial effects of this invention are as follows:

[0023] This invention is the first to discover and confirm the application value of trimetazidine in the prevention and control of diseases in crustaceans. Experimental results show that trimetazidine can significantly inhibit the replication process of white spot syndrome virus (WSSV) and decapod iridovirus 1 (DIV-1), improve the immunity of crustaceans, and alleviate viral damage. Specifically, its mechanism of action is manifested in at least three aspects: first, it effectively inhibits the transcription of WSSV and DIV-1 genomic mRNA; second, it reduces the generation of intact viral particles with infectious activity at the source; and third, it improves the survival rate of crustaceans infected with WSSV and DIV-1.

[0024] Therefore, this invention provides a novel and effective prevention and control solution for viral diseases in aquaculture, particularly for shrimp farming. Furthermore, this discovery provides a solid theoretical basis and valuable reference direction for the future application of trimetazidine in the prevention and control of related diseases in other crustaceans (such as crabs and lobsters). Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 This is a graph showing the results of confirming the optimal dosage of trimetazidine in shrimp according to Example 1 of the present invention.

[0027] Figure 2 This is a graph showing the effect of trimetazidine in Example 2 of the present invention on the copy number of intact white spot syndrome virus particles in shrimp after administration.

[0028] Figure 3 The figure shows the effect of trimetazidine in Example 3 of the present invention on the expression of the vp28 gene, a structural protein of white spot syndrome, after administration to shrimp.

[0029] Figure 4 The figure shows the effect of trimetazidine in Example 4 of the present invention on the copy number of intact viral particles of Decapoda iridovirus after administration to shrimp.

[0030] Figure 5 The figure shows the effect of trimetazidine in Example 5 of the present invention on the structural protein mcp001R of decapod iridovirus after administration to shrimp.

[0031] Figure 6 This is a graph showing the effect of trimetazidine in Example 6 of the present invention on shrimp mortality caused by white spot syndrome virus infection after administration to shrimp.

[0032] Figure 7 The figure shows the effect of trimetazidine in Example 7 of the present invention on shrimp mortality caused by Decapoda iridovirus infection after administration. Detailed Implementation

[0033] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples. Unless otherwise specified, experimental methods in the following embodiments are generally performed according to conventional biological methods and conditions in the art, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer. If specific experimental conditions are not specified in the embodiments, they are generally performed according to conventional conditions or the conditions recommended by the selling company; the materials, reagents, etc., used in the embodiments, unless otherwise specified, can be purchased commercially.

[0034] Example 1

[0035] To determine the optimal dosage of trimetazidine for shrimp: Trimetazidine (S4543, Selleck Chemicals) was dissolved in artificial seawater as a stock solution. 150 shrimp (average weight 8.5g per shrimp, ranging from 8-10g) were divided into 5 groups of 30 shrimp each. The trimetazidine stock solution was diluted with artificial seawater to prepare a series of dilutions with concentrations of 0.1, 0.2, 0.4, and 1.0 μg / μL. Each shrimp in each group was intramuscularly injected with 50 μL of artificial seawater or the dilution, resulting in dosages of 0 (control group, artificial seawater only), 5, 10, 20, and 50 μg, respectively. Survival rates of the shrimp in each group were observed and recorded every 12 hours post-injection.

[0036] The results are as follows Figure 1 As shown: Dosing 5 μg trimetazidine did not affect the survival of shrimp.

[0037] Example 2

[0038] Animal treatment: Trimetazidine was dissolved in artificial seawater as a stock solution. Before use, the stock solution was diluted to 100 μg / mL. Group 1 received an intramuscular injection of 50 μL of artificial seawater; Group 2 received an intramuscular injection of 50 μL of diluted trimetazidine. Shrimp in both groups were cultured for 3 hours after treatment. Each group of shrimp was then injected with 50 μL of WSSV, with a viral load of 10. 6Viral particles were cultured for 24 h and 48 h, and total DNA samples were extracted from blood cells using the QIAamp DNA Blood Mini Kit (51104; Qiagen). Changes in WSSV DNA were detected using qRT-PCR. The primers used were VP28-RTF: 5'-AGCTCCAACACCTCCTCCTTCA-3' (as shown in SEQ ID NO:1) and VP28-RTR: 5'-TTACTCGGTCTCAGTGCCAGA-3' (as shown in SEQ ID NO:2). The PCR program was 94 ℃ for 5 min; 94 ℃ for 10 s, 60 ℃ for 1 min, for 40 cycles. Melting curves were measured every 1 ℃ from 65 ℃ to 95 ℃.

[0039] Test results as follows Figure 2 As shown, trimetazidine can inhibit the replication of intact viral particles of vitiligo syndrome virus.

[0040] Example 3

[0041] The treatment and detection methods for experimental animals were the same as in Example 2. Two groups of shrimp were cultured for 3 hours after treatment. Each group of shrimp was injected with 50 μL of WSSV, with a viral load of 10. 6 Virus particles were cultured for 24 h and 48 h. RNA samples from shrimp hemocytes were obtained using an RNA extraction reagent. The RNA was then reverse transcribed into cDNA using a reverse transcription kit (R323-01; Vazyme). Changes in the mRNA of the WSSV structural protein vp28 were detected using qRT-PCR. The primers used were VP28-RTF: 5'-AGCTCCAACACCTCCTCCTTCA-3' (as shown in SEQ ID NO:1) and VP28-RTR: 5'-TTACTCGGTCTCAGTGCCAGA-3' (as shown in SEQ ID NO:2). The PCR program was 94 ℃ for 5 min; 94 ℃ for 10 s, 60 ℃ for 1 min, for 40 cycles. Melting curves were measured every 1 ℃ from 65 ℃ to 95 ℃.

[0042] Test results as follows Figure 3 As shown, trimetazidine can inhibit the mRNA transcription of the vitiligo syndrome virus structural protein vp28.

[0043] Example 4

[0044] The treatment and detection methods for experimental animals were the same as in Example 2. Two groups of shrimp were cultured for 3 hours after treatment. Each group of shrimp was injected with 50 μL of DIV-1, with a viral load of 10. 6Virus particles were cultured for 24 h and 48 h, and total DNA samples were extracted from blood cells using the QIAamp DNA BloodMini Kit (51104; Qiagen). Changes in DIV-1 DNA were detected using qRT PCR. The primers used were DIV1(MCP001R)-RTF: 5'-CGAGCACGTATGGGATGTGT-3' (as shown in SEQ ID NO:3) and DIV1(MCP001R)-RTR: 5'-CGCTCTGATCTGGGTCGAAA-3' (as shown in SEQ ID NO:4). The PCR program was 94 ℃ for 5 min; 94 ℃ for 10 s, 60 ℃ for 1 min, for 40 cycles. Melting curves were measured every 1 ℃ from 65 ℃ to 95 ℃.

[0045] Test results as follows Figure 4 As shown, trimetazidine can inhibit the replication of intact viral particles of decapod iridoviruses.

[0046] Example 5

[0047] The treatment and detection methods for experimental animals were the same as in Example 2. Two groups of shrimp were cultured for 3 hours after treatment. Each group of shrimp was injected with 50 μL of DIV-1, with a viral load of 10. 6 Virus particles were cultured for 24 h and 48 h. RNA samples from shrimp hemocytes were obtained using an RNA extraction reagent. The RNA was then reverse transcribed into cDNA using a reverse transcription kit. Changes in the mRNA of the DIV-1 structural protein MCP001R were detected using qRT-PCR. The primers used were DIV1(MCP001R)-RTF: 5'-CGAGCACGTATGGGATGTGT-3' (as shown in SEQ ID NO:3) and DIV1(MCP001R)-RTR: 5'-CGCTCTGATCTGGGTCGAAA-3' (as shown in SEQ ID NO:4). The PCR program was 94 ℃ for 5 min; 94 ℃ for 10 s, 60 ℃ for 1 min, for 40 cycles. Melting curves were measured every 1 ℃ from 65 ℃ to 95 ℃.

[0048] Test results as follows Figure 5 As shown, trimetazidine can inhibit the mRNA transcription of the MCP001R gene, a structural protein of decapod iridoviruses.

[0049] Example 6

[0050] The experimental animal treatment was the same as in Example 2, with both groups of shrimp cultured for 3 hours after treatment. Each group of shrimp was injected with 50 μL of WSSV, with a viral load of 10. 6Virus particles were used to record the survival rate of each group of shrimp every 12 hours.

[0051] Test results as follows Figure 6 As shown, trimetazidine can inhibit shrimp mortality caused by white spot syndrome virus.

[0052] Example 7

[0053] The experimental animal treatment was the same as in Example 2, with two groups of shrimp cultured for 3 hours after treatment. Each group of shrimp was injected with 50 μL of DIV-1, with a viral load of 10. 6 Virus particles were used to record the survival rate of each group of shrimp every 12 hours.

[0054] Test results as follows Figure 7 As shown, trimetazidine can inhibit shrimp mortality caused by decapod iridovirus.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of trimetazidine in the preparation of a broad-spectrum antiviral drug for aquaculture, wherein the virus is Decapoda iridovirus 1.

2. The application as described in claim 1, characterized in that, The antiviral drug is for decapod iridovirus diseases.

3. The application as described in claim 1, characterized in that, The active ingredient of the drug is trimetazidine.

4. The application as described in claim 1, characterized in that, The drug is administered to aquatic animals.

5. The application as described in claim 4, characterized in that, The aquatic animal in question is a shrimp.

6. The application as described in claim 1, characterized in that, The drug is a composition.

7. The application as described in claim 6, characterized in that, The composition includes pharmaceutical excipients.

8. The application as described in claim 1, characterized in that, Drugs are added to the feed of aquatic animals as feed additives.

9. The application as described in claim 1, characterized in that, The medication is administered via intramuscular injection.

10. The application as described in claim 1, characterized in that, The dosage of the drug is 0.1-0.65 mg / kg.

Images