Application of ningnanmycin in product for preventing and treating viral diseases of aquatic animals

Abstract

The invention relates to the technical field of aquaculture and prevention and treatment of aquatic diseases, and particularly discloses application of ningnanmycin in a product for preventing and treating viral diseases of aquatic animals. According to the application disclosed by the invention, the ningnanmycin is found to be capable of preventing or treating the iridovirus of the largemouth bass frog for the first time, and based on animal experiments, the ningnanmycin is verified to have remarkable antiviral activity, so that a new green and safe means is provided for preventing and treating the iridovirus of percodaceae species, and the new application of the ningnanmycin is expanded.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture and aquatic disease prevention and control, and specifically relates to the application of Ningnanmycin in products for preventing and controlling viral diseases of aquatic animals. Background Art

[0004] At present, there is little research on aquatic antiviral drugs at home and abroad. Since the drugs such as adamantane, guanine analogs and ribavirin, which were mainly used in human medicine, were banned in fishery production, there are basically no antiviral drugs for aquatic products; most vaccines can only be injected for immunization and only target a single viral disease, which greatly limits the application of vaccines in actual aquaculture production. Therefore, finding new therapeutic drugs has broad prospects.

[0005] Ningnanmycin (CAS: 156410-09-2) is a new cytidine nucleoside peptide antibiotic developed by the Chengdu Institute of Biology, Chinese Academy of Sciences. It was awarded the national invention patent (ZL93104287.9) in 1997. It has the advantages of low toxicity, low residue, no "three hazards" and accumulation problems, and no environmental pollution, which is in line with the current healthy and environmentally friendly breeding concept. This antibiotic has been widely promoted and applied in the prevention and treatment of plant viral diseases, showing obvious antiviral effects, especially for tobacco mosaic virus disease, tomato virus disease, pepper virus disease, etc. However, the current research on Ningnanmycin has not yet involved the antiviral activity of aquatic animals. Summary of the invention

[0006] The purpose of the present invention is to provide an application of Ningnanmycin in the preparation of products for preventing and treating viral diseases of aquatic animals, to explore the feasibility of Ningnanmycin in antiviral aquaculture, and to open up new avenues for innovative research on antiviral green fishery drugs.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The invention provides an application of Ningnanmycin in a product for preventing and treating viral diseases of aquatic animals.

[0009] In some embodiments, the aquatic animal is at least one of the order Perciformes.

[0010] In some embodiments, the product is a drug whose active ingredient includes ningnanmycin.

[0011] In some embodiments, the dosage form of the drug includes a liquid or a solid dose.

[0012] In some embodiments, the liquid agent includes an injection or an infusion.

[0013] The present invention proves, through animal experiments and cell experiments, that Ningnanmycin can be used as an antiviral drug for preventing and treating viral diseases in aquatic animals, and its effects specifically include enhancing the fish body's resistance to viral infection, inhibiting viral infection replication, and inhibiting viral infection proliferation.

[0014] In some embodiments, the product is fish feed supplemented with Ningnanmycin.

[0015] In some embodiments, the fish feed raw material comprises Ningnanmycin, a binder and feed.

[0016] In some embodiments, the mass ratio of Ningnanmycin to feed is (1450-1750) mg:1 kg.

[0017] In some embodiments, the feed is any one of aquatic feeds.

[0018] In some embodiments, the binder is any one of sodium hydroxymethylcellulose, gelatin or chitosan.

[0019] The invention mixes Ningnanmycin into feed for feeding. Experiments have proved that the feed containing Ningnanmycin will not suppress the feeding desire of fish and has good palatability. It has also been proved that Ningnanmycin treatment has no toxicity to the growth and development of sea bass. This feed feeding method simplifies the treatment of infected fish with Ningnanmycin and is convenient for farmers to use.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention is based on the results of animal experiments and cell experiments, and for the first time studies and verifies the biological activity of Ningnanmycin in resisting aquatic animal viruses, proving that Ningnanmycin has the effects of enhancing fish resistance to viral infection, inhibiting viral replication and proliferation, and improving the survival rate of infected fish. It can be effectively applied to the prevention and treatment of viral diseases in aquatic animal breeding, which provides a new way for the development and research of green aquaculture biological agents, and also expands the application field of Ningnanmycin.

[0022] 2. After proving that Ningnanmycin has an antiviral effect on fish, the present invention mixed Ningnanmycin into feed and fed it to fish. From the feeding phenomenon of adult fish, it can be seen that adult fish eat well, and there is no refusal to eat or weight loss; from the feeding phenomenon of growing and developing fish, it can be seen that Ningnanmycin treatment has no toxicity to the growth and development of sea bass. The two groups of experiments, on the one hand, show that feed mixed with Ningnanmycin can prevent and treat viral diseases of aquatic animals without the need to prepare special medicines, simplifying the treatment of infected fish with Ningnanmycin and facilitating the use of farmers; on the other hand, it shows that Ningnanmycin can be used in all stages of fish growth and development, further broadening the scope of use. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the survival status of largemouth bass in different experimental groups in Example 3 after being infected with largemouth bass frog iridovirus.

[0024] Figure 2 This is the change in viral load in the body of the largemouth bass after being infected with the largemouth bass frog iris virus in Example 4.

[0025] Figure 3 The cytotoxicity evaluation of different concentrations of Ningnanmycin on EPC cells in Example 5, (A) microscopic observation of the effect of Ningnanmycin on EPC cell morphology, (B) MTT staining observation, (C) cell viability calculation results.

[0026] Figure 4 The inhibition rates of different concentrations of Ningnanmycin on LMBV virus in the two prevention and treatment schemes in Example 5 are shown as follows: (A) prevention administration method, and (B) treatment administration method. DETAILED DESCRIPTION

[0027] The present invention will be described below in conjunction with specific embodiments. It should be noted that the following embodiments are examples of the present invention and are only used to illustrate the present invention, but not to limit the present invention. Other combinations and various modifications within the concept of the present invention may be performed without departing from the spirit or scope of the present invention.

[0028] It is worth noting that the Ningnanmycin powder described in the following examples is a 10% Ningnanmycin soluble powder purchased from Deqiang Biological Co., Ltd.

[0029] Example 1

[0030] The feeding experiment of sea bass on feed containing different amounts of Ningnanmycin was conducted as follows:

[0031] (1) The experiment was conducted in a 1.5m×1.5m×1.5m cage. During the culture period, the water temperature was 28±2℃, the dissolved oxygen was higher than 5.0mg / L, and the ammonia nitrogen was lower than 0.5mg / L. Adult largemouth bass purchased from a regular farm with a body length of 30±5cm were put into the cage for one week to adapt to the water environment, and unhealthy fish were eliminated to obtain normal fish;

[0032] (2) Four treatment groups were set up. Twenty adult fish were selected from each group and placed in a 1.5 m × 1.5 m × 1.5 m net cage. The initial weight of each fish was recorded.

[0033] (3) Ningnanmycin powder was weighed according to the amount of 0, 100, 400, and 1600 mg / kg feed, mixed with feed and fed three times a day. The daily feeding amount was 4% of the fish body weight for 7 days. After the feeding was completed, the post-experimental weight of each fish was weighed, and the difference in mass change before and after each group was calculated.

[0034] Results: Compared with the control group of 0 mg / kg Ningnanmycin, the weight change difference of the 100, 400, and 1600 mg / kg Ningnanmycin treatment groups was ≤20g, indicating that Ningnanmycin had no significant effect on the feeding and body weight of adult largemouth seabass.

[0035] Example 2

[0036] The experiment on the toxicity of different amounts of Ningnanmycin on the growth and development of sea bass was carried out as follows:

[0037] (1) The experiment was conducted in a 50L aquarium. The water was changed every day and the water temperature was maintained at 28±2℃. Largemouth bass weighing 20±5g purchased from a regular breeding farm were put into the aquarium and kept in the aquarium for 1 week to adapt to the water environment. Unhealthy fish were eliminated and normal fish were obtained.

[0038] (2) Four treatment groups were set up, with three replicates in each group. Twenty normal fish were selected and placed in a 50-L aquarium as one replicate;

[0039] (3) Ningnanmycin powder was weighed according to the amount of 0, 100, 400, and 1600 mg / kg feed, mixed with feed and fed three times a day, with a daily feeding amount of 4% of the fish body weight. The feeding was continued for 7 days, and the individual behavior and mortality of largemouth bass were continuously observed.

[0040] Results: Compared with the control group of 0 mg / kg Ningnanmycin, 100, 400, and 1600 mg / kg Ningnanmycin had no significant effect on the feeding and individual behavior of growing largemouth bass.

[0041] Example 3

[0042] Ningnanmycin was used to improve the survival rate of largemouth bass infected with LMBV. The steps are as follows:

[0043] (1) Largemouth bass were selected by the method described in Example 1, and 20 normal fish were placed in a 50L aquarium as one replicate, with 3 replicates in each group. A total of five treatment groups were set up, namely a negative control group, a positive control group, and groups with different amounts of Ningnanmycin added (100, 400, and 1600 mg / kg). The negative and positive control groups were fed with a basic feed, and the Ningnanmycin group was fed with a feed mixed with Ningnanmycin according to the amount of Ningnanmycin added. The feed was fed 3 times a day, and the daily feeding amount was 4% of the fish body weight, and the feeding was carried out for a total of 5 days;

[0044] (2) After 5 days of feeding, LMBV virus infection was performed: the negative control group was injected with 100 μL PBS buffer solution into the chest; the positive control group and Ningnanmycin group were injected with 100 μL of iridovirus dilution solution;

[0045] (3) The negative and positive control groups continued to be fed with the basic feed, and the Ningnanmycin group was fed with feed mixed with different amounts of Ningnanmycin;

[0046] (4) Observe and record the mortality of largemouth bass every 24 hours until the 7th day.

[0047] Results: Figure 1 As shown, the mortality rate of largemouth bass in the 1600 mg / kg Ningnanmycin group was significantly lower than that in the positive control group, and the death occurred later, which indicates that Ningnanmycin has a significant protective effect on largemouth bass infection with LMBV.

[0048] Example 4

[0049] Ningnanmycin inhibits the replication and proliferation of the virus in largemouth bass infected with largemouth bass frog iris virus (LMBV) in the fish body. The steps are as follows:

[0050] (1) Treat the control group and the experimental group separately according to the method described in Example 3;

[0051] (2) On the 1st, 4th, and 7th days after virus infection, liver samples from three fish in each group were collected for viral load detection. Viral DNA was extracted using the Aquatic Animal Pathogen Genomic DNA / RNA Extraction Kit (FAST), and the viral load was detected using a fluorescent probe amplification kit.

[0052] Results: Figure 2 As shown, the increase in viral load in the 1600 mg / kg Ningnanmycin group was lower than that in other groups, indicating that high concentrations of Ningnanmycin can inhibit the proliferation of LMBV.

[0053] Example 5

[0054] Ningnanmycin inhibits the replication and proliferation of LMBV in carp epithelioma cells (EPC) in vitro, the steps are as follows:

[0055] (1) Cell, virus, and drug treatment:

[0056] EPC cells were used as the research model and cultured in M199 medium at 28°C. LMBV was isolated and preserved from LMBV-infected largemouth bass in our laboratory. Ningnanmycin powder was diluted with M199 medium to a concentration of 1×10 -1 , 1×10 -2 , 1×10 -3 , 1×10-4 , 1×10 -5 , 1×10 -6 , 1×10 -7 , 1×10 -8 g / mL of solution;

[0057] (2) Determination of safe drug concentration:

[0058] To determine the cellular safety concentration of Ningnanmycin, EPC cells were seeded in a 96-well cell culture plate and cultured at 28°C for 24 h. The next step of the experiment was performed after the monolayer of cells covered the bottom of the well;

[0059] Eight drug groups, as well as a negative control group and a positive control group were set up; the drug concentrations of the eight drug groups were 1×10 -1 , 1×10 -2 , 1×10 -3 , 1×10 -4 , 1×10 -5 , 1×10 -6 , 1×10 -7 , 1×10 -8 g / mL, 8 samples in each drug group, the original culture medium was removed, 100 μL of different concentrations of drug solution was added to each well and cultured for 48 h, then MTT staining was performed, and the absorbance at 490 nm was measured with an enzyme marker, and the cell survival rate was calculated by the absorbance value to obtain the maximum safe concentration of the drug solution;

[0060] Cell survival rate = (average D490 nm value of the drug solution group - D490 nm value of the blank group) / (D490 nm value of the cell group - D490 nm value of the blank group) × 100%.

[0061] Results: Figure 3 As shown in A, when the concentration of Ningnanmycin is ≤1×10 -4 g / L, the EPC cell morphology was consistent with that of the control group, while when a higher concentration (≥1×10 -2 g / L), the cytotoxicity was obvious, resulting in cell shrinkage and cell detachment; the cell viability was determined by MTT method (see Figure 3 B and 3C), the results were consistent with those observed under the microscope; the results showed that the concentration of Ningnanmycin was 1×10 -4 g / mL or less has no cytotoxic effect on EPC cells and can be used in drug research.

[0062] (3) Two prevention and treatment programs were used to explore the mechanism of Ningnanmycin against LMBV:

[0063] Scheme 1-Prevention: EPC cells were grown to a monolayer in a 96-well plate, 100 μL of different concentrations of drug solution was added to each well, cultured at 28°C for 24 hours, and then inoculated with LMBV virus for 4 hours. M199 culture medium containing 2V% FBS was added for culture, and the OD490nm value was measured by MTT kit after 72 hours to calculate the inhibitory activity;

[0064] Scheme 2-treatment: EPC cells were grown to a monolayer in a 96-well plate, inoculated with LMBV virus at 28°C for 4 h, 100 μL of drug solution of different concentrations was added to each well, and the OD490nm value was measured by MTT kit after incubation at 28°C for 72 h to calculate the inhibitory activity;

[0065] Inhibitory activity (%) = (OD490nm value of drug group - OD490nm value of virus control group) / (OD490nm value of blank control group - OD490nm value of virus control group) / × 100%.

[0066] Results: Figure 4 As shown in Figure 2, Ningnanmycin has a good in vitro antiviral effect at a concentration of 1×10 -5 g / m, the inhibition rate of virus replication and proliferation can reach 60%.

[0067] The embodiments described above do not impose any form of limitation on the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. Application of Ningnanmycin in products for preventing and treating viral diseases of aquatic animals.

2. The use according to claim 1, characterized in that: The aquatic animal is at least one of the order Perciformes.

3. The use according to claim 1, characterized in that: The product is a medicine whose active ingredient includes Ningnanmycin.

4. The use according to claim 3, characterized in that: The dosage form of the drug includes liquid or solid.

5. The use according to claim 4, characterized in that: The liquid preparation includes an injection or an immersion preparation.

6. The use according to claim 1, characterized in that: The product is fish feed added with Ningnanmycin.

7. The use according to claim 6, characterized in that: The fish feed raw materials include Ningnanmycin, a binder and feed.

8. The use according to claim 7, characterized in that: The mass ratio of Ningnanmycin to feed is (1450-1750) mg:1 kg.

9. The use according to claim 7, characterized in that: The feed is any one of aquatic feeds.

10. The use according to claim 7, characterized in that: The binder is any one of sodium hydroxymethyl cellulose, gelatin or chitosan.

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