Antiviral active drugs for fish and use thereof

Abstract

The present application relates to the cross field of aquatic disease prevention and control and natural product antiviral drug research, and in particular to a fish antiviral active drug and application thereof.The present application firstly explicitly defines the antiviral activity of prodigiosin on IHNV and SVCV, expands the application field of prodigiosin, and can provide a new lead compound for the development of IHNV and SVCV virus drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of aquatic disease prevention and control and natural product antiviral drug research, in particular to a fish antiviral active drug and its application. BACKGROUND

[0002] Prodigiosin (PG) is a natural secondary metabolite synthesized by bacteria such as Serratia marcescens. Its chemical structure is C 20 H 25 N3O, with a molecular weight of 323.43 and a core skeleton of a tripyrrole ring structure. One of the pyrrole rings is substituted with a methyl group at C-2 and a pentyl group at C-3, and the other pyrrole ring has a methoxy modification. It is a dark red solid and is soluble in organic solvents such as methanol, ethanol, and acetonitrile. It has good chemical modification and preparation basis.

[0003] In recent years, the biological activity of prodigiosin has attracted much attention, and it has shown significant potential in the fields of antibacterial, antitumor, immunomodulation, and antiviral. As a natural monomer compound with a clear structure, prodigiosin has multi-target antiviral activity, environmental compatibility, and industrial production feasibility, which can break through the limitations of existing prevention and control technologies and provide innovative solutions for the green prevention and control of infectious hematopoietic necrosis virus (IHNV) and spring viremia of carp virus (SVCV).

[0004] In the field of aquaculture, IHNV and SVCV are members of the Rhabdoviridae family and are important pathogens that harm the aquaculture industry. IHNV mainly infects salmonids such as rainbow trout and salmon, and is particularly pathogenic to fry and juvenile fish, with a mortality rate of 80-100%, resulting in serious economic losses to the aquaculture industry. SVCV has a wide host range and can infect various cyprinids such as carp, crucian carp, and grass carp, causing symptoms such as systemic hemorrhage and edema, with a mortality rate of over 70%, and is listed as an important aquatic animal disease.

[0005] Currently, the prevention and control of IHNV and SVCV have significant technical bottlenecks: 1) Fish vaccine development is limited by factors such as viral serotype diversity and rapid mutation rate, and cross-protection effect is not good; 2) Chemical synthesis of antiviral drugs can cause water pollution, drug residues, and viral resistance, which violates the concept of ecological aquaculture; 3) Existing plant extract preparations have complex components, making quality control difficult, and the antiviral effect is unstable.

[0006] In the field of antiviral, studies have confirmed that prodigiosin has high inhibitory effect on MSRV, PVY and other viruses, and plays an antiviral effect through mechanisms such as stabilizing host p53 protein, inducing lysosome alkalization or regulating host immune pathways, and has the characteristics of natural source, low toxicity and low residue, good environmental compatibility, which meets the demand of green development of aquaculture.

[0007] However, the systematic study and application of prodigiosin in the prevention and control of important pathogenic viruses IHNV and SVCV in fish are still blank. Therefore, the research on the resistance of prodigiosin to IHNV and SVCV has important significance for breaking through the bottleneck of existing technology and developing green antiviral drugs. SUMMARY

[0008] The present application aims to provide a new use of prodigiosin, i.e. fish antiviral active drugs and their applications.

[0009] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0010] The present application provides the use of prodigiosin related substances in the preparation of a drug for improving the survival rate of fish infected with infectious hematopoietic necrosis virus and / or spring viremia of carp virus, wherein the administration concentration of the prodigiosin related substances is 0.1-0.8 mg / kg (based on the weight of the fish).

[0011] The present application provides the use of prodigiosin related substances in the preparation of a drug for reducing the viral load of fish infected with infectious hematopoietic necrosis virus and / or spring viremia of carp virus, wherein the administration concentration of the prodigiosin related substances is 0.1-0.8 mg / kg (based on the weight of the fish).

[0012] Preferably, the administration concentration of the prodigiosin related substances is 0.2 mg / kg (based on the weight of the fish).

[0013] Preferably, the administration concentration of the prodigiosin related substances is 0.8 mg / kg (based on the weight of the fish).

[0014] Preferably, the prodigiosin related substances include prodigiosin and pharmaceutically acceptable salts of prodigiosin.

[0015] In the present application, prodigiosin related substances include two categories, one is prodigiosin itself (prodigiosin), and the other is its pharmaceutically acceptable salt.

[0016] Preferably, the dosage form of the drug is selected from at least one of injection, soaking agent and oral preparation.

[0017] The present application provides a pharmaceutical composition for resisting infectious hematopoietic necrosis virus and / or spring viremia of carp virus, comprising a safe and effective amount of prodigiosin-related substances, wherein the prodigiosin-related substances include prodigiosin and pharmaceutically acceptable salts of prodigiosin.

[0018] Preferably, the prodigiosin-related substances are administered at a concentration of 0.1-0.8 mg / kg (based on the weight of the fish).

[0019] Preferably, the prodigiosin-related substances are administered at a concentration of 0.2 mg / kg or 0.8 mg / kg (based on the weight of the fish).

[0020] The present application provides a pharmaceutical composition for resisting fish rhabdovirus, comprising a safe and effective amount of active ingredients and a pharmaceutically acceptable carrier, wherein the active ingredients are selected from prodigiosin and pharmaceutically acceptable salts of prodigiosin.

[0021] Preferably, the pharmaceutical composition is administered by injection, immersion or oral route.

[0022] Preferably, the pharmaceutically acceptable carrier is adapted to the administration needs of aquatic animals and is selected from at least one of a buffer, a cosolvent, a stabilizer and a dispersant.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] (1) The antiviral activity of prodigiosin against IHNV and SVCV is first determined, which expands the application field of prodigiosin and provides a new lead compound for the development of drugs against IHNV and SVCV viruses.

[0025] (2) Prodigiosin plays a role by inhibiting viral infection, replication and regulating host immune response, and has a multi-element mechanism of action, which is not easy to induce viral drug resistance and has stable prevention and control effect.

[0026] (3) Prodigiosin is derived from microbial metabolism and has the characteristics of low toxicity, low residue and easy degradation, and will not pollute the water environment, which meets the development needs of green aquaculture.

[0027] (4) The pharmaceutical composition containing prodigiosin has flexible dosage forms, which is suitable for different breeding scenes and fish varieties, and is convenient to operate and easy to promote and apply on a large scale. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the absolute quantitative standard curve of prodigiosin against IHNV; the abscissa represents the logarithmic value of the copy number of IHNV, and the ordinate represents the Ct value;

[0029] Figure 2Absolute standard curve of prodigiosin for quantification of SVCV; the abscissa represents the logarithmic value of the copy number of SVCV, and the ordinate represents the Ct value;

[0030] Figure 3 Inhibition rate of IHNV by rainbow trout treated with different concentrations of prodigiosin (0.2 and 0.8 mg / kg);

[0031] Figure 4 Cumulative survival curve of rainbow trout treated with different concentrations of prodigiosin (0.2 and 0.8 mg / kg);

[0032] Figure 5 Inhibition rate of SVCV by carp treated with different concentrations of prodigiosin (0.2 and 0.8 mg / kg);

[0033] Figure 6 Cumulative survival curve of carp treated with different concentrations of prodigiosin (0.2 and 0.8 mg / kg). DETAILED DESCRIPTION

[0034] Hereinafter, the technical solutions of the present application will be described in conjunction with examples, but the present application is not limited to the following examples. The experimental methods and detection methods described in each example are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0035] As a natural secondary metabolite, prodigiosin has a unique tripyrrole ring structure, which endows it with high antiviral activity, and has rich chemical modification sites, so that its bioavailability can be further improved through structure optimization. Through in vivo and in vitro experiments, it has been verified that prodigiosin can significantly inhibit the replication of IHNV and SVCV, reduce the pathogenicity of viruses to hosts, and improve the survival rate of infected fish, and is safe for farmed fish without obvious toxic side effects.

[0036] Example 1 This example illustrates the determination of the IHNV resistance of prodigiosin

[0037] (1) Test materials

[0038] Virus material: IHNV was isolated and identified by the Aquatic Disease Laboratory of Northwest A&F University and preserved;

[0039] Preparation of test solution: accurately weigh the prodigiosin standard, prepare a 100 mg / mL mother liquor with chromatographic grade DMSO, and then dilute it to 10, 20, 40, 60 and 80 mg / mL working solution. After filtration through a 0.22 μm organic filter, it is stored in a brown chromatographic bottle in the dark;

[0040] Experimental animals: rainbow trout (body weight 6±0.3 g, for IHNV infection test), purchased from Gansu Shihesi Reservoir, after temporary cultivation for 1 week, healthy and disease-free individuals were selected for the experiment.

[0041] (2) Safety evaluation

[0042] Healthy rainbow trout were randomly selected and divided into 8 groups, 15 in each group, and placed in a 16℃ (rainbow trout) circulating water aquaculture tank. The blank control group TM (100 mM Tris-HCl, 10 mM MgCl2, pH=7.5) buffer and drug treatment group (injected with 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mg / kg concentration of prodigiosin), each injection volume was 100 μL. Continuous observation for 72 h, record fish survival and abnormal symptoms.

[0043] The results showed that: in the range of 0.1-0.8 mg / kg concentration, there was no death of rainbow trout, and no abnormal swimming, loss of appetite and other discomfort symptoms; at 1.6 mg / kg concentration, the survival rate of rainbow trout was 84.6%, indicating that the safe concentration of prodigiosin for experimental fish was higher than 0.8 mg / kg, but not higher than 1.6 mg / kg, and the subsequent experiment selected the concentration range of 0.1-0.8 mg / kg.

[0044] (3) Antiviral activity detection

[0045] Group design: rainbow trout was divided into 4 groups, 33 in each group, setting blank control group (injected with buffer, that is, Control group), positive control group (injected with virus liquid, that is, IHNV group), low-dose prodigiosin group (0.2 mg / kg), high-dose prodigiosin group (0.8 mg / kg), each group set 3 parallel. Among them, the buffer of the blank control group was TM (100 mM Tris-HCl, 10 mM MgCl2, pH=7.5) buffer.

[0046] Treatment method: each fish was injected with 100 μL of virus liquid (IHNV dose was 5.2×10 6 copies / fish) in the abdomen, and 100 μL of prodigiosin was injected in the back muscle.

[0047] Sample collection and detection: 48 h, 72 h after injection, 3 were randomly selected from each group, kidney, spleen tissue was collected, nucleic acid was extracted by genomic DNA / RNA extraction kit, virus copy number was detected by RT-qPCR (primer sequence see Table 1), and virus load was calculated by absolute quantitative standard curve. Figure 1

[0048] Table 1 IHNV quantitative detection primer sequence ​

[0049]

[0050] RT-qPCR reaction system: 7.5 μL 2×SYBR Green Master Mix, 0.3 μL each of forward and reverse primers (10 μM), 1 μL template, and ddH2O to a final volume of 15 μL. Reaction program: 95℃ pre-denaturation for 5 min; 95℃ for 10 s, 56℃ for 30 s, 40 cycles.

[0051] The results show that ( Figure 3 ): 72 h after injection, the low- and high-dose groups of styraxin showed inhibition rates of 60.6% and 73.6% against IHNV, respectively, significantly reducing the replication level of IHNV in fish. The control group, however, showed an inhibition rate of 0% against IHNV.

[0052] (4) Survival rate detection

[0053] The fish in the above groups were observed continuously for 14 days, and the number of deaths was recorded daily to calculate the cumulative survival rate.

[0054] The results show that ( Figure 4 ):

[0055] Positive control group: Rainbow trout had a survival rate of 0% at 14 days;

[0056] Low-dose styraxone group: Rainbow trout survival rate was 14.8%;

[0057] High-dose styraxone group: Rainbow trout survival rate was 25.9%;

[0058] The survival rate of the blank control group was 100%.

[0059] Example 2 This example illustrates the determination of the anti-SVCV activity of styraxone.

[0060] (1) Test materials

[0061] Viral material: SVCV was isolated, identified and preserved by the Aquatic Diseases Laboratory of Northwest A&F University;

[0062] Preparation of test solutions: Accurately weigh the styrax rubigin standard, prepare a stock solution of 100 mg / mL with chromatographic grade DMSO, and then dilute it to 10, 20, 40, 60, and 80 mg / mL working solutions. After filtration through a 0.22 μm organic filter, store the solution in a brown chromatographic bottle protected from light.

[0063] Experimental animals: juvenile carp (weight 8.3±0.4 g, used for SVCV infection test), purchased from the Zhuque Road Flower, Bird, Fish and Insect Market in Xi'an, Shaanxi Province. After being temporarily raised for 1 week, healthy and disease-free individuals were selected for the experiment.

[0064] (2) Safety evaluation

[0065] Healthy juvenile carps were randomly selected and divided into 8 groups, 15 in each group, and placed in a circulating water culture tank at 18°C (carp). A blank control group TM (100 mM Tris-HCl, 10 mM MgCl2, pH=7.5) buffer and a drug treatment group (injected with 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mg / kg concentrations of prodigiosin) were set up, with a volume of 100 μL injected per fish. Continuous observation was made for 72 h, and fish survival and abnormal symptoms were recorded.

[0066] The results showed that in the concentration range of 0.1-0.8 mg / kg, no juvenile carps died, and there were no abnormal symptoms such as abnormal swimming and reduced appetite; at a concentration of 1.6 mg / kg, the survival rate of juvenile carps was 79.3%, indicating that the safe concentration limit of prodigiosin for experimental fish was higher than 0.8 mg / kg, but should not be higher than 1.6 mg / kg, and the subsequent experiment selected the concentration range of 0.1-0.8 mg / kg.

[0067] (3) Detection of antiviral activity

[0068] Grouping design: The carps were divided into 4 groups, 33 in each group, and a blank control group (injected with buffer, i.e. Control group), a positive control group (injected with virus liquid, i.e. SVCV group), a low-dose prodigiosin group (0.2 mg / kg), and a high-dose prodigiosin group (0.8 mg / kg) were set up, with 3 parallels in each group. Among them, the buffer of the blank control group was TM (100 mM Tris-HCl, 10 mM MgCl2, pH=7.5) buffer.

[0069] Treatment method: Each carp was injected with 100 μL of virus liquid (SVCV dose was 3.7×10 6 copies / fish) in the abdomen, and 100 μL of prodigiosin was injected into the dorsal muscle.

[0070] Sample collection and detection: 48 h and 72 h after injection, 3 fish were randomly selected from each group, and kidney and spleen tissues were collected for extraction of nucleic acids using a genomic DNA / RNA extraction kit. The virus copy number was detected by RT-qPCR (primer sequences are shown in Table 2), and the viral load was calculated using the absolute quantitative standard curve. Figure 2

[0071] Table 2 SVCV quantitative detection primer sequences

[0072]

[0073] ​RT-qPCR reaction system: 2x SYBR Green Master Mix 7.5 μL, upstream and downstream primers (10 μM) 0.3 μL each, template 1 μL, ddH2O to 15 μL. Reaction program: 95℃ pre-denaturation 5 min; 95℃ 10 s, 56℃ 30 s, 40 cycles.

[0074] The results show that (Fig. 2) Figure 5 ): 48 h after injection, the inhibition rates of prodigiosin low and high dose groups on SVCV were 62.2% and 75.5% respectively, which significantly reduced the replication level of SVCV in fish. The inhibition rate of the Control group on SVCV was 0%.

[0075] (4) Survival rate detection

[0076] The above grouped fish were continuously observed for 14 d, and the number of deaths was recorded daily to calculate the cumulative survival rate. The results show that (Fig. 3) Figure 6

[0077] Positive control group: the survival rate of carp was 11.1% at 14 d.

[0078] Prodigiosin low dose group: the survival rate of carp was 29.6%;

[0079] Prodigiosin high dose group: the survival rate of carp was 37%;

[0080] The survival rate of the blank control group was 100%.

[0081] In summary, prodigiosin can significantly inhibit the replication of IHNV and SVCV in fish, and the survival rate of infected fish is increased in a dose-dependent manner, which has good activity against the two rhabdoviruses.

[0082] The results of the embodiments of the present application fully prove that prodigiosin can effectively prevent and control fish IHNV and SVCV infection, and its low toxicity, greenness and high efficiency make it an ideal candidate preparation for replacing chemical drugs in aquaculture, providing a new technical means for the prevention and control of two important aquatic viral diseases, and laying a foundation for the application of prodigiosin in the prevention and control of other aquatic viral diseases.

[0083] It should be understood that the disclosed application is not limited to the specific methods, schemes and substances described, as these can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiment schemes only and are not intended to limit the scope of the application, which is limited only by the appended claims.​

Claims

1. The use of styrax-related substances as active ingredients in the preparation of a drug for improving the survival rate of fish infected with infectious hematopoietic necrosis virus and / or carp spring viremia virus, characterized in that, The styraxone-related substance is styraxone or a pharmaceutically acceptable salt of styraxone, and the dosage concentration of the styraxone-related substance is 0.1-0.8 mg / kg.

2. The use of lecithin-related substances as active ingredients in the preparation of drugs for reducing viral load in fish infected with infectious hematopoietic necrosis virus and / or carp spring viremia virus, characterized in that, The styraxone-related substance is styraxone or a pharmaceutically acceptable salt of styraxone, and the dosage concentration of the styraxone-related substance is 0.1-0.8 mg / kg.

3. The application according to claim 1 or 2, characterized in that, The dosage concentration of the lecithin-related substance is 0.2 mg / kg.

4. The application according to claim 1 or 2, characterized in that, The dosage concentration of the lecithin-related substance is 0.8 mg / kg.

5. The application according to claim 1 or 2, characterized in that, The dosage form of the drug is selected from at least one of injections, soaking solutions, and oral preparations.

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