Use of sodium salinomycin in combating fish rhabdovirus

Abstract

The application discloses application of sodium salinomycin in resisting fish rhabdovirus. The application claims protection of application of sodium salinomycin or a derivative thereof or a pharmaceutically acceptable salt thereof or a substance taking sodium salinomycin or the derivative thereof or the pharmaceutically acceptable salt thereof as an active ingredient in preparation of a product for resisting fish rhabdovirus. The application proves that sodium salinomycin can inhibit relative expression of viral mRNA of IHNV, VHSV and SVCV in EPC cells, meanwhile, the titer of IHNV, VHSV and SVCV viruses is reduced; and the inhibiting effect of sodium salinomycin on IHNV, VHSV and SVCV presents a dose-dependent relationship. The sodium salinomycin and the salt thereof are expected to become a new type of drug for resisting fish rhabdovirus.

Description

Technical Field

[0001] This invention relates to the field of aquaculture, and in particular to the application of sodium salinomycin in the treatment of fish rhabdoviruses. Background Technology

[0002] Infectious hematopoietic necrosis (IHN), viral hemorrhagic septicemia (VHS), and spring viremia of carp (SVC) are viral infectious diseases that seriously affect the growth rate of global aquaculture production and jeopardize the green and sustainable development of aquaculture. Therefore, the research and application of drugs for the prevention and treatment of these diseases are particularly urgent.

[0003] Infectious hematopoietic necrosis virus (IHNV), belonging to the Rhabdoviridae family, primarily infects juvenile and young fish of salmonids such as rainbow trout, salmon, and Atlantic salmon. Infected fish typically exhibit lethargy, reluctance to move, and avoidance of water currents. Typical characteristics include protruding and darkened eyes, congested skin, abdominal distension, and the presence of opaque or brownish pseudo-tubular mucus-like feces from the anus. The gills are pale, and there is hemorrhage at the base of the fins. IHNV is mainly found in the kidneys, spleen, brain, and digestive tract of infected fish. Due to the high viral load in the digestive tract, the virus is often spread through feces, urine, and external mucous membranes. However, the virus can also be found in the sperm and egg fluids of parent fish. Fish that survive IHNV infection become asymptomatic carriers. Rainbow trout are the most severely affected, with mortality rates reaching up to 100%.

[0004] The pathogen of VHS is Viral Hemorrhagic Septicaemia Virus (VHSV), belonging to the Rhabdoviridae family. It has a wide host range, infecting farmed rainbow trout, turbot, flounder, and various wild freshwater and marine fish. The disease is characterized by hemorrhagic septicemia, with high viral loads in all tissues, including the skin and muscles. The kidneys, heart, and spleen have the highest viral loads and are the target organs of the virus, resulting in an extremely high mortality rate.

[0005] SVC is caused by Spring Viraemia of Carp Virus (SVCV), belonging to the Rhabdoviridae family. It can infect carp, koi, bighead carp, grass carp, silver carp, crucian carp, catfish, and other cyprinid fish, with carp and koi being the most susceptible, causing large-scale disease and mortality in these species. Fish of all ages can be infected, but younger fish are more susceptible. Infected fish often congregate at the pond inlet, exhibiting difficulty breathing, decreased vitality, and mortality. Diseased fish become sluggish in response to external stimuli, their swimming speed gradually decreases, and in the later stages, they almost stop swimming and lose their balance. Some die at the bottom of the pond, while others swim aimlessly or drift aimlessly at the pond edge. Diseased fish exhibit a darkening of body color, protruding eyes, swollen abdomen, and a red and swollen anus. The body surface (skin, fins, mouth) and gills are congested. The gills and body surface show congestion, and bleeding may occur in the eyes. The anus is inflamed, edematous, and protruding. Infected fish show slight or significant darkening of body color, pale gills, and sometimes skeletal muscle tremors. When the fish are removed from the water, abdominal fluid can be seen flowing out of the anus automatically. SVC is an acute, hemorrhagic viral infectious disease with a mortality rate as high as 90% in juvenile fish.

[0006] All three diseases mentioned above are currently listed as reportable diseases by the World Organisation for Animal Health (OIE), while IHN and SVC are classified as Class II animal diseases by my country's Ministry of Agriculture and Rural Affairs. Outbreaks of these diseases severely restrict the healthy and sustainable development of my country's fish farming industry.

[0007] Salinomycin, also known as salinomycin, was first discovered by Miyazaki et al. in 1968 in cultures of *Streptomyces albopictus*. It is a polyether ionotropic antibiotic. Salinomycin exhibits strong inhibitory and bactericidal effects against most Gram-positive bacteria and various coccidia, and is therefore mainly used to prevent and treat coccidiosis in poultry. It can also be used as a feed additive to improve feed utilization and promote growth in pigs. A research article published in *Cell* in 2009 reported that salinomycin can specifically kill breast cancer stem cells. Furthermore, there are reports that the sodium salt of salinomycin (C... 42 H 70 O 11 Na,SAL) has similar properties and anti-tumor stem cell effects to salinomycin, but is relatively cheaper and more cost-effective, so it can be used as a substitute for salinomycin in research. Summary of the Invention

[0008] The purpose of this invention is to provide the application of sodium salinomycin in the treatment of fish rhabdomyovirus.

[0009] In a first aspect, the present invention claims protection for the use of salinomycin or its derivatives or pharmaceutically acceptable salts or substances having salinomycin or its derivatives or pharmaceutically acceptable salts as active ingredients in the preparation of products for use against fish rhabdoviruses.

[0010] The fish bullet virus may be infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, and / or carp spring viremia virus.

[0011] Secondly, the present invention claims protection for the use of salinomycin or its derivatives or pharmaceutically acceptable salts or substances having salinomycin or its derivatives or pharmaceutically acceptable salts as active ingredients in the preparation of products for the prevention and / or treatment of diseases caused by fish rhabdovirus infection.

[0012] The fish rhabdovirus may be infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, and / or carp spring viremia virus. Correspondingly, the disease may be infectious hematopoietic necrosis disease, viral hemorrhagic septicemia, and / or carp spring viremia.

[0013] Thirdly, the present invention claims protection for the use of salinomycin or its derivatives or pharmaceutically acceptable salts, or substances having salinomycin or its derivatives or pharmaceutically acceptable salts as active ingredients, in the preparation of products for inhibiting the expression of fish rhabdovirus RNA (e.g., mRNA).

[0014] The fish bullet virus may be infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, and / or carp spring viremia virus.

[0015] Fourthly, the present invention claims protection for the use of salinomycin or its derivatives or pharmaceutically acceptable salts, or substances having salinomycin or its derivatives or pharmaceutically acceptable salts as active ingredients, in the preparation of products for inhibiting the expression of fish rhabdovirus RNA (e.g., mRNA) at the cellular level.

[0016] The fish bullet virus may be infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, and / or carp spring viremia virus.

[0017] Fifthly, the present invention claims protection for the use of salinomycin or its derivatives or pharmaceutically acceptable salts or substances having salinomycin or its derivatives or pharmaceutically acceptable salts as active ingredients in the preparation of products for reducing fish rhabdovirus titers.

[0018] The fish bullet virus may be infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, and / or carp spring viremia virus.

[0019] Sixthly, the present invention claims protection for the use of salinomycin or its derivatives or pharmaceutically acceptable salts or substances having salinomycin or its derivatives or pharmaceutically acceptable salts as active ingredients in the preparation of products for reducing fish rhabdovirus titers at the cellular level.

[0020] The fish bullet virus may be infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, and / or carp spring viremia virus.

[0021] In a specific embodiment of the present invention, the fish cells are bighead carp epithelial cells (EPC cells).

[0022] In a specific embodiment of the present invention, the pharmaceutically acceptable salt is salinomycin sodium (C 42 H 70 O 11 Na, SAL).

[0023] Experiments have shown that salinomycin sodium (C 42 H 70 O 11 Na, SAL, can inhibit the relative expression of IHNV, VHSV, and SVCV viral mRNA in EPC cells, while reducing the viral titers of IHNV, VHSV, and SVCV; furthermore, the inhibitory effect of SAL on IHNV, VHSV, and SVCV shows a dose-dependent relationship. These results suggest that salinomycin and its salts hold promise as novel drugs against fish rhabdoviruses. Attached Figure Description

[0024] Figure 1 The effect of different concentrations of salinomycin sodium on cell viability. In the figure, different lowercase letters indicate significant differences (P<0.05).

[0025] Figure 2 Effects of different concentrations of salinomycin sodium on the relative expression levels of viral mRNA after treating cells with IHNV, VHSV, and SVCV. In the figure, * indicates a significant difference compared to the control group (P<0.05).

[0026] Figure 3 The effect of different concentrations of salinomycin sodium on viral titers after treating cells with IHNV, VHSV, and SVCV. In the figure, * indicates a significant difference compared to the control group (P<0.05). Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] Example 1: Application of salinomycin sodium in the treatment of fish rhabdomyovirus

[0030] I. Experimental Materials and Methods

[0031] 1. Cells and viruses

[0032] Epithelial cells of the bighead carp—Epithelioma papulosum cyprini (EPC) cells (CRL-2872, ATCC); Infectious hematopoietic necrosis virus Sn1203 strain (IHNV-Sn1203) (Genbank accession number: KC660147.1); Viral hemorrhagic septicemia virus strain 1872 (VHSV-1872) (VR-1872, ATCC); Cypriniform viremia virus Shlj1 strain (SVCV-Shlj1) were preserved in our laboratory (Reference: "Ji Feng. Prediction of spatial structure of glycoprotein and B cell antigenic epitopes of Cypriniform viremia virus Shlj1 strain [J]. Journal of Dalian Ocean University, 2017, 32(4):440-446.").

[0033] 2. Reagents

[0034] Cell culture medium MEM (C11095500BT), trypsin (C25200072), fetal bovine serum (FB25015), and penicillin-streptomycin solution (C15140122) were purchased from Gibco; One Step SYBR PrimeScript PLUS PT-PCR kit (RR096A) was purchased from TaKaRa; Trizol reagent (10296028) was purchased from Invitrogen; CellCounting Kit-8 (CCK-8) kit (B34304) was purchased from Bimake; and salinomycin sodium (HY-17439) was purchased from MCE, hereinafter referred to as SAL.

[0035] 3. SAL cytotoxicity detection

[0036] EPC cells in good growth condition were collected, digested with trypsin, and then seeded into 96-well cell culture plates with 6 × 10⁶ cells per well in MEM cell culture medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. 4After incubating the 96-well plates at 25°C for 24 hours, fresh medium containing SAL was added. Five concentration gradients of SAL were set: 25 μM, 10 μM, 5 μM, 1 μM, and 0.5 μM. After 7 days of incubation under 0.5% CO2 conditions, SAL cytotoxicity was assessed according to the CCK-8 assay instructions. A negative control group (Mock) without SAL was also included.

[0037] 4. Study on the effects of different concentrations of SAL on fish rhabdoviruses (IHNV, VHSV, SVCV)

[0038] (1) Effects of different intracellular concentrations of SAL on the relative expression levels of fish rhabdovirus (IHNV, VHSV, SVCV) mRNA

[0039] EPC cells in good growth condition were harvested, digested with trypsin, and then seeded into 6-well cell culture plates in MEM cell culture medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, with 2 × 10⁶ cells per well. 6 Six-well plates were incubated at 25°C for 24 hours, after which the culture medium was discarded. IHNV-Sn1203 virus strain (MOI = 0.1), VHSV-1872 virus strain (MOI = 0.1), and SVCV-Shlj1 virus strain (MOI = 0.1) were added, respectively. After 1 hour, the culture medium was replaced with cell culture medium containing 2% fetal bovine serum (FBS) with SAL at final concentrations of 0.5 μM, 1 μM, 5 μM, and 10 μM. The plates were then incubated at 15°C and 0.5% CO2 for 48 hours. After this, the cell culture medium was discarded, and viral RNA was extracted according to the TRIzol reagent instructions to detect the effect of SAL on the relative expression level of fish rhabdovirus mRNA. A negative control group (Mock) without SAL was also included.

[0040] Using extracted viral RNA as a template, the mRNA expression of IHNV, VHSV, and SVCV in EPC cells was detected according to the One Step SYBR PrimeScript PLUS PT-PCR kit instructions. The primers used are shown in Table 1.

[0041] Table 1. Primers for Rhabdovirus Assay

[0042]

[0043] (2) The effect of different concentrations of SAL on the titers (TCID) of fish rhabdoviruses (IHNV, VHSV, SVCV) 50 The impact of

[0044] EPC cells in good growth condition were harvested, digested with trypsin, and then seeded into 6-well cell culture plates in MEM cell culture medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, with 2 × 10⁶ cells per well. 6 Six-well plates were incubated at 25°C for 24 hours, then the culture medium was discarded. IHNV-Sn1203 virus strain (MOI = 0.1), VHSV-1872 virus strain (MOI = 0.1), and SVCV-Shlj1 virus strain (MOI = 0.1) were added, respectively. After 1 hour, the medium was replaced with cell culture medium containing 2% fetal bovine serum (FBS) with SAL concentrations of 0.5 μM, 1 μM, 5 μM, and 10 μM. The plates were then incubated at 15°C and 0.5% CO2 for 48 hours. The cell culture supernatant was collected for virus titer (TCID). 50 The test was performed. A negative control group (Mock) without SAL was also included.

[0045] Take EPC cells in good growth condition, digest them, and then feed them at a rate of 6 × 10⁻⁶. 4 Inoculate 10 cells / well into a 96-well plate, incubate at 25°C for 24 hours, and then inoculate 10 cells / well. 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 Culture supernatant was prepared at each dilution, with 100 μL per well for every 8 wells. A blank control group was also included. Cultures were performed at 15°C and 0.5% CO2 for 7 days. TCID was calculated using the Reed-Muench method. 50 .

[0046] II. Results and Analysis

[0047] 1. Detection results of SAL's cytotoxicity

[0048] To determine the toxicity of SAL to EPC cells, this invention used five concentration gradients of SAL: 25 μM, 10 μM, 5 μM, 1 μM, and 0.5 μM, and cell proliferation was assessed 7 days after treatment. The results showed ( Figure 1 SAL at 25 μM and 10 μM significantly inhibited the proliferation of EPC cells; at 5 μM, 1 μM and 0.5 μM, it had no significant effect on the proliferation of EPC cells.

[0049] 2. Effects of different intracellular concentrations of SAL on the relative expression levels of fish rhabdovirus (IHNV, VHSV, SVCV) mRNA

[0050] To investigate the effect of SAL on the relative expression levels of fish rhabdoviruses (IHNV, VHSV, SVCV) mRNA in cells, this invention infected EPC cells with IHNV-Sn1203, VHSV-1872, and SVCV-Shlj1 virus strains at an MOI of 0.1. One hour after infection, cells were treated with SAL (final concentrations of 10 μM, 5 μM, 1 μM, and 0.5 μM), and viral RNA was extracted after 48 hours for mRNA expression level detection. The results showed that ( Figure 2 Treatment with 10 μM, 5 μM, and 1 μM SAL significantly inhibited the mRNA expression levels of IHNV, VHSV, and SVCV in cells. Compared with the control group (Mock), the mRNA expression level of IHNV in cells decreased by approximately 333, 33, and 2.6 times, respectively. In the study of VHSV, the mRNA expression level of VHSV in cells decreased by approximately 1000, 9.7, and 1.9 times, respectively, compared with the control group (Mock). In the study of SVCV, the mRNA expression level of SVCV in cells decreased by approximately 500, 22, and 2.0 times, respectively, compared with the control group (Mock). However, treatment with 0.5 μM SAL did not significantly inhibit the mRNA expression levels of VHSV and SVCV in cells, but only the mRNA expression level of IHNV decreased significantly (P<0.05). The above results indicate that SAL can significantly inhibit the relative expression of fish rhabdoviruses (IHNV, VHSV, SVCV) mRNA in cells, and this inhibition is dose-dependent within a certain dose range.

[0051] 3. The titer (TCID) of different concentrations of SAL against fish rhabdoviruses (IHNV, VHSV, SVCV) 50 The impact of

[0052] To investigate the effect of SAL on the titers of fish rhabdoviruses (IHNV, VHSV, SVCV), this invention infected EPC cells with IHNV-Sn1203, VHSV-1872, and SVCV-Shlj1 virus strains at an MOI of 0.1. One hour after infection, cells were treated with SAL (final concentrations of 10 μM, 5 μM, 1 μM, and 0.5 μM), and the cell culture supernatant was collected after 48 hours for virus titer determination (TCID). 50 ) detection. The results show that ( Figure 3 The IHNV virus titer is 10. 7 TCID 50 / mL, the IHNV virus titer after treatment with 10μM SAL was 10. 4 TCID 50The IHNV virus titer was 10 after treatment with 5 μM SAL at a concentration of 10 / mL. 5 TCID 50 The IHNV virus titer was 10 after treatment with 1 μM SAL at a concentration of 1 mL / mL. 6 TCID 50 The IHNV virus titer was 2 × 10⁶ / mL after treatment with 0.5 μM SAL. 6 TCID 50 / mL; VHSV virus titer is 10 8 TCID 50 / mL, VHSV viral titer after 10μM SAL treatment was 10. 5.2 TCID 50 The VHSV virus titer was 10 after treatment with 5 μM SAL at a concentration of 10 / mL. 6 TCID 50 The VHSV virus titer was 10 after treatment with 1 μM SAL at a concentration of 1 mL / mL. 7 TCID 50 The VHSV viral titer was 10 after treatment with 0.5 μM SAL at a concentration of 0.5 μmL. 8 TCID 50 / mL; SVCV virus titer is 10 7 TCID 50 / mL, the SVCV virus titer after treatment with 10μM SAL was 10. 4.2 TCID 50 The SVCV virus titer was 10 after treatment with 5 μM SAL at a concentration of 10 / mL. 6 TCID 50 The SVCV virus titer was 10 after treatment with 1 μM SAL at a concentration of 1 mL / mL. 7 TCID 50 The SVCV virus titer was 10 after treatment with 0.5 μM SAL at a concentration of 1 mL / mL. 7 TCID 50 / mL. The above results indicate that SAL has a significant inhibitory effect on the viral titer of fish rhabdoviruses (IHNV, VHSV, SVCV), and exhibits a dose-dependent effect within a certain range.

[0053] The present invention has been described in detail above. Those skilled in the art will recognize that 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. While specific embodiments have been provided, 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.

Claims

1. The use of salinomycin or a pharmaceutically acceptable salt thereof, or a substance having salinomycin or a pharmaceutically acceptable salt thereof as an active ingredient, in the preparation of products for use against fish rhabdoviruses; The fish rhabdoviruses mentioned are infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, and / or carp spring viremia virus.

2. The use of salinomycin or a pharmaceutically acceptable salt thereof, or a substance having salinomycin or a pharmaceutically acceptable salt thereof as an active ingredient, in the preparation of products for the prevention and / or treatment of diseases caused by fish rhabdovirus infection; The fish rhabdoviruses mentioned are infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus and / or carp spring viremia virus; The diseases mentioned are infectious hematopoietic necrosis, viral hemorrhagic septicemia, and / or carp viremia.

3. The use of salinomycin or a pharmaceutically acceptable salt thereof, or a substance having salinomycin or a pharmaceutically acceptable salt thereof as an active ingredient, in the preparation of a product for inhibiting the expression of fish rhabdovirus RNA; The fish rhabdoviruses mentioned are infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, and / or carp spring viremia virus.

4. The use of salinomycin or a pharmaceutically acceptable salt thereof, or a substance having salinomycin or a pharmaceutically acceptable salt thereof as an active ingredient, in the preparation of a product for inhibiting the expression of fish rhabdovirus RNA at the cellular level; The fish rhabdoviruses mentioned are infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, and / or carp spring viremia virus.

5. The application according to claim 4, characterized in that: The cells in question are epithelial cells of the bighead carp.

6. The use of salinomycin or a pharmaceutically acceptable salt thereof, or a substance having salinomycin or a pharmaceutically acceptable salt thereof as an active ingredient, in the preparation of products for reducing fish rhabdovirus titers; The fish rhabdoviruses mentioned are infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, and / or carp spring viremia virus.

7. The use of salinomycin or a pharmaceutically acceptable salt thereof, or a substance having salinomycin or a pharmaceutically acceptable salt thereof as an active ingredient, in the preparation of a product for reducing fish rhabdovirus titers at the cellular level; The fish rhabdoviruses mentioned are infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, and / or carp spring viremia virus.

8. The application according to claim 7, characterized in that: The cells in question are epithelial cells of the bighead carp.

9. The application according to any one of claims 1-8, characterized in that: The pharmaceutically acceptable salt is salinomycin sodium.

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