Use of isotretinoin in the preparation of a medicine for resisting African swine fever virus

Abstract

The application provides an application of isotretinoin in preparation of an anti-African swine fever virus drug, and belongs to the technical field of veterinary medicine. It is found that isotretinoin can effectively interfere with the replication process of ASFV by inhibiting the binding of F1055L protein to virus DNA. Experimental data show that isotretinoin can effectively inhibit the replication of ASFV while having low toxicity to host cells. The anti-ASFV method provided by the application effectively inhibits the replication of ASFV through isotretinoin, has high clinical application potential, and provides a new drug strategy for the prevention and treatment of African swine fever.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of veterinary medicine, in particular to a treatment method for African swine fever virus (ASFV). BACKGROUND

[0002] African swine fever (ASF) is a highly contagious and lethal disease caused by African swine fever virus (ASFV), mainly affecting domestic pigs (Sus scrofa domesticus) and wild boars (Sus scrofa). ASFV is a large double-stranded DNA virus belonging to the Asfarviridae family. The clinical manifestations of ASF vary greatly depending on the virulence of the infecting strain. Acute cases usually present with high fever, lethargy, and loss of appetite, often accompanied by typical skin changes such as redness or cyanosis on the ears, nose, and limbs. Severe infections can also be accompanied by digestive system symptoms (such as diarrhea, vomiting), rapid breathing, and nervous system manifestations (such as ataxia or convulsions). The infection of ASFV has a very high mortality rate, especially in areas that rely on pork production.

[0003] ASFV interferes with the cGAS-STING signaling pathway by encoding a series of proteins, inhibiting the host's innate immune response, and thus inhibiting the production of type I interferon (IFN). The only currently available vaccine, ASFV-G-ΔI177L, was developed by deleting the I177L gene, but this vaccine comes from the Georgia strain with high pathogenicity, and has not yet been approved for widespread use. The development of antiviral drugs is an alternative method for the prevention and control of African swine fever.

[0004] Currently, no effective antiviral drug for ASFV has been successfully developed, although some studies have explored vaccine candidates and other antiviral strategies for ASFV. Due to the variability and immune escape mechanisms of ASFV, the effects of existing vaccines and drugs are still unstable. The complexity of ASFV makes it an important research topic. ASFV is one of the largest and most complex animal viruses known, with a virus particle diameter of about 200 to 300 nanometers, much larger than other animal viruses such as coronaviruses (80-120 nanometers). Its unique structure and genomic complexity make it highly stable and persistent, capable of surviving in a variety of environmental conditions. The structure of ASFV presents a multi-level geometric symmetry, including the inner core, lipid membrane, capsid layer, and outer membrane. The genome of ASFV is 170 to 190 kilobase pairs (kbp) in length, encoding 150 to 200 proteins, demonstrating its remarkable biological complexity. These characteristics enable ASFV to interact complexly with host cells, making it an important topic in veterinary virology research.

[0005] The life cycle of ASFV is a complex multi-stage process, mainly occurring in the host's macrophages. ASFV uses various mechanisms of host cells for replication and transmission, involving the interaction of multiple virus-encoded proteins, and some key proteins, such as F1055L protein, play an important role in the replication process of the viral genome. Although there is some understanding of the replication mechanism of ASFV, specific inhibition strategies for the virus, especially by interfering with the function of key proteins in the replication process of the virus, are still important problems in the current research and development of anti-ASFV drugs. SUMMARY

[0006] The treatment of ASFV is in urgent need of breakthrough, especially the antiviral drugs that can effectively inhibit the replication of ASFV. The purpose of the present application is to provide the application of retinoic acid in the preparation of anti-African swine fever virus drugs, based on the potential of retinoic acid in antiviral, it is proposed to effectively interfere with the replication of ASFV by inhibiting the interaction of ASFV F1055L protein and DNA, thereby providing a new treatment idea for the prevention and treatment of African swine fever.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] In the first aspect, the present application provides the application of retinoic acid in the preparation of anti-African swine fever virus drugs. Further, the anti-African swine fever virus refers to that retinoic acid inhibits the binding of F1055L and viral DNA, and then interferes with the replication process of ASFV, thereby achieving antiviral. Still further, the anti-African swine fever virus drug is formulated into a dosage form suitable for administering retinoic acid at a dose of 10 μM to 200 μM.

[0009] In the second aspect, the present application provides an anti-African swine fever virus drug, and the effective component thereof comprises retinoic acid.

[0010] As a further optimization of the above-mentioned anti-African swine fever virus drug, the anti-African swine fever virus drug is formulated into a dosage form suitable for administering retinoic acid at a dose of 10 μM to 200 μM. Still further, the anti-African swine fever virus drug is formulated into a dosage form suitable for administering retinoic acid at a dose of 150 μM to 200 μM.

[0011] In the third aspect, the present application provides a method for inhibiting the proliferation of ASFV in cells in vitro, which is to co-incubate the cells in vitro with retinoic acid. Further, the cells are pig lung macrophages. The concentration of the retinoic acid is 10 μM-200 μM.

[0012] Compared with the prior art, the present application has the following beneficial effects:

[0013] 1. New antiviral mechanism: Current treatments for ASFV mostly rely on vaccine development, but existing vaccines are not stable due to the variability and immune escape characteristics of ASFV, and have limited application. The present application directly interferes with the replication process of ASFV by using isotretinoin to inhibit the binding of ASFV F1055L protein to DNA, thereby effectively preventing the replication of viral genomes. This direct targeting of viral replication mechanism breaks through the limitations of existing vaccines and indirect immune strategies, providing a new treatment option.

[0014] 2. Use of reporter virus to detect antiviral effect: The reporter virus integrates the easily detectable mCherry into the African swine fever virus genome, constructing a "labeled" virus, which greatly optimizes the screening process of antiviral drugs.

[0015] 3. Low cytotoxicity: Experimental data show that isotretinoin effectively inhibits ASFV replication while having low toxicity to host cells. Compared with existing antiviral drugs, this has a great advantage because many traditional drugs often have strong toxic side effects on host cells while effectively inhibiting viruses. The antiviral method provided by the present application can effectively reduce damage to normal cells, improving drug safety and applicability.

[0016] 4. Rapid response to ASF outbreak: The technical solution of the present application provides a rapid antiviral treatment method that can quickly control the spread of the virus without effective vaccines. Unlike existing vaccines with long development cycles and uncertain immune effects, the antiviral drug of the present application can serve as an emergency response measure and have a rapid effect.

[0017] 5. Broad application prospects: The treatment method of the present application not only has antiviral effects against ASFV, but also has good adaptability. Since isotretinoin is a drug that has been widely used for skin disease treatment and has a mature safety record and use history, its application in anti-ASFV has high clinical conversion potential. In addition, the treatment method of the present application has strong operability and can provide effective control means for different regions and different types of ASF.

[0018] 6. Supplement the shortcomings of existing treatment methods: Currently, the treatment methods for ASFV are relatively single, mainly relying on vaccination. Due to the slow development of vaccines and the influence of ASFV variation, the present application provides a new antiviral drug to effectively supplement the shortcomings of existing treatment methods, providing a new choice for the prevention and treatment of African swine fever. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1Figure 6 is a graph showing the results of the determination of the half-maximal inhibitory concentrations (IC50) of retinoids on ASFV;

[0020] Figure 2 Figure 6 is a graph showing the results of the determination of the half-maximal inhibitory concentrations (IC50) of retinoids on ASFV;

[0021] Figure 3 Figure 7 is a graph showing the results of the determination of the titer of the progeny ASFV virus after treatment with retinoids;

[0022] Figure 4 Figure 8 is a graph showing the results of the determination of the cytotoxicity of retinoids in PAMs cells;

[0023] Figure 5 Figure 9 is a fluorescence microscope image showing the infection of PAM cells (multiplicity of infection of 0.1) with the reporter virus ASFV-F055L-Flag-Strep 2 hours after treatment with retinoids (10 - 200 μM) for 24 hours. The scale bar is 200 μm. DETAILED DESCRIPTION

[0024] The following examples are intended to further illustrate the present application but not to limit the scope of the present application. Any changes, modifications, substitutions, combinations, simplifications within the scope of the claims made by those skilled in the art should be equivalent replacement methods, and some non-essential improvements and adjustments made to the present application without departing from the spirit and scope of the present application still belong to the protection scope of the present application.

[0025] In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the test materials used are purchased from conventional biochemical reagent companies unless otherwise specified. Retinoids are purchased from MedChemExpress (MCE) company.

[0026] The African swine fever virus strain is a genotype II African swine fever virus strain ASFV-F1055L-Flag-Strep (mCherry) recombinant strain constructed by the research team and stored in the African swine fever prevention and control research center of South China Agricultural University.

[0027] Currently, the prevention and control measures of ASFV mainly rely on the development of vaccines, but due to the variability of ASFV and the problem of vaccine immune escape, the existing vaccines have not been widely used. Therefore, it is urgent to develop new antiviral treatment methods. In addition, the existing antiviral treatment methods are mainly limited to laboratory research stage, and lack effective clinical application.

[0028] The present application aims to provide a new anti-ASFV treatment method by inhibiting the interaction between ASFV F1055L protein and DNA with isotretinoin, thereby inhibiting the replication of the virus. F1055L, as a key factor in the replication process of ASFV, its binding to DNA is crucial for the replication of the viral genome. Therefore, inhibiting the function of F1055L protein, especially its binding to DNA, can effectively prevent the replication process of ASFV, thereby providing a new treatment method for the prevention and control of ASF. The technical problems solved by the present application include:

[0029] 1. Lack of effective ASFV antiviral drugs: Currently, there is no specific antiviral drug for ASFV, and the existing treatment methods mostly rely on vaccines, and the effect of the vaccine is unstable. Therefore, it is urgent to develop new drug treatment programs to achieve effective control of ASFV.

[0030] 2. Limited efficacy of existing drugs: Although some chemical molecules show certain activity in antiviral research, due to the lack of inhibitors specific to the replication mechanism of ASFV, the clinical application effect of existing drugs is limited.

[0031] 3. ASFV replication mechanism is not completely clear: The replication mechanism of ASFV is complex, involving the synergistic action of multiple viral encoded proteins. Although some studies have revealed some replication factors of ASFV, the specific function of F1055L protein in viral replication is still not fully understood, and how to effectively intervene its action is still a technical problem.

[0032] The technical solution of the present application reveals that isotretinoin can inhibit the binding of F1055L protein to DNA, thereby effectively interfering with the replication of ASFV, providing a new treatment idea and providing a new target for the research and development of anti-ASFV drugs.

[0033] The core technical solution of the present application is based on the interference effect of isotretinoin on the replication process of ASFV. The replication of ASFV depends on a variety of viral encoded enzymes and auxiliary factors in host cells, among which F1055L protein is a key factor in the replication process of ASFV genome. F1055L belongs to ATPase protein and has DNA binding activity, which plays a crucial role in the replication process of ASFV genome. Existing studies have shown that F1055L protein promotes the replication of viral genome and maintains the life cycle of virus by binding to viral DNA. Therefore, the binding of F1055L protein to DNA is a key step in the replication process of ASFV.

[0034] The present application finds that isotretinoin can effectively interfere with the replication process of ASFV by inhibiting the binding of F1055L protein to viral DNA. Isotretinoin, as a drug widely used in the treatment of skin diseases, has attracted some attention in recent years for its antiviral activity. Studies have shown that isotretinoin can inhibit viral gene expression and replication by regulating various signaling pathways in host cells. However, the inhibitory effect of isotretinoin on ASFV, especially its impact on key steps in the ASFV life cycle, has not been thoroughly studied. Therefore, the present application provides a new idea for the development of anti-ASFV drugs by innovatively discovering the mechanism of isotretinoin inhibiting F1055L protein-DNA interaction.

[0035] In the present application, a recombinant reporter virus ASFV-F1055L-Flag-Strep (mCherry) is used to detect the inhibitory effect of isotretinoin on African swine fever virus.

[0036] In the technical solution of the present application, isotretinoin can significantly inhibit the replication of ASFV at a relatively low concentration without causing significant toxicity to host cells. This characteristic makes isotretinoin an ideal candidate drug against ASFV. Experimental data show that isotretinoin can effectively inhibit the proliferation of ASFV in porcine alveolar macrophages (PAMs) and its antiviral effect is significantly better than that of some existing drugs. In addition, the inhibitory effect of isotretinoin on F1055L protein further reveals its potential as an anti-ASFV drug. Example 1:

[0037] The present application provides a method for inhibiting the replication of African swine fever virus (ASFV) using isotretinoin. First, in order to determine the safe concentration range of isotretinoin for PAMs, cells were seeded in a 96-well plate at a density of 1x10 5 After 4 hours of recovery, isotretinoin was diluted in cell culture medium at a concentration gradient to generate nine different concentrations (10 μM, 25 μM, 50 μM, 100 μM, 150 μM, and 200 μM). 100 μL of each concentration was added to the cells. The control group received 100 μL of complete medium containing 0.05% DMSO, while the blank control group received 100 μL of cell-free complete medium. Each concentration was tested in six replicate wells.

[0038] After incubation at 37°C and 5% CO2 for 24 hours, 10 μL of CCK-8 reagent was added to each well, and then incubation was continued for 1 hour. The optical density (OD) of each well was measured using a microplate reader at a reference wavelength of 450 nm. The experimental results are as follows:Figure 4 The results showed that the cytotoxicity of isotretinoin on PAMs cells was low in the concentration range of 10 μM to 200 μM, indicating that it was safe and effective in this concentration range. Example 2:

[0039] Antiviral activity of isotretinoin

[0040] After the PAMs were recovered, they were seeded in a 12-well plate at a density of 5x10 5 cells per well. After 12 hours, the cells were infected with the reporter virus ASFV-F1055L-Flag-Strep at a MOI of 0.1 for 2 hours to allow the virus to enter the cells. Subsequently, the virus inoculum was removed and replaced with RPMI-1640 medium supplemented with 10% serum replacement, with the addition of different concentrations of isotretinoin (10 μM, 25 μM, 50 μM, 100 μM, 150 μM and 200 μM). After 24 hours of incubation, the percentage of fluorescent cells was evaluated under a fluorescence microscope. Subsequently, the supernatant and cells were collected for further analysis. The titer of the progeny virus was determined using the endpoint dilution method (TCID50), and the half maximal inhibitory concentration (IC50) value of isotretinoin on ASFV infection was determined by RT-qPCR. The results showed that the spread of ASFV-F1055L-Flag-Strep was significantly reduced in isotretinoin-treated PAMs ( Figure 5 ). Isotretinoin significantly inhibited the transcription levels of the CP204L gene and the B646L gene at 24 hours post-infection (Figure 1). The half maximal inhibitory concentration (IC50) was 24.47 μM using the assay based on the CP204L gene and 23.66 μM when evaluated using the assay based on the B646L gene (Figure 2). Isotretinoin significantly reduced the titer of the reporter virus (ASFV-F1055L-Flag-Strep) at concentrations of 150 μM and 200 μM (Figure 3).

[0041] In summary, the anti-ASFV treatment method provided by the present application effectively inhibits the replication of ASFV through isotretinoin, has high clinical application potential, and provides a new drug strategy for the prevention and treatment of African swine fever.

Claims

1. Application of isotretinoin in the preparation of drugs against African swine fever virus.

2. The application according to claim 1, characterized in that: The anti-African swine fever virus refers to the antiviral effect achieved by isotretinoin inhibiting the binding of F1055L to viral DNA, thereby interfering with the replication process of ASFV.

3. The application according to claim 1, characterized in that: The anti-African swine fever virus drug is formulated into a dosage form suitable for administering isotretinoin at doses from 10 μM to 200 μM.

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