Application of GW4064 in the preparation of anti-polysporic reproductive tract virus drugs

Abstract

The present invention relates to the field of biomedicine technology, and in particular to the use of GW4064 in the preparation of anti-polysporic reproductive tract virus (PRRSV) drugs. The present invention discloses a new use of the farnesoid X receptor agonist GW4064 in the preparation of anti-polysporic reproductive tract virus (PRRSV) drugs. GW4064 has strong antiviral activity against PRRSV, and in particular shows a significant inhibitory effect on highly pathogenic strains (HP-PRRSV). GW4064 interferes with the viral life cycle by targeting the NSP3 protein of PRRSV, thereby inhibiting the replication and spread of the virus. GW4064 has a significant antiviral effect on various variants of PRRSV, showing a broad-spectrum antiviral effect. At the same time, GW4064 has a small molecular weight and is easy to enter cells, and can effectively exert its effect without the need for carrier delivery. The invention also provides a pharmaceutical composition with GW4064 as the main active ingredient, which provides a theoretical basis and practical basis for the development of PRRSV antiviral drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to the application of GW4064 in the preparation of anti-polysporic reproductive tract virus (PRRSV) drugs. Background Art

[0002] my country is the world's largest pig producer and pork consumer, accounting for over half of both global production and consumption. Pork accounts for 60% of my country's meat consumption. Disease is a major factor impacting the healthy development of the pig industry. Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ear disease, first appeared in the United States in 1987 and was subsequently reported in Europe. In 1996, Guo Baoqing et al. first isolated PRRSV in my country, naming the isolated strain CH-1a and confirming its presence in my country. Currently, PRRS is one of the most threatening infectious diseases to the healthy development of the global pig industry. PRRSV primarily causes reproductive failure in sows, respiratory symptoms in pigs of various ages, especially piglets, and secondary infections such as Haemophilus parasuis and Streptococcus, resulting in significant economic losses to the global pig industry. In particular, in 2006, outbreaks of porcine high fever syndrome (PHFS) occurred in most provinces of my country and Vietnam, causing devastating losses. According to incomplete statistics, PRRS causes annual economic losses of up to $650 million in the United States, €1.5 billion in Europe, and 40 billion yuan in China. Currently, the pathogenic mechanism of the virus and its mechanism of inducing host immune responses remain unclear.

[0003] The biggest difference between PRRSV and other viruses is that it primarily infects immune cells—swine alveolar macrophages—and disrupts the immune system, similar to HIV. The virus exhibits the following characteristics: ① Immunosuppression, interfering with other vaccine immunizations; ② High variability, prone to mutation and recombination, producing more pathogenic strains; ③ No / low neutralizing antibodies, as the GP5 protein, the primary antigenic epitope that decoys neutralizing antibodies, is not fully exposed on the surface of the virus particles; ④ Inflammatory storm, excessive induction of cytokine expression; ⑤ Antibody-dependent enhancement (ADE), where low / non-neutralizing antibodies promote viral invasion; ⑥ Persistent infection, with prolonged viremia and intermittent detoxification; ⑦ Secondary infection, including secondary infections with Haemophilus parasuis and Streptococci.

[0004] Based on the above characteristics of PRRSV, combined with years of practical experience, the commercial vaccines currently developed, including attenuated and inactivated vaccines, are not ideal and cannot prevent the occurrence and spread of the disease. These vaccines also have the following problems: slow production of neutralizing antibodies, poor cross-protection against heterologous strains, prolonged vaccine virus viremia, and recombination with field strains to produce recombinant viruses with stronger pathogenicity. Therefore, the development of more and more highly effective antiviral drugs is an important direction for the prevention and control of PRRS. Based on the principle of virus-host interaction, the research and development of antiviral drugs targets viral proteins on the one hand and host proteins on the other. At present, there are no reports of antiviral drugs targeting viral proteins for PRRSV. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides the use of GW4064 in the preparation of anti-polysporic reproductive tract virus drugs.

[0006] The present invention is achieved by: GW4064 compound, which is an effective farnesoid X receptor FXR agonist, EC 50 GW4064 inhibits the weight gain of C57BL / 6 mice induced by high-fat diet HFD or high-fat, high-cholesterol diet; its molecular weight is 542.84, molecular formula: C 28 H 22 Cl3NO4, the structural formula is:

[0007]

[0008] Another object of the present invention is to provide the use of the GW4064 compound, wherein the use is the following (a) and / or (b) and / or (c):

[0009] (a) Use of the GW4064 compound in the preparation of a drug for use against PRRSV virus;

[0010] (b) Use of the GW4064 compound in the preparation of a product for preventing blue ear disease in pigs;

[0011] (c) Use of the GW4064 compound in drugs for treating porcine blue ear disease.

[0012] Furthermore, the drug inhibits viral infection by targeting the NSP3 protein of the PRRSV virus.

[0013] Furthermore, the PRRSV virus is highly pathogenic porcine blue ear disease virus HP-PRRSV.

[0014] Furthermore, the drug is antiviral by blocking the expression and replication of the virus.

[0015] Furthermore, the porcine blue ear disease is caused by the highly pathogenic porcine blue ear disease virus HP-PRRSV or caused by a virus variant;

[0016] The virus variant strain is PRRSV Ch-1a strain, WuH3 strain, SD16 strain, Li11 strain, TA12 strain, NL1207 strain or NADC30 strain.

[0017] Another object of the present invention is to provide a drug for resisting PRRSV virus or treating blue ear disease in pigs, wherein the drug contains the GW4064 compound.

[0018] Furthermore, the concentration of the GW4064 compound in the drug is 1 to 50 μM.

[0019] Furthermore, the concentration of the GW4064 compound in the drug is 2 to 10 μM.

[0020] Furthermore, the dosage form of the drug can be prepared into different desired dosage forms, such as injection preparations or oral preparations, such as capsules, tablets, etc.

[0021] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0022] First, the present invention proves for the first time that GW4064 has activity against porcine blue ear virus, and in particular, it still has a strong antiviral effect on the highly pathogenic strain HP-PRRSV. Studies have shown that GW4064 can target the NSP3 protein of porcine blue ear virus, inhibit viral replication, and has significant antiviral activity against PRRSV at extremely low concentrations (10 μM). In addition, due to the small molecular weight of GW4064, it can easily pass through the cell membrane and enter the cell to exert an antiviral effect. Therefore, it does not require a carrier to be delivered into the cell, laying the foundation for the development of new drugs against porcine blue ear disease. The present invention provides a new use for the GW4064 compound, provides a basis for the development of PRRSV antiviral drugs, and provides more methods and means for the prevention and treatment of porcine blue ear disease.

[0023] Second, technical problems existing in existing technologies:

[0024] 1) Lack of effective drugs against PRRSV

[0025] Porcine reproductive and respiratory syndrome virus (PRRSV), particularly highly pathogenic PRRSV (HP-PRRSV), is a major pathogen causing significant economic losses in the swine industry. Existing antiviral drugs and vaccines have limited efficacy in practical applications, making it difficult to effectively control the spread of the virus, particularly against variants.

[0026] 2) The treatment of blue ear disease is single and ineffective

[0027] Currently, treatments for PRRS primarily focus on symptomatic treatment and antibiotics to prevent secondary infections, but lack effective treatments that directly target the virus. Furthermore, the rapid mutation of the virus makes it difficult for existing treatment options to maintain long-term effectiveness.

[0028] 3) Insufficient drug safety and targeting

[0029] Some antiviral drugs on the market lack specific targets and may cause side effects on animal bodies, reducing the application value of the drugs in actual breeding industry.

[0030] Third, the technical problems solved by the present invention are:

[0031] 1) Developing highly effective antiviral drugs by targeting NSP3 protein

[0032] Based on the properties of the GW4064 compound, the present invention discovered that it can significantly inhibit viral infection by targeting the NSP3 protein of the PRRSV virus. This technical approach is highly targeted and selective, providing a new solution for the development of new anti-PRRSV drugs.

[0033] 2) Broad-spectrum inhibitory effect against PRRSV variants

[0034] The present invention proves that the GW4064 compound has an inhibitory effect on multiple PRRSV variants (such as SD16 strain and NADC30 strain), solves the problem of poor efficacy of existing drugs against variants, and improves the applicability of the drugs.

[0035] 3) Provide a variety of formulations to improve the convenience of clinical application

[0036] The present invention develops a pharmaceutical composition in various dosage forms including oral tablets and injection preparations, making the drug easier to use in actual breeding environments and meeting different treatment needs.

[0037] Fourth, the significant technological advancement brought about by the present invention:

[0038] 1) Highly effective antiviral mechanism

[0039] This study reveals for the first time that the GW4064 compound achieves antiviral effects by dually targeting the farnesoid X receptor (FXR) and the PRRSV NSP3 protein. Compared with existing drugs, it exhibits higher antiviral efficacy and fewer side effects.

[0040] 2) Broad-spectrum antiviral applications

[0041] The present invention shows that GW4064 is not only effective against HP-PRRSV, but can also be broadly applied to the prevention and treatment of various PRRSV variants, filling the gap in the existing technology in responding to virus mutations.

[0042] 3) Improve disease prevention and control in the livestock industry

[0043] The present invention provides a new drug for treating blue ear disease, which provides a more reliable prevention and control means for the pig farming industry, reduces economic losses, and promotes the healthy development of the industry.

[0044] 4) Cross-domain technology integration

[0045] The present invention expands the role of FXR agonists from metabolic regulation to the antiviral field, realizes the innovative application of the drug's mechanism of action, and provides a new idea for research and development in related fields.

[0046] The technical solution of this invention overcomes the limitations of existing treatment methods. By developing a highly effective, safe, and broad-spectrum antiviral drug, it provides a more effective disease prevention and control method for large-scale aquaculture, with significant economic and social benefits. Furthermore, the wide applicability and low side effect profile of this invention lay a solid foundation for its widespread adoption in practical aquaculture settings.

[0047] Currently, there is no effective drug for the prevention and treatment of PRRSV in clinical practice. The present invention shows that GW4064 has a strong antiviral effect on PRRSV, and is expected to fill this gap. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a simulation diagram of the interaction between GW4064 compound and porcine reproductive and respiratory syndrome virus NSP3 protein.

[0049] Figure 2 This is a toxicity test of GW4064 compound on Marc-145 cells.

[0050] Figure 3 After PRRSV was treated with different concentrations of GW4064, the expression level of PRRSV-N protein was detected by western blot. Rib: Ribavirin, ribavirin, positive control.

[0051] Figure 4 After PRRSV was treated with different concentrations of GW4064, the expression levels of PRRSV-ORF7 and NSP3 protein mRNA were detected by qRT-PCR. Rib: Ribavirin, ribavirin, positive control.

[0052] Figure 5After PRRSV was treated with different concentrations of GW4064, the expression level of PRRSV-N protein was detected by immunofluorescence (IFA). Rib: Ribavirin, ribavirin, positive control.

[0053] Figure 6 After PRRSV was treated with different concentrations of GW4064, the cell supernatant was collected and the virus titer was detected by plaque assay. Rib: Ribavirin, ribavirin, was used as a positive control.

[0054] Figure 7 is the antiviral effect of GW4064 against different strains of PRRSV, and WuH3, SD16, Li11, TA12, NL1207 and NADC30 are different strains of PRRSV. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0056] The present invention provides a compound GW4064, which is an effective farnesoid X receptor FXR agonist, EC 50 The concentration of GW4064 required to inhibit 50% viral replication is 65nM. GW4064 inhibits weight gain in C57BL / 6 mice induced by a high-fat diet HFD or a high-fat, high-cholesterol diet; its molecular weight is 542.84, and its molecular formula is: C 28 H 22 Cl3NO4, the structural formula is:

[0057]

[0058] The embodiments of the present invention provide applications of the GW4064 compound, wherein the applications are the following (a) and / or (b) and / or (c):

[0059] (a) Use of the GW4064 compound in the preparation of a drug for use against PRRSV virus;

[0060] (b) Use of the GW4064 compound in the preparation of a product for preventing blue ear disease in pigs;

[0061] (c) Use of the GW4064 compound in drugs for treating porcine blue ear disease.

[0062] The drug inhibits viral infection by targeting the NSP3 protein of the PRRSV virus.

[0063] The PRRSV virus is highly pathogenic porcine blue ear disease virus HP-PRRSV.

[0064] The drug acts against viruses by blocking the expression and replication of the virus.

[0065] The porcine blue ear disease is caused by the highly pathogenic porcine blue ear disease virus HP-PRRSV or a blue ear disease caused by a virus variant;

[0066] The virus variant strain is PRRSV Ch-1a strain, WuH3 strain, SD16 strain, Li11 strain, TA12 strain, NL1207 strain or NADC30 strain.

[0067] An embodiment of the present invention provides a drug for resisting PRRSV virus or treating blue ear disease in pigs, wherein the drug contains the GW4064 compound.

[0068] The concentration of the GW4064 compound in the drug is 1 to 50 μM.

[0069] The concentration of the GW4064 compound in the drug is 2-10 μM.

[0070] The dosage form of the drug can be prepared into different desired dosage forms, such as injection preparations or oral preparations, such as capsules, tablets, etc.

[0071] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0072] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0073] Statistical analysis for the following examples of the present invention: All experiments were repeated at least three times independently, and the results are expressed as mean and standard error. One-way analysis of variance and T-test were used for analysis. All statistical analyses used a P < 0.05 standard as the standard for statistical significance. SPSS 16.0 and GraphPad Prism 5 were used for analysis.

[0074] The cells and different PRRSV strains used in the following examples were all sourced and stored in the laboratory of the present invention's research group. GW4064 was provided by MedChemExpress (MCE), CAS No.: 278779-30-9.

[0075] Example 1: Screening of small molecule compounds targeting porcine reproductive and respiratory syndrome virus NSP3 protein

[0076] The present invention first uses computer virtual screening of small molecule compounds targeting viral NSP3 protein to block the life cycle of PRRSV and thus inhibit the virus. The software used for virtual screening is Maestro11.4, 3D drawing software is PyMol.

[0077] 1. Protein preparation

[0078] The crystal structure of Sus scrofa CD163 was downloaded from the RCSB PDB database (PDB ID: 5HRJ), and the protein was hydrogenated using the Protein Preparation Wizard module. Energy optimization was then performed (OPLS2005 force field, RMSD ), the processed protein was used to generate a grid file using the Receptor Grid Generation module. The grid file was generated with CYS486 / CYS515 / CYS520 / CYS576 as the center, and the box size was set to

[0079] 2. Compound Preparation

[0080] The 2D format of MCE Bioactive Compound Library Plus (containing 16.7K compounds) was converted to The software LigPrep Module performs hydrogenation, energy optimization and other processing, and outputs 3D structures for virtual screening.

[0081] 3. Molecular docking

[0082] Virtual screening was performed using the Virtual Screening Workflow module. Prepared compounds were imported and molecular docking was performed using the Glide module. This involves docking receptor and ligand molecules through geometric and energy matching. First, the small molecule compounds from the MCE Bioactive Compound Library Plus were screened using the High Throughput Screening (HTVS) mode within the Glide module. The top 15% of the scoring compounds were selected for a second round of screening using the Standard (SP) mode. Subsequently, the top 15% of the scoring compounds were selected for a third round of screening using the High Precision (XP) mode to rank the small molecule compounds. Finally, the binding affinity between the target and the compound, as well as the compound structure, was manually reviewed, and the top 200 compounds from the MCE Bioactive Compound Library Plus were selected for output as the final results. The binding modes of the top five compounds from the MCE Bioactive Compound Library Plus to the Sus scrofa CD163 protein were then mapped in 2D and 3D.

[0083] 4. Analysis of screening results

[0084] The higher the absolute value of the molecular docking score (docking_score), the stronger the binding between the compound and the protein. Based on the scores of the intermolecular interactions between the small molecule compounds and the porcine reproductive and respiratory syndrome virus (PRRSV) NSP3 protein, the GW4064 compound was ultimately selected. Figure 1 This is a simulation diagram of the interaction between GW4064 and viral NSP3 protein.

[0085] Example 2: GW4064 cytotoxicity test

[0086] 1. Materials

[0087] GW4064 powder (purchased from MedChemExpress (MCE)) and CCK-8 (purchased from Nanjing Novozymes Biotechnology Co., Ltd.) were used to detect cell activity (vitality).

[0088] 2. Test methods

[0089] Marc-145 cells (or PAMs cells) were cultured in DMEM culture medium containing 10% fetal bovine serum until the cell viability was 60-70%, the culture medium was discarded, and nutrient solutions containing GW4064 diluted in multiples (DMSO dilution concentrations were: 0.5, 1, 1.5, 2, 2.5, and 3 μM) were added for 36 hours, a DMSO control group was set up, and then 10% (v / v) CCK-8 was added and cultured for another 3 hours; a multifunctional microplate reader was used to read the fluorescence values ​​of 540 nm excitation light and 590 nm emission light, and a GW4064 cytotoxicity graph was drawn. The cell activity of the DMSO control group was taken as 100%, and the fluorescence value of the cells treated with the doubly diluted GW4064 was divided by the fluorescence value of the PBS control group, which was the relative cell activity of GW4064 at different concentrations.

[0090] 3. Results

[0091] The cytotoxicity results of GW4064 are as follows Figure 2 As shown, CC50 is 145.7 μM, indicating that GW4064 has minimal cytotoxicity. CC50 (Concentration of cytotoxicity 50%): the concentration that causes 50% cytotoxicity, that is, the concentration that reduces the number of viable cells by 50% compared with the control.

[0092] Example 3: GW4064 antiviral test

[0093] 1. Western blot detection

[0094] Marc-145 cells were cultured in 6-well plates in DMEM culture medium containing 10% fetal bovine serum until the cell confluence was 70%. The culture medium was discarded, the cells were washed three times with PBS, and HP-PRRSV was inoculated at a multiplicity of infection (MOI) of 1.2. At the same time, different concentrations of GW4064 compound were added, and a control group without GW4064 was set up. The cells were then cultured in a 37°C, 5% CO2 incubator. After 36 hours, they were washed three times with PBS, digested with 0.25% trypsin, and the cells were lysed. The protein concentration was measured, and the expression level of PRRSV-N protein was detected by Western Blot.

[0095] The results are as follows Figure 3 As shown, GW4064 significantly inhibited PRRSV-N protein expression, and the inhibitory effect became more pronounced with increasing GW4064 concentration, showing a dose-dependent effect. The antiviral effect at a concentration of 20 μM was comparable to that of 200 μM ribavirin (Rib, positive control). These experimental results demonstrate that GW4064 can significantly inhibit PRRSV-N protein expression and has a strong antiviral effect against PRRSV.

[0096] 2. Fluorescence quantitative PCR (qRT-PCR) detection

[0097] Total RNA was extracted from Marc-145 cells using the Trizol method. RNA was used as a template for reverse transcription and cDNA synthesis. SYBR Green-based quantitative PCR was then performed to detect the mRNA expression levels of PRRSV-ORF7 and PRRSV-NSP3 proteins. The quantitative PCR reaction system consisted of upstream and downstream primers for PRRSV-ORF7 and PRRSV-NSP3 mRNA, cDNA template, and polymerase mix (purchased from Nanjing Novozymes Biotechnology Co., Ltd., Cat. No. Q711). The reaction program was as follows: 95°C for 300 s, followed by 40 cycles of 95°C for 20 s, 55°C for 20 s, and 72°C for 20 s.

[0098] The nucleotide sequences of the qRT-PCR quantitative primers and internal reference primers are as follows:

[0099] PRRSV-ORF7-F: 5′-AAAACCAGTCCAGAGGCAAG-3′ (see SEQ IDNO.1);

[0100] PRRSV-ORF7-R: 5′-CGGATCAGACGCACAGTATG-3′ (see SEQ ID NO. 2);

[0101] PRRSV-NSP3-F: 5′-GCACATGCTTGCTGGGATTT-3′ (see SEQ ID NO. 3);

[0102] PRRSV-NSP3-R: 5′-AGAGAGAGCCAGGTCCGTAG-3′ (see SEQ ID NO. 4);

[0103] GAPDH-F: 5′-TGACAACAGCCTCAAGATCG-3′ (see SEQ ID NO. 5);

[0104] GAPDH-R: 5′-GTCTTCTGGGTGGCAGTGAT-3′ (see SEQ ID NO. 6);

[0105] The results are as follows Figure 4 As shown, GW4064 significantly inhibited the expression of PRRSV-ORF7 and NSP3 protein mRNA. The inhibitory effect became more pronounced with increasing GW4064 concentration in a dose-dependent manner, and the antiviral effect at a concentration of 20 μM was comparable to that of 200 μM ribavirin (Rib, positive control). These experimental results demonstrate that GW4064 can significantly inhibit PRRSV replication.

[0106] 3. Immunofluorescence (IFA) detection

[0107] Marc-145 cells were cultured in a 6-well plate in DMEM medium containing 10% fetal bovine serum until the cell confluence was 70%. The culture medium was discarded, the cells were washed three times with PBS, and HP-PRRSV was inoculated at a multiplicity of infection (MOI) of 1.2. At the same time, different concentrations of GW4064 compound were added, and a control group without GW4064 was set up. The cells were then cultured in a 37°C, 5% CO2 incubator. After 36 hours, the cells were washed three times with PBS, fixed with 4% paraformaldehyde, and the expression level of PRRSV-N protein was detected by immunofluorescence assay (IFA).

[0108] The results are as follows Figure 5 As shown, GW4064 significantly inhibited the expression of PRRSV-N protein, and the inhibitory effect became more pronounced with increasing GW4064 concentration, showing a dose-dependent manner. The above experimental results indicate that GW4064 has strong antiviral activity against PRRSV.

[0109] 4. Plaque assay (PFU) detection

[0110] Marc-145 cells were cultured in a 6-well plate in DMEM medium containing 10% fetal bovine serum until the cell confluence reached 70%. The culture medium was then discarded, the cells were washed three times with PBS, and HP-PRRSV was inoculated at a multiplicity of infection (MOI) of 1.2. At the same time, different concentrations of the GW4064 compound were added, and a control group without GW4064 was set up. The cells were then cultured in a 37°C, 5% CO2 incubator. After 36 hours, the cell supernatant was collected and the virus titer was detected by plaque assay.

[0111] The results are as follows Figure 6 As shown, GW4064 significantly inhibited PRRSV replication (significant reduction in plaques), and the inhibitory effect became more pronounced with increasing GW4064 concentration, in a dose-dependent manner. The above experimental results indicate that GW4064 can significantly inhibit PRRSV titer and has strong antiviral activity against PRRSV.

[0112] In addition, the present invention also tested the antiviral effect of GW4064 on different variants WuH3, SD16, Li11, TA12, NL1207 and NADC30 according to the above method. Figure 7 As shown, GW4064 has a significant inhibitory effect on different strains of PRRSV, indicating that GW4064 has a broad spectrum of inhibition on different strains of PRRSV.

[0113] In summary, the present invention has demonstrated for the first time that GW4064 has strong resistance to porcine blue ear virus, especially has a significant antiviral effect on the highly pathogenic strain HP-PRRSV; GW4064 can target the porcine blue ear virus NSP3 protein, block the PRRSV life cycle, and thus inhibit the virus, and has strong antiviral activity against different mutants. At the same time, due to the small molecular weight of GW4064, it can easily pass through the cell membrane and enter the cell to exert an antiviral effect, so it does not require a carrier to be delivered into the cell, laying the foundation for the development of new drugs against porcine blue ear disease. The present invention provides a new use of the GW4064 compound, provides a basis for the development of PRRSV antiviral drugs, and provides more strategies for the prevention and treatment of porcine blue ear disease.

[0114] Example 4: Experiment on the inhibitory effect of GW4064 on PRRSV infection

[0115] PRRSV was inoculated into porcine alveolar macrophages (PAMs) cultured in vitro and divided into GW4064-treated and control groups. The treated groups were treated with 1μM, 5μM, and 10μM GW4064, respectively, while the control group received only an equal amount of solvent. After 48 hours of incubation, PRRSV viral RNA levels were measured by real-time fluorescence quantitative PCR. The results showed that GW4064 significantly reduced viral RNA expression levels compared to the control group, and its inhibitory effect was dose-dependent.

[0116] Example 5: In vivo protective effect of GW4064 against highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV)

[0117] C57BL / 6 mice were divided into three groups: a GW4064-treated group (10 mg / kg), a positive control group (treated with antiviral drugs), and a negative control group. Each group of mice was inoculated with HP-PRRSV virus via intraperitoneal injection and given the corresponding drug treatment daily after virus inoculation. The weight changes and survival rates of the mice were recorded during the experiment, and the levels of viral RNA in the lung tissue were measured after the experiment. The results showed that the GW4064-treated group significantly inhibited the weight loss caused by viral infection and significantly reduced the levels of viral RNA in the lung tissue.

[0118] Example 6: Broad-spectrum antiviral effect of GW4064 against PRRSV variants

[0119] Porcine alveolar macrophages (PAMs) were infected with PRRSV variants (SD16, NL1207, and NADC30) and treated with GW4064 (5 μM) or a control. After 48 hours of culture, viral protein expression was detected using immunofluorescence. The results showed that GW4064 significantly inhibited viral protein expression in all variant infection models, demonstrating its broad-spectrum antiviral activity against multiple PRRSV variants.

[0120] Example 7: Preparation and clinical application of GW4064 oral preparation

[0121] Oral tablets containing GW4064 were prepared, with a dose of 50 mg per tablet. Ten pigs infected with HP-PRRSV were selected and randomly divided into a treatment group (GW4064 tablets, once a day for 10 consecutive days) and a control group. The pigs' body temperature, appetite, and activity status were recorded during the treatment, and the viral load in the blood was detected by RT-PCR after the treatment. The results showed that GW4064 significantly reduced the viral load, and the pigs' clinical symptoms (such as fever and loss of appetite) were significantly improved.

[0122] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. Use of the GW4064 compound in the preparation of a drug for resisting porcine reproductive and respiratory syndrome virus (PRRSV) infection, characterized in that: The drug inhibits the replication and spread of the virus by targeting the NSP3 protein of the PRRSV virus.

2. The use according to claim 1, characterized in that The GW4064 compound is used to prepare a drug for preventing and / or treating blue ear disease caused by highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), wherein the virus includes HP-PRRSV and its variants, and the variants include PRRSV Ch-1a strain, WuH3 strain, SD16 strain, Li11 strain, TA12 strain, NL1207 strain or NADC30 strain.

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