Application of chrysanthemum ketone in the preparation of drugs for the prevention and treatment of viral diseases in animals

Abstract

This invention discloses the application of chrysanthemum ketone or its pharmaceutically acceptable salts in the preparation of drugs for the prevention and treatment of viral diseases in animals. This invention is the first to propose and verify that chrysanthemum ketone or its pharmaceutically acceptable salts can effectively inhibit porcine pseudorabies virus, porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, influenza A virus H1N1 subtype, Newcastle disease virus, porcine circovirus type 2, and porcine transmissible gastroenteritis virus, exhibiting broad-spectrum antiviral activity. Simultaneously, experimental data show that when chrysanthemum ketone is used to treat animals suffering from viral diseases, it not only reduces tissue viral load, improves clinical symptoms and histopathological damage, and increases survival rate, but also has excellent safety and promising prospects for clinical translation.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and in particular relates to the application of chrysanthemum ketone in the preparation of drugs for the prevention and treatment of viral diseases in animals. Background Technology

[0002] Animal viral diseases are diverse, rapidly spreading, and have a high rate of asymptomatic infection, which has long been a significant factor hindering the healthy development of the livestock industry. Currently, African swine fever is prevalent, the NADC30 strain of porcine reproductive and respiratory syndrome virus has become the dominant circulating strain with frequent recombination, PEDV is the dominant cause of viral diarrhea in piglets, and variant strains such as S-INDEL have weakened the cross-protective efficacy of existing vaccines. Avian influenza virus continues to expand its host range and shows a trend of cross-species transmission. The global situation for animal disease prevention and control remains severe. At the same time, mixed infections with multiple pathogens are becoming increasingly complex, with secondary infections of viruses and bacteria overlapping. Furthermore, the continuous mutation of pathogens, the window period for vaccine protection, and the lack of effective interventions for emerging and re-emerging diseases pose multiple challenges to the traditional prevention and control system centered on vaccination and biosecurity.

[0003] Against this backdrop, developing host-targeted small molecule drugs with broad-spectrum antiviral activity has become an important complementary strategy. Unlike direct antiviral drugs that target only specific viral proteins, host-targeted drugs, by intervening in host cellular processes on which the replication of multiple viruses depends, can theoretically exert inhibitory effects across viral species boundaries and significantly reduce the probability of drug resistance due to mutations at a single viral target. This is particularly suitable for aquaculture scenarios where multiple viruses coexist and pathogens mutate rapidly. Halofuginone (HF), a derivative of halofuginone, has a well-defined chemical structure and a certain foundation for veterinary applications. Previous studies have mainly focused on its antiparasitic, antifibrotic, and immunomodulatory effects, indicating that it can regulate multiple biological processes within host cells. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to address the problems of limited selection of antiviral drugs and lack of broad-spectrum candidate preparations that can be used to intervene in multiple viral infections in existing animal viral disease prevention and control methods, and to provide an application of chrysanthemum ketone in the preparation of drugs for the prevention and treatment of animal viral diseases.

[0005] Technical solution: The application of the present invention, namely, chrysanthemum ketone or its pharmaceutically acceptable salt, in the preparation of drugs for the prevention and treatment of viral diseases in animals.

[0006] Preferably, the pharmaceutically acceptable salt is selected from one or more of hydrobromide, hydrochloride, sulfate, phosphate, methanesulfonate, acetate, lactate, citrate, tartrate, maleate, fumarate, or succinate.

[0007] Preferably, the animal viral disease is a disease caused by one or more of the following viruses: porcine pseudorabies virus, porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, influenza A virus H1N1 subtype, Newcastle disease virus, porcine circovirus type 2, and porcine transmissible gastroenteritis virus.

[0008] Preferably, the application is in the preparation of drugs that reduce viral load during the course of viral diseases in animals.

[0009] Preferably, the application is in the preparation of a medicament for improving tissue damage caused by viral diseases in animals.

[0010] Preferably, the application is in the preparation of a drug that improves animal survival rate.

[0011] Preferably, the drug contains styraxone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] Preferably, the drug further contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more of diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0013] Preferably, the dosage form of the drug includes capsules, granules, powders, tablets, microcapsules, injections, infusions, oral liquids, suspensions, patches, suppositories, microemulsions, liposomes, nanoparticles, and lyophilized powder injections.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention is the first to propose and verify that styraxone or its pharmaceutically acceptable salt can effectively inhibit porcine epidemic diarrhea virus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, influenza A virus H1N1 subtype, Newcastle disease virus, porcine circovirus type 2 and porcine transmissible gastroenteritis virus, and has broad-spectrum antiviral activity; 2. Experimental data show that when styraxone is used to treat animals with viral diseases, it can not only reduce tissue viral load, improve clinical symptoms and histopathological damage, and increase survival rate, but also has excellent safety and excellent prospects for clinical translation. Attached Figure Description

[0015] Figure 1 The image shows the results of the verification of the anti-swine pseudorabies virus activity of chrysanthemum ketone. Figure 2 The image shows the results of the verification of the broad-spectrum antiviral activity of chrysanthemum ketone. Figure 3 The figure shows the effect of styraxone on the survival rate and body weight changes of PRV / XJ5 infected mice. Figure 4The figure shows the effect of styraxone on brain and lung lesions in PRV / XJ5 infected mice. Figure 5 Figure 1 shows the effect of styraxone on viral load in lung and brain tissues of PRV / XJ5-infected mice. Figure 6 The figure shows the effect of styraxone on the survival rate and weight changes of H1N1-infected mice. Figure 7 Figure 1 shows the effect of styraxone on pathological damage in lung tissue of H1N1-infected mice. Figure 8 The figure shows the effect of styraxone on viral load in lung tissue of H1N1-infected mice. Figure 9 The figure shows the effect of styraxone on the survival rate and clinical manifestations of PEDV-infected piglets. Figure 10 Figure 1 shows the effect of styraxone on changes in body weight and body temperature in PEDV-infected piglets. Figure 11 Figure 1 shows the effect of styraxone on small intestinal tissue lesions in PEDV-infected piglets. Figure 12 Figure 1 shows the effect of styraxone on the viral load of PEDV-infected piglets. Figure 13 The graph shows the effects of styraxone on mouse survival rate and weight changes. Figure 14 Figure 1 shows the effect of styraxone on the morphology of major organs and tissues in mice. Figure 15 The graph shows the effects of styraxone on complete blood count and serum biochemical indicators in mice. Figure 16 The graph shows the effect of fentanyl on piglet survival rate. Figure 17 The graph shows the effects of styraxone on the weight and body temperature of piglets. Figure 18 Figure 1 shows the effect of styraxone on the morphology of major organs and tissues in piglets. Figure 19 The figure shows the effect of fentanyl on routine blood tests and serum biochemical indicators in piglets. Detailed Implementation

[0016] The technical solution of the present invention will be further described below.

[0017] Example 1: Validation of the broad-spectrum antiviral activity of halofuginone (HF) 1. Validation of the anti-porcine pseudorabies virus (PRV) activity of chrysanthemum ketone 1.1 Validation of the anti-PRV / XJ5 activity of styraxone at a multiplicity of infection (MOI) of 0.1 PK-15 B6 cells (purchased from the China Institute of Veterinary Drug Control) were used at a concentration of 2×10⁶ cells / year. 5 PK-15 B6 cells were seeded at a density of 1 cells / well in 12-well plates and cultured overnight in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO2. The original medium was then replaced with DMEM complete medium (containing 1‰ DMSO) or fresh DMEM complete medium containing 0, 20, 40, 80 or 100 µM styraxone (purchased from MedChemExpress LLC., catalog number HY-N1584) at a final concentration of 1‰ or 100 µM styraxone (all containing 1‰ DMSO) or fresh DMEM complete medium (Mock). The cells were then cultured for 1 h as usual. Subsequently, PK-15 B6 cells were infected with PRV / XJ5 (GenBank accession number: OP512542, provided by the College of Veterinary Medicine, Yangzhou University) with an MOI of 0.1 in the presence of different concentrations of HF.

[0018] 1.1.1 Determination of PRV gB protein expression level 24 h after infection, cells were collected and total protein was extracted. Loading buffer was added in proportion and the mixture was heated to boiling in a metal bath for 10 min before SDS-PAGE electrophoresis. After transfer and blocking, PRV gB primary antibody (provided by the College of Veterinary Medicine of Yangzhou University) diluted 1:1000 or β-actin primary antibody diluted 1:10000 was added and incubated overnight at 4℃. After rinsing, the cells were incubated at room temperature for 1 h with the corresponding species secondary antibody diluted 1:10000. Finally, ECL method was used for development and image acquisition.

[0019] Western blot analysis was performed. The results are as follows: Figure 1 As shown in Figure A, HF significantly downregulated the expression level of gB protein in a dose-dependent manner.

[0020] 1.1.2 Virus titer determination Collect cell culture supernatant 24 h after infection, centrifuge to remove cell debris, and then perform 10-fold serial dilutions to obtain 10 0 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7The virus solution was diluted to various concentrations; each dilution was inoculated into Vero cells, with 8 replicates per dilution. Cells were cultured at 37°C and 5% CO2, and the cytopathic effect (CPE) was observed. The viral titer was calculated using the Reed-Muench method based on the number of positive wells at each dilution, and expressed as TCID50. 50 / mL indicates.

[0021] The results are as follows Figure 1 As shown in B, HF can significantly reduce viral titers.

[0022] 1.1.3 Virus copy number determination 24 h after infection, cell culture supernatant was collected, centrifuged at 12000 rpm for 15 min to remove cell debris, and 400 μL of supernatant was added with 10% SDS and proteinase K to bring the final proteinase K concentration to 3 mg / mL. After thorough mixing, the mixture was incubated at 56℃ for 2 h. After incubation, an equal volume of phenol-chloroform was added, and the mixture was vigorously shaken and centrifuged at 12000 rpm for 15 min at 4℃. The upper aqueous phase was transferred to a new 1.5 mL centrifuge tube, and 2 volumes of pre-cooled 75% ethanol were added. The mixture was precipitated at -20℃ for 30 min, followed by centrifugation at 12000 rpm for 10 min. The supernatant was discarded and the precipitate was dried. An appropriate amount of ddH2O was added to dissolve the DNA, and the mixture was stored at -20℃ for subsequent viral genome copy number detection.

[0023] Using extracted viral DNA as a template for real-time quantitative PCR, and recombinant plasmid pMD19-T-PRV ( gB A standard curve was established using plasmid copy number 94 (provided by the College of Veterinary Medicine, Yangzhou University) as a standard. The standard curve was plotted with the cycle threshold Ct as the ordinate and the common logarithm of plasmid copy number lgC as the abscissa. Based on this, the PRV in the sample was calculated. gB Gene copy number. The primer and probe sequences are as follows: gB 94-F: 5'-acaagttcaaggcccacatctac-3'; gB 94-R: 5'-gtccgtgaagcggttcgtgat-3'; gBProbe: 5'-FAM-acgtcatcgtcacgacc-TAMRA-3'; qPCR reaction system: 10 μL of 2×AceQ qPCR Probe Master Mix (Novizan, Q112-03), 0.8 μL of upstream primer (10 μM), 0.8 μL of downstream primer (10 μM), 0.4 μL of probe (10 μM), 0.4 μL of ROX II, 2.6 μL of H2O, 5 μL of DNA template, total volume 20 μL; qPCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 62℃ annealing and extension for 30 s, for a total of 45 cycles.

[0024] The results are as follows Figure 1 As shown in Figure C, the viral genome copy number decreased significantly after HF treatment, further confirming the inhibitory effect of HF on PRV infection.

[0025] 1.2 Verification of the anti-PRV / XJ5 activity of chrysanthemumine at different MOIs PK-15 B6 cells at 2×10 5 PK-15 B6 cells were seeded at a density of 1 cells / well in 12-well plates and cultured overnight in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% carbon dioxide. The original medium was then replaced with DMEM complete medium (containing 1‰ DMSO) or fresh DMEM complete medium containing 0, 80, or 100 µM styraxone (all containing 1‰ DMSO) or fresh DMEM complete medium (Mock). After routine culture for 1 h, PK-15 B6 cells were infected with PRV / XJ5 with MOI=0.5, 1, or 2 in the presence of different concentrations of HF for 24 h.

[0026] 1.2.1 Determination of PRV gB protein expression level Cells were collected 24 h after infection, and total protein was extracted and gB protein expression level was determined using the aforementioned method.

[0027] The results are as follows Figure 1 As shown in Figure D, HF significantly inhibited the expression of gB protein under different MOI conditions.

[0028] 1.2.2 Virus titer determination Cell culture supernatant was collected 24 h after infection, and the viral titer in the supernatant was determined using the aforementioned method.

[0029] The results are as follows Figure 1 As shown in E, HF significantly inhibited viral titers under different MOI conditions.

[0030] 1.3 Verification of the anti-PRV / Ra activity of chrysanthemum ketone PK-15 B6 cells at 2×105 PK-15 B6 cells were seeded at a density of 1 cells / well in 12-well plates and cultured overnight in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% carbon dioxide. The original medium was then replaced with DMEM complete medium (containing 1‰ DMSO) or fresh DMEM complete medium containing final concentrations of 0, 20, 40, 80, or 100 µM styraxone. After routine culture for 1 h, PK-15 B6 cells were infected with PRV / Ra (provided by the College of Veterinary Medicine, Yangzhou University) at an MOI of 0.1 for 24 h in the presence of different concentrations of HF.

[0031] Cells and cell culture supernatant were collected 24 h after infection. Total protein in the cells was extracted and gB protein expression level was measured using the aforementioned method. At the same time, the viral titer in the cell culture supernatant was measured using the aforementioned method.

[0032] The results of gB protein expression level determination are as follows: Figure 1 As shown in F, the virus titer determination results are as follows: Figure 1 As shown in G, HF also significantly inhibited gB protein expression and viral titer during PRV / Ra strain infection.

[0033] 1.4 Validation of the antiviral activity of chrysanthemum ketone after PRV infection PK-15 B6 cells at 2×10 5 PK-15 B6 cells were seeded at a density of cells / well in 12-well plates and cultured overnight in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO2. PK-15 B6 cells were then infected with PRV / Ra (provided by Yangzhou University) at an MOI of 0.1. At 0, 2, 4, 6, or 8 h post-infection, PK-15 B6 cells were added to a final concentration of 0, 80, or 100 µM styraxone or fresh DMEM complete medium to replace the original medium (Mock), and cultured for another 24 h.

[0034] Cells were then collected, and the total protein in the cells was extracted using the aforementioned method, and the expression level of gB protein was determined.

[0035] The results of gB protein expression level determination are as follows: Figure 1 As shown in H, adding HF at different time points after infection can effectively inhibit gB protein expression, and the earlier the drug is added, the more obvious the antiviral effect is.

[0036] 2. Validation of the anti-porcine epidemic diarrhea virus (PEDV) activity of chrysanthemum ketone Vero cells (provided by the College of Veterinary Medicine, Yangzhou University) were used at 2 × 10⁻⁶. 5Cells were seeded at a density of 1 cells / well in 12-well plates and cultured overnight in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% carbon dioxide. Then, the original medium was replaced with DMEM complete medium (containing 1‰ DMSO) or fresh DMEM complete medium containing final concentrations of 0, 20, 40, 80 or 100 µM thiazoline (all containing 1‰ DMSO) or fresh DMEM complete medium (Mock). After routine culture for 1 h, Vero cells were infected with PEDV / HLJBY (provided by the College of Veterinary Medicine, Yangzhou University) with an MOI of 1 for 24 h in the presence of different concentrations of HF.

[0037] Cells and cell culture supernatant were collected 24 h after infection. Total protein was extracted from the cells and the expression level of PEDV N protein was measured using the method described above. The primary antibody used was replaced with PEDV N primary antibody diluted 1:1000 (prepared and provided by the laboratory of the College of Veterinary Medicine, Yangzhou University). At the same time, the viral titer in the cell culture supernatant was measured using the method described above.

[0038] The results of PEDV N protein expression level determination are as follows: Figure 2 As shown in Figure A, the virus titer determination results are as follows: Figure 2 As shown in Figure D, HF also significantly inhibited N protein expression and viral titer during PEDV infection.

[0039] 3. Validation of the anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity of styraxone Marc-145 cells (provided by the College of Veterinary Medicine, Yangzhou University) were used at 2×10 5 Marc-145 cells were seeded at a density of 1 cells / well in 12-well plates and cultured overnight in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% carbon dioxide. Subsequently, the original medium was replaced with DMEM complete medium (containing 1‰ DMSO) or fresh DMEM complete medium containing final concentrations of 0, 20, 40, 80 or 100 µM styraxone, and cultured for 1 h. Then, Marc-145 cells were infected with PRRSV (provided by the College of Veterinary Medicine of Yangzhou University) with an MOI of 0.1 for 24 h in the presence of different concentrations of HF.

[0040] 24 h after infection, cells and cell culture supernatant were collected. Total protein was extracted from the cells and the expression level of PRRSV N protein was measured using the method described above. The primary antibody used was replaced with PRRSV N primary antibody diluted 1:1000 (prepared and provided by the laboratory of the College of Veterinary Medicine, Yangzhou University). At the same time, the viral titer in the cell culture supernatant was measured using the method described above.

[0041] The results of PRRSV N protein expression level determination are as follows: Figure 2 As shown in B, the virus titer determination results are as follows: Figure 2 As shown in E, HF also significantly inhibited N protein expression and viral titer during PRRSV infection.

[0042] 4. Verification of the anti-H1N1 subtype activity of styraxone against influenza A virus. MDCK cells (provided by the College of Veterinary Medicine, Yangzhou University) were used at 2×10 5 Cells were seeded at a density of cells / well in 12-well plates and cultured overnight in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% carbon dioxide. Then, the original medium was replaced with DMEM complete medium (containing 1‰ DMSO) or fresh DMEM complete medium containing final concentrations of 0, 20, 40, 80 or 100 µM styraxone (all containing 1‰ DMSO) or fresh DMEM complete medium (Mock). After routine culture for 1 h, MDCK cells were infected for 24 h with H1N1 / PR8 (provided by the College of Veterinary Medicine, Yangzhou University) at MOI=1 in the presence of different concentrations of HF.

[0043] Twenty-four hours after infection, cells and cell culture supernatants were collected. Total protein was extracted from the cells using the methods described above, and the expression level of H1N1 NP protein was measured. The primary antibody used was replaced with a 1:1000 dilution of H1N1 NP primary antibody (prepared and provided by the Veterinary College Laboratory of Yangzhou University). Simultaneously, the hemagglutination assay was used to determine the HA titer in the cell culture supernatant. The cell culture supernatant was serially diluted twofold, and then an equal volume of 1% chicken erythrocyte suspension was added to each well. After gentle mixing, the mixture was incubated at 37°C for 15 min, and the hemagglutination of erythrocytes was observed. The highest dilution that produced complete hemagglutination was taken as the HA titer.

[0044] The results of H1N1 NP protein expression level determination are as follows: Figure 2 As shown in C, the results of the HA potency assay are as follows: Figure 2 As shown in F, HF also significantly inhibited NP protein expression and HA titer during H1N1 infection.

[0045] 5. Validation of the anti-Newcastle disease virus (NDV) activity of chrysanthemum ketone Vero cells at 2 × 10 5 Cells were seeded at a density of cells / well in 12-well plates and cultured overnight in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% carbon dioxide. Then, the original medium was replaced with DMEM complete medium (containing 1‰ DMSO) or fresh DMEM complete medium containing final concentrations of 0, 20, 40, 80 or 100 µM thiazoline, and cultured for 1 h. Vero cells were then infected with NDV (provided by the College of Veterinary Medicine, Yangzhou University) with an MOI of 0.01 for 24 h in the presence of different concentrations of HF.

[0046] 24 h after infection, cells and cell culture supernatant were collected. Total protein was extracted from the cells using the method described above, and the expression level of NDV HN protein was measured. The primary antibody used was replaced with NDV HN primary antibody diluted 1:1000 (prepared and provided by the laboratory of the College of Veterinary Medicine, Yangzhou University). At the same time, the viral titer in the cell culture supernatant was measured using the method described above.

[0047] The results of NDV HN protein expression level determination are as follows: Figure 2 As shown in G, the virus titer determination results are as follows: Figure 2 As shown in J, HF also significantly inhibited HN protein expression and viral titer during NDV infection.

[0048] 6. Validation of the anti-porcine circovirus type 2 (PCV2) activity of styraxone PK-15 B6 cells at 2×10 5 PK-15 B6 cells were seeded at a density of 1 cells / well in 12-well plates and cultured overnight in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% carbon dioxide. Subsequently, the original medium was replaced with DMEM complete medium (containing 1‰ DMSO) or fresh DMEM complete medium containing final concentrations of 0, 20, 40, 80 or 100 µM styraxone, and cultured for 1 h. Then, PK-15 B6 cells were infected with PCV2 (provided by the College of Veterinary Medicine of Yangzhou University) with an MOI of 0.5 for 24 h in the presence of different concentrations of HF.

[0049] Cells and cell culture supernatant were collected 24 h after infection. Total protein was extracted from the cells and the expression level of PCV2 Cap protein was measured using the method described above. The primary antibody used was replaced with PCV2 Cap primary antibody diluted 1:1000 (prepared and provided by the laboratory of the College of Veterinary Medicine, Yangzhou University). At the same time, the viral titer in the cell culture supernatant was measured using the method described above.

[0050] The results of PCV2 Cap protein expression level determination are as follows: Figure 2 As shown in H, the virus titer determination results are as follows: Figure 2 As shown in K, HF also significantly inhibited Cap protein expression and viral titer during PCV2 infection.

[0051] 7. Validation of the anti-transmissible gastroenteritis virus (TGEV) activity of styraxone ST cells (preserved by the laboratory of the College of Veterinary Medicine, Yangzhou University) were used at 2×10 5The cells were seeded at a density of cells / well in 12-well plates and cultured overnight in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% carbon dioxide. The original medium was then replaced with DMEM complete medium (containing 1‰ DMSO) or fresh DMEM complete medium at final concentrations of 0, 20, 40, 80 or 100 µM thiazoline (all containing 1‰ DMSO) or fresh DMEM complete medium (Mock). The cells were then cultured for 1 h using standard methods. Subsequently, the ST cells were infected with TGEV (provided by the College of Veterinary Medicine of Yangzhou University) with an MOI of 0.01 for 24 h in the presence of different concentrations of HF.

[0052] Cells and cell culture supernatant were collected 24 h after infection. Total protein was extracted from the cells and the expression level of TGEV N protein was measured using the method described above. The primary antibody used was replaced with TGEV N primary antibody diluted 1:1000 (prepared and provided by the laboratory of the College of Veterinary Medicine, Yangzhou University). At the same time, the viral titer in the cell culture supernatant was measured using the method described above.

[0053] The results of TGEV N protein expression level assay are as follows: Figure 2 As shown in Figure I, the virus titer determination results are as follows: Figure 2 As shown in L, HF also significantly inhibited N protein expression and viral titer during TGEV infection.

[0054] Example 2: Validation of the therapeutic effect of styraxone on PRV / XJ5 infected mice SPF-grade female BALB / c mice aged 6-8 weeks (purchased from the Experimental Animal Center of Yangzhou University) were selected and randomly divided into the following groups after acclimatization: healthy control group (Mock), PRV / XJ5 challenge group (PRV / XJ5), HF administration group (0.02 mg / kg), HF administration group (0.04 mg / kg), and HF administration group (0.1 mg / kg), with 9 mice in each group.

[0055] The PRV / XJ5 challenge group and all HF administration groups were inoculated via nasal drops for 10 days. 3.5 TCID 5050 μL of PRV / XJ5 virus solution was administered, and the healthy control group received an equal volume of DMEM culture medium via intranasal drops. The HF treatment group received an appropriate dose of HF (prepared with PBS containing 1% DMSO as the solvent) via intraperitoneal injection. The PRV / XJ5 challenge group received an equal volume of PBS containing 1% DMSO as a solvent control. The healthy control group received an equal volume of PBS containing 1% DMSO via intraperitoneal injection. Treatment was administered once daily for a total of four times. Mice were monitored for seven consecutive days post-challenge, recording survival rate, weight changes, and clinical symptoms. On day 4 post-infection, three mice from each group were randomly euthanized, and brain and lung tissues were collected for necropsy, pathological photography, and viral load detection. On day 7 post-infection, the remaining mice were euthanized, and brain and lung tissues were collected, fixed, and stained with H&E.

[0056] 1. Evaluation of mouse survival rate Mice in the PRV / XJ5 challenge group gradually developed typical infection symptoms such as lethargy, ruffled fur, rapid breathing, frequent turning, and itching within a 4-day observation period, accompanied by a 100% mortality rate. Survival status is as follows... Figure 3 As shown in Figure A, compared with the challenge control group, the survival rate of mice after HF administration was significantly improved, and the protective effect was more obvious with increasing dose: the survival rate was 16.6% in the 0.02 mg / kg dose group, increased to 50% in the 0.04 mg / kg dose group, and reached 100% in the 0.1 mg / kg dose group, indicating that HF ​​has a dose-dependent protective effect against PRV infection in vivo.

[0057] 2. Evaluation of mouse body weight changes Changes in weight, such as Figure 3 As shown in Figure B, after calculating the rate of weight change in each group based on the body weight on day 0 of infection, it was found that the body weight of mice challenged with PRV / XJ5 decreased significantly, indicating that acute infection can lead to significant systemic wasting and stress. Although HF treatment did not significantly stop the trend of weight loss in the early stage of infection, the body weight of mice protected by HF began to gradually recover after day 4 of infection, and eventually approached the level of the Mock group.

[0058] 3. Evaluation of protective effects on mouse brain and lung tissue Mouse brain and lung tissues collected on day 7 post-infection were fixed in 10% neutral formaldehyde solution for 24 h, dehydrated in a gradient manner, embedded in paraffin, and cut into 3 μm sections. After dewaxing and hydration, H&E staining was performed, and the sections were mounted with neutral resin and observed and images were acquired under a microscope.

[0059] Mouse brain and lung tissues collected on day 4 post-infection, as shown in... Figure 4As shown in Figure A, the brain and lung tissues of mice in the Mock group appeared normal, with uniform color and no obvious abnormalities. Mice in the PRV / XJ5 challenge group showed lung congestion and edema, exhibiting dark red consolidation; brain tissue was congested, and meningeal blood vessels were dilated. Compared with the challenge group, the above lesions were alleviated after HF treatment, with more significant improvements observed in the 0.04 mg / kg and 0.1 mg / kg dose groups.

[0060] H&E staining results are as follows Figure 4 As shown in Figure B, mice in the PRV / XJ5 challenge group exhibited widened alveolar septa, thickened alveolar walls, disordered alveolar structure, and significant inflammatory cell infiltration. Neuronal damage and inflammatory cell infiltration were also observed in the brain tissue. Treatment with 0.1 mg / kg HF significantly alleviated these pathological changes, resulting in more intact alveolar structure, reduced inflammatory cell infiltration, and no significant pathological damage in the brain tissue. These results indicate that HF ​​has a significant protective effect against PRV-induced brain and lung tissue damage, consistent with its effect of improving mouse survival.

[0061] 4. Evaluation of the inhibitory effect of HF on PRV replication in vivo On day 4 post-infection, mouse brain and lung tissues were collected and homogenized thoroughly using a multi-sample cryo-grinding device to prepare tissue homogenates. After three freeze-thaw cycles, the tissue homogenates were centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. Viral DNA was then extracted using the phenol-chloroform extraction method described in Example 1.1.3, and qPCR was used to detect and calculate the PRV in lung and brain tissues. gB Gene copy number.

[0062] The results are as follows Figure 5 As shown, the viral copy number in the lung and brain tissues of mice challenged with PRV / XJ5 remained at a high level. Although the viral load in the 0.02 mg / kg HF group showed a decreasing trend, it did not reach statistical significance. The 0.04 mg / kg and 0.1 mg / kg HF groups exhibited significantly lower viral loads in both lung and brain tissues, more than 100-fold lower than the challenged control group. These results indicate that HF ​​can significantly inhibit PRV replication in mouse lung and brain tissues in a dose-dependent manner.

[0063] Example 3: Validation of the therapeutic effect of chrysanthemum ketone on H1N1-infected mice SPF-grade male C57BL / 6 mice aged 6-8 weeks (purchased from the Experimental Animal Center of Yangzhou University) were selected and randomly divided into the following groups after acclimatization: healthy control group (Mock), H1N1 / PR8 challenge group (H1N1 / PR8), HF administration group (0.02 mg / kg), HF administration group (0.04 mg / kg), and HF administration group (0.1 mg / kg), with 9 mice in each group.

[0064] The H1N1 / PR8 challenge group and all HF administration groups were inoculated via nasal drops for 10 days. 4.5 EID 50 50 μL of H1N1 / PR8 virus solution was administered, and a healthy control group received an equal volume of DMEM culture medium via intranasal drops. The HF treatment group received an appropriate dose of HF (prepared with PBS containing 1% DMSO as the solvent) via intraperitoneal injection. The H1N1 / PR8 challenge group received an equal volume of PBS containing 1% DMSO as a solvent control. The healthy control group received an equal volume of PBS containing 1% DMSO via intraperitoneal injection. Administration was once daily for a total of 8 days. Mice were monitored for 14 consecutive days after challenge, and survival rate, weight changes, and clinical symptoms were recorded.

[0065] 1. Evaluation of mouse survival rate Mouse survival status as follows Figure 6 As shown in Figure A, the survival rate of mice in the H1N1 / PR8 challenge group gradually decreased with the progression of infection, with deaths beginning on day 8, and a survival rate of 33.3% at the end of the experiment. Compared with the challenge group, the survival rate of the HF treatment group was significantly increased, showing a dose-dependent protective trend. Specifically, the survival rate of mice in the 0.04 mg / kg group increased to 50.0%, and the survival rate in the 0.1 mg / kg group further increased to 66.7%.

[0066] 2. Evaluation of mouse body weight changes Changes in weight, such as Figure 6 As shown in Figure B, the body weight of mice in the H1N1 / PR8 challenge group decreased significantly. The trend of body weight loss in the early stage of infection was not significantly reversed after HF treatment, but it promoted the recovery of body weight in the later stage of infection to some extent.

[0067] 3. Evaluation of protective effect on mouse lung tissue Lung tissues from mice collected on day 14 post-infection were fixed in 10% neutral formaldehyde solution for 24 h, dehydrated in a gradient manner, embedded in paraffin, and cut into 3 μm sections. After dewaxing and hydration, hematoxylin-eosin (H&E) staining was performed, and the sections were mounted with neutral resin and observed and images were acquired under a microscope.

[0068] H&E staining results are as follows Figure 7 As shown, the lung tissue morphology of mice in the Mock group was basically normal, with clear alveolar cavities, uniform alveolar septa, and no obvious inflammatory response. After H1N1 / PR8 challenge, the lung tissue of mice showed obvious pathological damage, manifested as alveolar structure destruction, alveolar septal thickening, localized lung parenchyma densification, and numerous inflammatory cell infiltrations. Compared with the challenge group, the 0.1 mg / kg HF treatment group showed reduced lung tissue lesions, better preservation of alveolar structure, and decreased alveolar septal thickening and inflammatory cell infiltration. This indicates that HF ​​has a protective effect against lung tissue damage caused by H1N1 / PR8 infection.

[0069] 4. Evaluation of the inhibitory effect of HF on H1N1 replication in vivo Lung tissues from mice collected on day 8 post-infection were homogenized using a multi-sample cryo-mortar and prepared into tissue homogenates. Total RNA was extracted from the lung tissues using the Novizan RNA isolater Total RNA Extraction Reagent Kit (R401-01), and the RNA was reverse transcribed into cDNA using the Novizan HiScript III RT SuperMix for qPCR (+gDNA wiper) Kit (R323-01). Relative quantitative PCR was then performed using the Novizan UniPeak U+ One Step RT-qPCR SYBRGreen Kit (Q226-01) to detect H1N1 infection. NP Genes are the detection targets, with GAPDH As an internal reference gene, through 2 -ΔΔCt Method Calculation NP Relative gene expression levels are used to reflect the H1N1 viral load in mouse lung tissue. Among them, H1N1... NP The gene primer sequences are as follows: NP -F: 5'-gggcagaacgtctgacatga-3'; NP-R: 5'-gggttcgttgccttttcgtc-3'; GAPDH The primer sequences are: GAPDH -F:5'-gtcggttgtggatctgacct-3'; GAPDH -R:5'-agcttgacgaagtggtcgtt-3'.

[0070] The results are as follows Figure 8 As shown, in the lung tissue of mice in the H1N1 / PR8 challenge group NP Gene expression levels were significantly increased, reaching more than 200-fold above baseline. After HF treatment, in the lung tissue of mice in the 0.04 mg / kg group... NP Genetic levels were significantly reduced, with the 0.1 mg / kg group showing the most significant inhibitory effect, decreasing by more than 150 times compared to the challenge control group. These results indicate that HF ​​can effectively inhibit the replication of H1N1 / PR8 in mouse lung tissue, and its inhibitory effect increases with increasing dosage.

[0071] Example 4: Verification of the therapeutic effect of styraxone on PEDV-infected piglets Five-day-old piglets were selected and randomly divided into three groups after acclimatization: a healthy control group (Mock, n=2), a PEDV challenge group (PEDV, n=5), and a HF administration group at a dose of 0.06 mg / kg (0.06 mg / kg, n=5).

[0072] Piglets in both the PEDV challenge group and the HF administration group were orally vaccinated 10 times. 6.0 TCID 50 2 mL of PEDV F3 generation virus solution (provided by the College of Veterinary Medicine, Yangzhou University) was administered per piglet; healthy controls were orally administered an equal volume of DMEM culture medium. The HF administration group received an appropriate dose of HF (prepared with PBS containing 1% DMSO as the solvent) via intraperitoneal injection. The PEDV challenge group and healthy controls received an equal volume of PBS containing 1% DMSO, once daily for a total of 4 doses. In the PEDV-challenged piglet model, clinical examinations were performed daily after virus inoculation, including observation of mental status, activity level, body size changes, walking ability, respiratory status, conjunctival condition, appetite, and defecation, and clinical scores were calculated. Disease progression was dynamically recorded based on cumulative scores. When the clinical score reached 14 points, euthanasia was immediately performed and relevant samples were collected. On day 7 of the experiment, all remaining piglets were euthanized, and duodenal, jejunal, and ileal tissues were collected for viral load detection and H&E staining to observe intestinal histopathological changes. In addition, anal swabs were collected from piglets daily, and the viral nucleic acid copy number was detected by RT-qPCR to analyze the dynamic changes in PEDV shedding levels at different time points.

[0073] 1. Evaluation of piglet survival rate and clinical manifestations The survival status of piglets is as follows Figure 9 As shown in Figure A, the changes in clinical scores are as follows: Figure 9 As shown in B, the perianal area and necropsy findings are as follows: Figure 9As shown in Figure C, piglets in the Mock group maintained good mental condition, normal feed intake and defecation throughout the experiment, with no diarrhea, vomiting, or death observed, and the survival rate remained at 100%. Piglets in the PEDV group began to show obvious clinical symptoms on day 2 after challenge, with some experiencing vomiting and diarrhea; by day 3, all piglets had diarrhea, diarrhea scores rapidly increased, and mortality began, with the survival rate dropping to 20% by the end of the experiment. Compared to the challenge control group, piglets in the 0.06 mg / kg HF group showed significantly reduced clinical symptoms, with only a few experiencing vomiting and diarrhea on day 2 after challenge. Their overall diarrhea score was lower than the PEDV group, and they largely recovered by day 7 after challenge. The survival rate of piglets in the 0.06 mg / kg HF group dropped to 80% on day 3 after challenge, and no further deaths occurred thereafter. It is noteworthy that the diarrhea score in the PEDV group decreased later; this should be considered in conjunction with changes in survival rate, as it may be related to the fact that severely affected piglets were not included in subsequent scoring. Gross observation and necropsy results were largely consistent with clinical manifestations. In the Mock group, piglets showed clean perianal area, natural distribution of abdominal organs, uniform small intestine diameter, smooth serosa, normal intestinal wall thickness, and no obvious abnormalities within the intestinal lumen. In the PEDV group, piglets showed significant yellow, watery fecal contamination around the anus. Autopsy revealed significant small intestinal dilation, thinning of the intestinal wall with hemorrhage, and the intestinal lumen filled with yellow contents. While piglets in the 0.06 mg / kg HF group still exhibited some degree of perianal contamination and intestinal abnormalities, the overall lesions were significantly reduced, manifested by decreased perianal fecal contamination and reduced small intestinal dilation. These results are consistent with the trends in clinical symptoms and survival rate. In conclusion, 0.06 mg / kg HF administration can alleviate clinical symptoms and intestinal lesions in PEDV-infected piglets and improve their survival rate, indicating that HF ​​has a protective effect against PEDV-challenged piglets.

[0074] 2. Evaluation of changes in piglet weight and body temperature Changes in weight, such as Figure 10 As shown in Figure A, the overall weight change of piglets in the Mock group showed an upward trend. In the PEDV group, weight gain was inhibited after challenge, showing a significant decrease. Compared with the challenged control group, the weight loss of piglets in the 0.06 mg / kg HF administration group was smaller, and the overall trend was improved compared to the challenged control group. These results indicate that PEDV challenge and HF administration had no effect on piglet body temperature, and HF administration can alleviate, to some extent, the diarrhea-related weight loss caused by PEDV infection.

[0075] Changes in body temperature during the entire observation period, such as Figure 10 As shown in Figure B, the body temperature of piglets in each group was within the normal physiological range, and no abnormal fluctuations were observed, indicating that neither PEDV challenge nor HF administration had a significant impact on the body temperature of piglets.

[0076] 4. Evaluation of the protective effect on the digestive tract of piglets Piglet intestinal tissue collected on day 7 post-infection was fixed in 10% neutral formaldehyde solution for 24 h, dehydrated in a gradient manner, embedded in paraffin, and cut into 3 μm sections. After dewaxing and hydration, hematoxylin-eosin (H&E) staining was performed, and the sections were mounted with neutral resin and observed and images were acquired under a microscope.

[0077] H&E staining results are as follows Figure 11 As shown, the duodenum, jejunum, and ileum of piglets in the PEDV group exhibited varying degrees of pathological damage: the duodenum showed villus atrophy, blurred outlines, and disintegration at the apex; the jejunum showed necrosis and dissolution of villus epithelial cells, villus disintegration, and a large number of sloughed epithelial cells were visible in the intestinal lumen; the ileum showed shortened villus and degeneration, necrosis, and shedding of villus epithelial cells. Compared with the PEDV group, the villus structure of each intestinal segment of piglets in the 0.06 mg / kg HF treatment group was relatively intact, and the degree of villus atrophy and shedding was less severe than in the PEDV group. These results indicate that 0.06 mg / kg HF can effectively alleviate the pathological damage to the small intestinal tissue of piglets caused by PEDV infection.

[0078] 5. Evaluation of the inhibitory effect of HF on PEDV replication in vivo Intestinal tissue collected from piglets on day 7 post-infection was thoroughly homogenized using a multi-sample cryo-grinder to prepare tissue homogenate. The tissue homogenate was subjected to three freeze-thaw cycles and then centrifuged at 12000 rpm for 15 min, and the supernatant was collected. Subsequently, total RNA was extracted from lung tissue using the Novizan RNA isolater Total RNA Extraction Reagent kit (R401-01) as described in Example 3, and the RNA was reverse transcribed into cDNA using the Novizan HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (R323-01) with PEDV- N A standard curve was established using recombinant plasmids (provided by the College of Veterinary Medicine, Yangzhou University) as standards. The standard curve was plotted with the cycle threshold Ct on the ordinate and the common logarithm of plasmid copy number lgC on the abscissa. Based on this, the PEDV in the samples was calculated. N Gene copy number. The primer and probe sequences are as follows: PEDV- N -F:5'-gaattcccaagggcgaaaat-3'; PEDV- N -R:5'-ttttcgacaaattccgcatct-3'; PEDV- N probe: 5'-FAM-cgtagcagcttgcttcggaccca-BHQ-3'; the reaction system and conditions are the same as in Example 3.

[0079] Viral RNA was extracted from daily anal swabs collected from piglets using the Novizan RNA isolater Total RNA Extraction Reagent kit (R401-01), and the PEDV content was determined using the aforementioned method. N Gene copy number.

[0080] The results of the viral load assay in the small intestine are as follows: Figure 12 As shown in Figure A, high viral loads were detected in the duodenum, jejunum, and ileum of the PEDV group, with the highest level in the jejunum; the viral load in the small intestine of the 0.06 mg / kg HF group was lower than that of the PEDV group. Fecal viral shedding results are as follows... Figure 12 As shown in Figure B, the PEDV group began to show significant viral shedding on day 2 after challenge, maintaining a high level from day 3 to 5, then gradually declining, but a high viral load was still detectable on day 7. In the 0.06 mg / kg HF treatment group, some piglets shed the virus on day 2 after challenge, but the overall viral shedding level was lower than that in the PEDV group, with a more significant decrease in the later stages. These results indicate that HF ​​treatment showed an overall decreasing trend in viral load in the intestinal tissue and viral shedding in feces in piglets.

[0081] Example 5: Biosafety evaluation of styraxone in mice SPF-grade male C57BL / 6 mice aged 5–6 weeks (purchased from the Experimental Animal Center of Yangzhou University) were selected and, after acclimatization, randomly divided into a negative control group (NC) and a HF experimental group, with 6 mice in each group. The HF experimental group was administered 0.5 mg / kg HF (prepared with PBS containing 1% DMSO as the solvent) via intraperitoneal injection, while the negative control group was given an equal volume of PBS containing 1% DMSO. Administration was once daily for 8 consecutive days. The experimental period was 14 days. During this period, the mice's mental state, activity level, coat color changes, and food and water intake were observed daily, and weight changes were recorded regularly.

[0082] After the experiment, orbital blood was collected from mice and placed in either anticoagulant or procoagulant blood collection tubes. Anticoagulant whole blood was used for complete blood count (CBC) analysis, while serum samples were used for serum biochemical assays. CBC and serum biochemical assays were performed by the College of Veterinary Medicine, Yangzhou University. The mice were then euthanized and dissected, and the heart, liver, spleen, lungs, and kidneys were harvested. After fixation using the methods described above, the organs were stained with Hematoxylin and eosin (H&E) and observed under a light microscope to assess the potential toxic effects of HF on major organs. The reference ranges for mouse CBC and biochemical assay data were derived from the Chinese Veterinary Pharmacopoeia and relevant literature.

[0083] Survival curves Figure 13As shown in Figure A, no mice in any experimental group died during the entire biosafety evaluation period, indicating that HF ​​at a dose of 0.5 mg / kg did not cause acute toxicity in mice. Weight monitoring results are as follows: Figure 13 As shown in Figure B, the weight gain of mice in the HF group was slower and less pronounced in the early stages of administration compared to the NC group. After drug withdrawal, the weight of mice in the HF group gradually recovered and returned to levels close to those of the control group. These results indicate that intraperitoneal injection of 0.5 mg / kg HF inhibits weight gain in mice, but this inhibition is only transient and does not lead to a sustained decrease in weight.

[0084] Histopathological examination results as follows Figure 14 As shown, the organ structures of mice in the HF group were basically intact, and no obvious pathological abnormalities were observed compared with those in the NC group; no obvious pathological changes such as necrosis, degeneration, edema, congestion or inflammatory cell infiltration were observed in the heart, liver, spleen, lungs and kidneys.

[0085] The results of blood tests and serum biochemical marker tests performed on mice are as follows: Figure 15 As shown in the statistical chart, the gray areas represent the reference ranges for each indicator, all of which are determined based on the Chinese Veterinary Pharmacopoeia and relevant literature. Specifically, blood routine test results showed that the white blood cell count (WBC), lymphocyte count (LYMPH), monocyte count (MONO), neutrophil count (Gran), red blood cell count (RBC), and hemoglobin concentration (HGB) remained relatively stable between the HF and NC groups, and all were within the reference range. Biochemical test results showed that the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and urea (UREA) in the HF group did not show significant abnormalities and were generally within the reference range. Although the AST levels of some mice were higher than the reference range, they were not accompanied by a synchronous increase in ALT, and histopathological examination showed no obvious liver damage. This suggests that the change is more likely related to individual differences, sampling process, or sample processing factors, and is not sufficient to conclude that HF ​​is hepatotoxic to mice at this dose.

[0086] Example 6: Biosafety evaluation of styraxone in piglets Five-day-old piglets (purchased from a farm in Jiangsu Province) were randomly divided into a negative control group (NC) and a HF experimental group, with three piglets in each group. The HF experimental group received 0.18 mg / kg HF (prepared with PBS containing 1% DMSO as the solvent) via intraperitoneal injection, while the negative control group received an equal volume of PBS containing 1% DMSO. Administration was once daily for four consecutive days. The experimental period was 11 days. During this period, the piglets' mental state, activity level, coat color changes, and food and water intake were observed daily, and changes in weight and body temperature were recorded regularly. One day after the last administration, blood was collected via the anterior vena cava and placed in anticoagulant and procoagulant blood collection tubes, respectively. Anticoagulant whole blood was used for complete blood count (CBC) analysis, and serum samples were used for serum biochemical assays. CBC and serum biochemical assays were performed by the relevant testing platform of the Veterinary College of Yangzhou University. After the experiment, the piglets were euthanized and dissected. The heart, liver, spleen, lungs, and kidneys were removed, fixed according to the aforementioned experimental methods, and then subjected to Hematologic & Escherichia coli (H&E) staining. The reference ranges for blood routine and biochemical indicators in piglets are based on data from the Chinese Veterinary Pharmacopoeia and related literature.

[0087] Survival curves Figure 16 As shown, no obvious abnormalities were observed in the clinical manifestations of piglets in each group during the observation period, and no deaths occurred during the entire biosafety evaluation period, indicating that HF ​​at a dose of 0.18 mg / kg did not cause acute toxicity in piglets.

[0088] Weight monitoring results as follows Figure 17 As shown in Figure A, the body temperature monitoring results are as follows: Figure 17 As shown in B, after administration of 0.18 mg / kg HF, the weight of piglets was not significantly different from that of the NC group, and the overall trend remained the same; no abnormalities were observed in body temperature changes either.

[0089] Histopathological examination results as follows Figure 18 As shown, the organ structures of piglets in the HF group were basically intact, and no obvious pathological abnormalities were observed compared with those in the NC group.

[0090] Blood samples were collected from piglets for routine blood tests and serum biochemical marker analysis. The results are as follows: Figure 19As shown in the chart, the gray areas represent the reference ranges for each indicator, all determined according to the Chinese Veterinary Pharmacopoeia and relevant literature. Blood routine tests showed that WBC, LYMPH, middle cell (Mid) absolute count, Gran, RBC, and HGB remained generally stable between the HF and NC groups, with no consistent abnormal changes, and most indicators were within the reference range. Although some piglets had WBC, RBC, and HGB values ​​exceeding the reference range, the fluctuations were small, suggesting they were more likely related to individual differences or differences in baseline physiological state. Biochemical tests showed that ALT, AST, and UREA levels in the HF group were not significantly abnormal and remained within the reference range overall.

Claims

1. The use of a styraquinone or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and treatment of viral diseases in animals.

2. The application according to claim 1, characterized in that, The pharmaceutically acceptable salt is selected from one or more of hydrobromide, hydrochloride, sulfate, phosphate, methanesulfonate, acetate, lactate, citrate, tartrate, maleate, fumarate, or succinate.

3. The application according to claim 1, characterized in that, The animal viral diseases mentioned are diseases caused by one or more of the following viruses: porcine pseudorabies virus, porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, influenza A virus H1N1 subtype, Newcastle disease virus, porcine circovirus type 2, and porcine transmissible gastroenteritis virus.

4. The application according to claim 1, characterized in that, The application is in the preparation of drugs that reduce viral load during the course of viral diseases in animals.

5. The application according to claim 1, characterized in that, The application is in the preparation of drugs to improve tissue damage caused by viral diseases in animals.

6. The application according to claim 1, characterized in that, The application is in the preparation of drugs that improve animal survival rates.

7. The application according to claim 1, characterized in that, The drug contains styraxone or a pharmaceutically acceptable salt thereof as its active ingredient.

8. The application according to claim 7, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients include any one or more of the following: diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

10. The application according to claim 1, characterized in that, The dosage forms of the drugs include capsules, granules, powders, tablets, microcapsules, injections, infusions, oral liquids, suspensions, patches, suppositories, microemulsions, liposomes, nanoparticles, and lyophilized powder injections.

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