Use of UH15-38 in the preparation of drugs for preventing and treating PEDV-PoRVA co-infection

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of UH15-38 in preparation of a medicine for preventing and treating PEDV-PoRVA co-infection. The application first systematically establishes a PEDV and PoRVA co-infection piglet model, and in combination with a high-throughput transcriptome method, deeply analyzes an enhancement mechanism of the co-infection on host pathogenicity from an immune regulation level. Based on the mechanism, the application finds that UH15-38 can effectively treat PEDV and PoRVA co-infection piglets. Experimental data show that UH15-38 significantly relieves clinical diarrhea symptoms caused by the co-infection, reduces PoRVA and PEDV virus copy numbers, effectively inhibits replication of the viruses in the intestinal tract, and significantly reduces IL-1beta and p-MLKL expression levels in jejunum tissue, and reduces necrotic apoptosis and inflammation. UH15-38 can provide new application value for treatment of porcine viral diarrhea.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of UH15-38 in the preparation of drugs for the prevention and treatment of PEDV-PoRVA co-infection. Background Technology

[0002] Porcine viral diarrhea is a diarrheal disease caused by viral infection in pigs. Porcine epidemic diarrhea virus (PEDV) and porcine rotavirus A (PoRVA) are among the main pathogens causing porcine diarrhea. Newborn piglets infected with these pathogens experience severe diarrhea, dehydration, and even death, with extremely high morbidity and mortality rates. In actual production, multiple pathogens often co-circulate in porcine diarrhea cases, and viral recombination and co-infection further exacerbate the severity of the disease and increase the difficulty of prevention and control. PEDV belongs to the genus Coronavirus of the family Coronaviridae and is an enveloped, single-stranded, positive-sense RNA virus. Porcine rotavirus (PoRV) belongs to the family Reoviridae and the genus Rotaviruses. Based on serological classification, it can be divided into five groups: A, B, C, E, and H. PoRVA is considered the most widely reported and most pathogenic group of porcine rotaviruses, especially in young pigs, where infection often manifests as highly contagious diarrheal symptoms. Existing PEDV and PoRVA vaccines offer limited protection, and there is a lack of effective targeted treatment strategies for swine viral diarrhea caused by co-infection with multiple viruses. This co-infection of PEDV and PoRVA has become a significant factor threatening the pig industry. Summary of the Invention

[0003] The first aspect of the present invention is to provide the use of UH15-38 or a pharmaceutically acceptable salt thereof in the preparation of a drug for the prevention and treatment of porcine viral diarrhea.

[0004] A second aspect of the present invention aims to provide the use of UH15-38 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing co-infection with porcine epidemic diarrhea virus and porcine rotavirus.

[0005] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: A first aspect of the invention provides the use of UH15-38 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and treatment of porcine viral diarrhea; The chemical formula of UH15-38 is C 26 H 27 N5O2; The structural formula is shown in equation (Ⅰ): Equation (Ⅰ).

[0006] In some embodiments of the present invention, the swine viral diarrhea is caused by infection with any one of groups of viruses 1) to 3): 1) Porcine epidemic diarrhea virus (PEDV); 2) Porcine Rotavirus A (PoRVA); 3) Porcine epidemic diarrhea virus and porcine rotavirus A.

[0007] In some embodiments of the present invention, the prevention and / or treatment include prevention and / or treatment.

[0008] In some embodiments of the present invention, the swine viral diarrhea is caused by co-infection with swine epidemic diarrhea virus and swine rotavirus.

[0009] In some embodiments of the present invention, the porcine rotavirus includes porcine rotavirus A, porcine rotavirus B, porcine rotavirus C, porcine rotavirus E, and porcine rotavirus H.

[0010] In some embodiments of the present invention, the porcine rotavirus is porcine rotavirus A.

[0011] In some embodiments of the present invention, the pharmaceutically acceptable salt includes: acid addition salts and base addition salts.

[0012] In some embodiments of the present invention, a "pharmaceutically acceptable acid addition salt" refers to a salt that retains the biological effectiveness and properties of the free base, is not undesirable in biological or other respects, and is formed from an inorganic acid and an organic acid, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and such as, but not limited to, acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor 10 sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, dodecyl sulfate, ethane 1,2 disulfonic acid, ethane sulfonic acid, 2-hydroxyethane sulfonic acid, fumaric acid, galactopyric acid, gentian acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, glutaric acid, 2 Oxoglutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, etc.

[0013] In some embodiments of the present invention, a "pharmaceutically acceptable base addition salt" refers to a salt that retains the biological effectiveness and properties of the free acid and is not undesirable in biological or other respects. These salts are prepared by the addition of an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, thiazoline, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0014] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients.

[0015] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, release inhibitors, and carriers.

[0016] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0017] In some embodiments of the present invention, the dosage form of the drug includes one of the following: powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0018] In some embodiments of the present invention, the administration of the drug includes administration via the gastrointestinal tract or non-gastrointestinal route.

[0019] In some embodiments of the present invention, the gastrointestinal administration includes one of oral administration, sublingual administration, and rectal administration.

[0020] In some embodiments of the present invention, the non-gastrointestinal administration includes one of intravenous injection, subcutaneous injection, and mucosal administration.

[0021] In some embodiments of the present invention, the product is applied to mammals; preferably pigs.

[0022] In some embodiments of the present invention, when UH15-38 is administered to pigs, its concentration is 1~10 mg / kg.

[0023] A second aspect of the present invention aims to provide the use of UH15-38 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating co-infection with porcine epidemic diarrhea virus and porcine rotavirus.

[0024] The beneficial effects of this invention are: This invention establishes, for the first time, a piglet model co-infected with PEDV and PoRVA. Using high-throughput transcriptomics, it delves into the mechanism by which co-infection enhances host pathogenicity at the level of immune regulation. Based on this mechanism, this invention discovers that UH15-38 can effectively treat piglets co-infected with PEDV and PoRVA. Experimental data show that UH15-38 significantly alleviates clinical diarrhea symptoms caused by co-infection, reduces the viral copy number of PoRVA and PEDV, effectively inhibits viral replication in the intestine, and significantly reduces the expression levels of IL-1β and p-MLKL in jejunal tissue, as well as alleviating necrosis, apoptosis, and inflammatory responses. UH15-38 offers new application value for the treatment of porcine viral diarrhea. Attached Figure Description

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 Clinical symptom scores were assigned to pigs infected with PEDV, PoRVA, PEDV, and PoRVA, respectively, and to healthy pigs.

[0026] Figure 2Clinical diarrhea photographs of pigs infected with PEDV, PoRVA, PEDV and PoRVA respectively, where: (A) PEDV; (B) PoRVA; (C) PEDV and PoRVA; (D) healthy piglets.

[0027] Figure 3 Results of IHC and HE staining analysis of the jejunum of piglets infected with PEDV, PoRVA, and PEDV and PoRVA.

[0028] Figure 4 The results of the PEDV and PoRVA co-infection of a 7-day-old piglet transcriptome model are as follows: (A) swab copy number; (B) tissue viral load; (C) clinical score; (D) survival rate (significance is indicated by an asterisk. * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P < 0.001).

[0029] Figure 5 Clinical anatomical lesions of piglets co-infected with PEDV and PoRVA were identified as follows: (A) PoRVA group infected with 7-day-old piglets; (B) PEDV group infected with 7-day-old piglets; (C) PEDV and PoRVA co-infected group infected with 7-day-old piglets; (D) Control group 7-day-old piglets.

[0030] Figure 6 This is a schematic diagram of the transcriptomics sample preparation process.

[0031] Figure 7 The results show the statistical analysis of the number of differentially expressed genes and the volcano plots for different treatment groups, including: (A) PCA analysis; (B) statistical analysis of differentially expressed genes in the PEDV, PoRVA, and PEDV and PoRVA co-infection groups; (C) Venn diagram of the intersection of differentially expressed genes in the PEDV, PoRVA, and PEDV and PoRVA co-infection groups; and (D) volcano plots of differentially expressed genes in the PEDV, PoRVA, and PEDV and PoRVA co-infection groups.

[0032] Figure 8To explore the necroptosis pathway using clinical pathological sections combined with transcriptomics data, including: (A) pathological lesions in HE sections; (B) the ratio of villous height to crypt depth (VH:CD) in HE sections; (C) GSEA enrichment curves for necroptosis in the PoRVA, PEDV, and PEDV-PoRVA co-infection groups; (D) GSEA enrichment curves for apoptosis; (E) GSEA enrichment curves for ferroptosis; (F) the distribution of core enriched genes in the GSEA enrichment curves for necroptosis in the PEDV-PoRVA co-infection group; (G) a heatmap showing gene expression levels in the necroptosis pathway in the PoRVA, PEDV, and PEDV-PoRVA co-infection groups; and (H) a network diagram of core gene interactions in the necroptosis pathway (significance is indicated by asterisks. * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P < 0.001).

[0033] Figure 9 To validate the expression results of p-RIPK3 and p-MLKL in IPEC-J2 cells at the protein level, the results were as follows: (A) Western blot analysis to validate the expression of differentially expressed genes; (B) grayscale scan results of p-RIPK3 and p-MLKL genes (significance is indicated by asterisks. * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P < 0.001).

[0034] Figure 10 The results of UH15-38 inhibition of IPEC-J2 cells co-infected with PEDV and PoRVA were validated, including: (A) protein level validation of the expression of PEDV N, PoRVA VP6, RIPK3, p-RIPK3, MLKL and p-MLKL; (B) grayscale scanning results of p-RIPK3 and p-MLKL genes (significance is indicated by asterisks. * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P < 0.001).

[0035] Figure 11 The results of UH15-38 in improving the clinical symptoms of piglets co-infected with PEDV and PoRVA were as follows: (A) Flowchart; (B) Dynamic changes in PEDV and PoRVA viral load in piglet feces; (C) Comparison of viral load in tissues; (D) Trends in clinical symptom scores of piglets in each experimental group; (E) Survival analysis of piglets after challenge in each experimental group (significance is indicated by an asterisk. * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P < 0.001).

[0036] Figure 12The results of IHC and HE staining analysis of PEDV and PoRVA in the jejunal tissue of co-infected piglets after UH15-38 treatment.

[0037] Figure 13 The results of IHC analysis show the effects of UH15-38 on the expression of IL-1β and p-MLKL in the jejunal tissue of piglets co-infected with PEDV and PoRVA. Detailed Implementation

[0038] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0039] The reagents and consumables used in this invention are as follows: UH15-38 was purchased from MedChemExpress; Phospho-MLKL (Ser345) (D6E3G) RabbitmAb antibody was purchased from Cyxin Biotech; Anti-BRoVV / P6 mAb (M100055) and Anti-PEDV / NP mAb (M100048) were purchased from Beijing Subenyuan Biotechnology Co., Ltd.; Phospho-RIPK3 (Ser316) Antibody and RIPK3 Antibody were purchased from Affinity Biosciences; Anti-MLKL Antibody was purchased from Huaan Biotechnology; Hifair® V C58P2 Multiplex One Step RT-qPCR Probe Kit (UDG Plus) was purchased from Shanghai Yisheng Biotechnology Co., Ltd.; 1× Phosphate Buffered Salt Solution... Saline (PBS) was purchased from Hyclone; DNA molecular weight marker was purchased from TaKaRa; DMEM medium, FBS fetal bovine serum, and 0.25% trypsin-EDTA were purchased from Gibco; penicillin-streptomycin antibiotics and DAPI staining solution were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; Trizol was purchased from Invitrogen; and disposable cotton swabs were purchased from Hangzhou Optipro Biotechnology Co., Ltd.

[0040] All biological experiments in this invention were independently repeated three times. The data are presented in the form of "mean ± standard error (mean ± SEM)". Statistical tests for differences between groups were performed using a two-tailed t-test, with a significance level set at P < 0.05. The significance of the results is expressed by the following symbols: ns indicates no significant difference; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0041] Example 1: Statistical Analysis of Clinical Symptoms of Co-infection with PEDV and PoRVA 1. Hematoxylin-eosin staining (H&E) (1) Dewax the paraffin slices in xylene 2-3 times, each time for about 5 minutes.

[0042] (2) Gradually transfer the sections to an ethanol gradient: 100%, 95%, 90%, and 80% ethanol, soaking for 3 minutes in each concentration, gradually removing the ethanol and introducing the aqueous phase. Finally, soak the sections in distilled water for 3-5 minutes.

[0043] (3) Place the sections in the hematoxylin solution and stain for 5-10 minutes. Gently wash the sections with distilled water to remove excess hematoxylin.

[0044] (4) Immerse the sections in 1% hydrochloric acid ethanol for a short time (about 10-20 seconds). Rinse the sections with water.

[0045] (5) Immerse the sections in eosin solution and stain for 3-5 minutes. Wash the sections with distilled water to remove excess eosin.

[0046] (6) Gradually immerse the sections in ethanol solutions of progressively increasing concentrations: 70%, 80%, 90%, 95%, and 100%, soaking for 3-5 minutes in each concentration. Immerse the sections in xylene 2-3 times, 5 minutes each time, until the sections become transparent.

[0047] (7) Remove the slide from the xylene, apply a mounting medium, and cover with a coverslip. Observe the staining results using a microscope.

[0048] 2. Immunohistochemistry (1) Fix the tissue with 10% neutral formalin, usually for 4–24 hours.

[0049] (2) The tissue was dehydrated by ethanol of different concentrations and then transparentized with xylene.

[0050] (3) Immerse the transparent tissue in paraffin wax, and cut it into thin slices after the paraffin wax solidifies.

[0051] (4) Use a microtome to cut the tissue into 4-5 μm thin slices and attach them to a glass slide.

[0052] (5) After deparaffining with xylene, the sections were gradually hydrated with ethanol of different concentrations (100%, 95% and 70%), and finally washed with distilled water.

[0053] (6) Heat retrieval with appropriate buffer solution, usually by heating at 90-100°C for 10-20 minutes in a microwave oven or pressure cooker to restore the detectability of the antigen.

[0054] (7) Treat the sections with hydrogen peroxide (H2O2) to remove endogenous peroxidase in the tissue and prevent background interference.

[0055] (8) Block the sections with a blocking solution containing normal animal serum or BSA (bovine serum albumin), and incubate at room temperature for 30 minutes to reduce nonspecific binding.

[0056] (9) Incubate the slide with a specific primary antibody (antibody against the target antigen). The incubation time varies depending on the source and brand of the antibody. Usually, it is incubated overnight at 4°C or for 1-2 hours at room temperature.

[0057] (10) Wash the slides multiple times with PBS (phosphate buffer) or TBS (Tris buffer) to remove unbound primary antibodies.

[0058] (11) Incubate the slices with a secondary antibody that matches the source of the primary antibody for 1 hour.

[0059] (12) Wash the sections with PBS or TBS to remove unbound secondary antibodies.

[0060] (13) Using diaminobenzidine (DAB) as a chromogenic substrate, DAB reacts with HRP to generate a visible brown product that indicates the location of the antigen. If a fluorescently labeled secondary antibody is used, it can be observed directly with a fluorescence microscope.

[0061] (14) Use hematoxylin to reverse stain the sections. Usually, reverse staining takes 1-3 minutes. Hematoxylin can stain cell nuclei, making it convenient for observation.

[0062] (15) Sections were dehydrated with ethanol of different concentrations (70%, 95% and 100%). Sections were cleared with xylene. Mounted with neutral resin.

[0063] 3. Experimental Results In the clinical production follow-up, this example found that PEDV and PoRVA single infection, PEDV and PoRVA co-infection, and healthy piglets showed higher clinical scores in clinically infected piglets. Figure 1 ) and more severe clinical symptoms ( Figure 2 This indicates that co-infection with both viruses leads to more severe intestinal damage, manifested as more severe diarrhea and higher mortality. However, existing conventional antiviral drugs are not effective in treating co-infected cases, and co-infection further complicates the condition. Dissection of pigs infected with PoRVA and PEDV alone and in combination with PoRVA and PEDV revealed severe necrosis and shedding of intestinal villi in piglets with co-infection. IHC and HE staining were performed to detect viral infection and pathological damage. Figure 3 ).

[0064] Clinical symptom scores (CSS) assess the severity of diarrhea based on stool consistency: 0 points, normal (no diarrhea); 1 point, soft stool; 2 points, mild watery stool; 3 points, moderate mucous or watery diarrhea; 4 points, severe watery or projectile diarrhea.

[0065] Example 2 Experimental design for co-infection of piglets with PEDV and PoRVA 1. Experimental design for co-infection of piglets with PEDV and PoRVA All experimental animals were sourced from a well-established, conventionally raised farm with a good health record. Prior to the experiment, all piglets were tested to confirm they were free of porcine enteroviruses. Twenty-four 7-day-old piglets were selected and randomly divided into four groups of six, housed in separate rooms. The piglets were fed fresh milk (heated to approximately 39°C) five times daily and had free access to distilled water throughout the experiment. The groups were: PEDV group, PoRVA group, PEDV and PoRVA group, and PBS group. The PEDV group received 1 mL of 1×10⁻⁶ PBS orally. 5 TCID 50 PEDV XY strain; PoRVA group orally administered 1 mL of 1×10 6 TCID 50 The PoRVA / CHN / GD / 2024 strain was used. Piglets in both the PEDV and PoRVA groups were orally administered 1 mL of each virus. Clinical symptoms, such as diarrhea, vomiting, and loss of appetite, were monitored daily in infected piglets. Rectal swabs were collected daily, and viral RNA loads of the PEDV N gene and PoRVA VP4 gene in pig feces were detected using quantitative real-time RT-qPCR. All surviving pigs were euthanized at the end of the experiment (day 7 post-infection).

[0066] The Clinical Symptom Score (CSS) assesses the severity of diarrhea based on stool consistency: 0 points, normal (no diarrhea); 1 point, soft stool; 2 points, mild watery stool; 3 points, moderate mucous or watery diarrhea; 4 points, severe watery or projectile diarrhea.

[0067] 2. Experimental Results Previous epidemiological investigations revealed a surge in the co-infection of PEDV and PoRVA in pig herds, leading to faster onset of clinical diarrhea in piglets, particularly higher mortality rates in newborn piglets. To investigate the impact of PEDV and PoRVA co-infection in piglets, this study established a co-infected piglet animal model. This model is crucial for elucidating the mechanisms underlying the accelerated disease progression and high mortality caused by co-infection, thus providing a solid foundation for further research into the pathological changes, immune responses, and potential therapeutic interventions induced by co-infection. Seven-day-old piglets were infected with PEDV and PoRVA separately or simultaneously, while negative control piglets remained normal throughout the experiment. RT-qPCR (Hifair® V C58P2 Multiplex One Step RT-qPCR Probe Kit (UDG Plus) purchased from Shanghai Yisheng Biotechnology Co., Ltd.) detected a peak PoRVA swab copy number of 10 on day 1 in the co-infected group. 6.3 Genome copy number / mL, PoRVA group 10 2.8 Genome copy number / mL, subsequently decreased in the co-infection group, and reached its highest level of 10 on day 4 in the PoRVA group. 5.7 Genome copy number / mL, co-infection group: 10 3.98 Genome copy number / mL ( Figure 4 In PoRVA fecal swabs and jejunal tissue samples from the infected group (A), Figure 4 In the co-infection group (B), viral copy number was suppressed compared to the PoRVA group. There was no significant difference in viral copy number in fecal swabs and jejunal tissue between the co-infection and PEDV groups. Seven-day-old piglets infected with PEDV developed mild diarrhea on day 2, severe diarrhea from day 3 to day 6, and began to recover on day 7. Figure 4 (C) The survival rate of piglets 7 days after infection was 83.3% ( Figure 4 (D). In the 7-day-old PoRVA group, the survival rate of infected piglets was 100%. The co-infected group showed mild diarrhea on day 1 and severe diarrhea from day 2 to day 6. Figure 4 In the co-infected group (C), the survival rate was only 33.3%, and co-infection resulted in a higher mortality rate in 7-day-old piglets. Figure 4 (D).

[0068] The results indicate that the animal model of co-infection with PEDV and PoRVA is consistent with the clinical co-infection results, providing a valuable tool for further research on the molecular mechanisms and immune responses caused by co-infection.

[0069] Example 3: Transcriptomic study of 7-day-old piglets co-infected with PEDV and PoRVA 1. Preparation of intestinal tissue transcriptomic samples from 7-day-old piglets co-infected with PEDV and PoRVA Forty piglets were randomly selected and divided into four groups of ten, representing ten biological replicates. On day 4 post-infection, six piglets were randomly selected for dissection, and fresh infected jejunal tissue was harvested. Connective tissue and other non-research-required tissue types were immediately removed. The tissue surface was quickly rinsed with pre-cooled PBS (RNase-free) or 0.9% saline to remove any residual blood. The tissue was cut into small pieces (approximately the size of a soybean) with dimensions ≤0.5 mm (length, width, and height). Sufficient liquid nitrogen was prepared in advance for freezing the tissue, and samples were pre-loaded into cryovials. The frozen tissue samples were thoroughly mixed and stored in 2 mL or larger screw-cap cryovials (RNase-free), labeled, and immediately (within 20 seconds) frozen in liquid nitrogen for 3–4 hours. Afterward, they were transferred to -80 °C for long-term storage and transported to Biomarker Biotech Ltd. on dry ice.

[0070] 2. Library construction and sample sequencing Nucleic acid was extracted using TRIzol reagent, and its concentration was detected using Nanodrop 2000. Integrity was assessed using Agient 2100 and LabChip GX. mRNA Capture Beads were thoroughly mixed on a four-dimensional gyroscope and equilibrated for 30 min before use. The mRNA Capture Beads were then added to the prepared total RNA sample and incubated at 65°C to denature the RNA. Reagents required for reverse transcription of the first strand were added to the centrifuge tube from the previous step, mixed, centrifuged, and then programmed for cDNA first-strand synthesis in a PCR instrument. Reagents for second-strand synthesis were added sequentially to the synthesized cDNA first-strand product, mixed, centrifuged, and then programmed for second-strand synthesis in a PCR instrument. All reagents required for the PCR reaction were added to the centrifuge tube, mixed, centrifuged, and then amplified using a PCR instrument according to the PCR reaction conditions. The constructed library was sequenced using an Illumina Novaseq 6000.

[0071] PE150 sequencing platform: Illumina NovaSeq 6000 platform, manufactured in San Diego.

[0072] PE150 Sequencing Reagent: NovaSeq 6000 S4 Reagent Kit, made in San Diego.

[0073] 3. HE slice analysis HE sections were analyzed using ImageJ software to determine the villus height to crypt depth (VH:CD) ratio. The intestinal pathological damage scoring criteria are shown in Table 1 below.

[0074] Table 1. Intestinal pathological damage score

[0075] 4. Bioinformatics Analysis The raw expression data were analyzed using R software. Differentially expressed genes were identified using the DESeq2 package with log2|Fold Change|≥ 1.5 and p<0.001 as the screening criteria. GSEA (Gene Set Enrichment Analysis) analysis was performed using the clusterProfiler package, and plotting was done using ggplot2 and ggrepel. Gene interaction networks were plotted using Cytoscape.

[0076] 5. Experimental Results In animal experiments involving co-infection of piglets with PEDV and PoRVA, co-infection resulted in more severe pathological damage and a higher mortality rate, and co-infection inhibited PoRVA replication. This finding suggests that co-infection accelerates the disease progression in piglets through some mechanism. To further explore the molecular mechanism of this clinical phenomenon, this embodiment will analyze the effects of PEDV and PoRVA co-infection on gene expression in the piglet intestine using transcriptomics. Transcriptomics analysis will help understand the molecular regulatory network within host cells under viral co-infection conditions, reveal changes in gene expression in the piglet intestine under co-infection conditions, and explore related pathogenic mechanisms. To assess the pathogenicity of different infections during the acute infection phase, the differences in small intestinal damage in piglets from different infection groups were evaluated. The PoRVA group showed swelling and fluid accumulation (…). Figure 5 In the PEDV group, the intestines showed significant dilation, with some areas exhibiting a pale appearance, indicating mucosal damage. Figure 5 (Medium B). The co-infected group showed more severe tissue damage, villous atrophy, and hemorrhage in the intestines. The intestines were swollen and lost their normal structural integrity. Figure 5 (C) The intestinal anatomy of the blank control group was normal. Figure 5 (D). Transcriptome analysis workflow as follows: Figure 6 As shown.

[0077] Results of gene differential expression analysis as follows Figure 7As shown, differentially expressed genes were screened using log2Fold Change ≥ 1.5 and P < 0.001 as the screening criteria. PCA analysis results showed good intra-group repeatability and large inter-group differences. Figure 7 (A). By comparing the PEDV-infected group and the control group, a total of 2275 significantly differentially expressed genes were found in the jejunum, of which 1447 genes were upregulated and 828 genes were downregulated. Figure 7 (B) Comparison between the PoRVA infection group and the control group revealed 137 significantly different genes in the jejunum, of which 86 genes were upregulated and 51 genes were downregulated. Comparison between the PEDV and PoRVA co-infection group and the control group revealed 1869 significantly different genes in the jejunum, of which 853 genes were upregulated and 1016 genes were downregulated. Furthermore, there were 93 overlapping genes in the jejunum among the PEDV infection group, the PoRVA infection group, and the PEDV and PoRVA co-infection group. Figure 7 The five most significant genes in the jejunum of PoRVA infection were ADAM7, LARP6, CPO, FAM151A, and TMEM252; the five most significant genes in the jejunum of the PEDV infection group were ADAM7, FGFBP1, TGM3, GHRL, and FTCD; the five most significant genes in the jejunum of the PoRVA and PEDV co-infection group were KDM5D, ADAM7, SPAI-2, F11, and USP9Y. Figure 7 (D). Significantly downregulated genes increased during co-infection. These data indicate that PoRVA and PEDV, when infecting the host alone and co-infecting the host, respectively, result in different and widespread changes in the host's gene expression patterns.

[0078] We observed the damage to each infected group in the intestinal segment using HE sections, and quantified the degree of pathological damage ( Figure 8 The ratio of intestinal villus length (VH) to crypt depth (CD) (A) Figure 8 (B) Under normal physiological conditions, a healthy intestine has longer villi, shallower crypts, a higher VH:CD ratio, sufficient mucosal absorptive area, and balanced cell renewal. Viral infection leads to necrosis and shedding of villous epithelial cells, shortening, fusion, and even disappearance of villi, resulting in a sharp reduction in absorptive area. To compensate for the loss of villous cells, stem cells at the base of the crypts accelerate proliferation and differentiation, leading to increased crypt depth. The lower the VH:CD ratio, the more severe the mucosal damage. Both the infection group and the control group showed a decrease in VH:CD, with the co-infection group exhibiting the most severe pathological damage and the greatest decrease in VH:CD, manifesting as signs of necrosis.

[0079] Further GSEA analysis of death-related pathways in each infection group revealed upregulation of necroptosis and apoptosis pathways, while ferroptosis was inhibited. Figure 8(C, D, E). Furthermore, we visualized the trend of higher upregulation of the necroptosis pathway and its core genes in the GSEA diagram during co-infection. Genes such as RIPK3, VDAC2, and AIFM1 are key drivers of enrichment. Figure 8 (F). A heatmap was used to illustrate the differentially expressed genes in the necroptosis pathway compared to the control group in the three infection groups. Figure 8 (G). A core gene interaction network of the necrosis-apoptosis pathway was plotted using Cytoscape, and the top 10 genes by priority were shown. RIPK3 is the most critical gene, indicating that this molecule may play a key regulatory role. Figure 8 Co-infection with H (H) causes severe tissue damage by activating the necroptotic pathway.

[0080] Example 4: Validation based on transcriptomics results 1. Establishment of an IPEC-J2 cell model co-infected with PEDV and PoRVA IPEC-J2 cells in the cell culture and passage growth phase were trypsinized and seeded into 12-well plates. Cells were cultured to 80%-90%. The PEDV strain was diluted with sterile PBS to adjust the viral titer to an appropriate concentration (MOI=0.1). IPEC-J2 cells were infected with the virus solution for 1 hour. After infection, the virus solution was replaced with RPMI-1640 medium containing 10 v / v% fetal bovine serum, and the cells were cultured until the desired time point. The PoRVA virus solution was diluted to the required MOI (MOI=0.1). IPEC-J2 cells were infected with the PoRVA virus solution for 1 hour. After infection, the medium was replaced with RPMI-1640 medium containing 10 v / v% fetal bovine serum, and the cells were cultured until the desired time point. Co-infection with PEDV and PoRVA: The concentrations of PEDV and PoRVA were adjusted separately to maintain their respective MOIs (MOI=0.1). The two virus solutions were mixed and used to infect IPEC-J2 cells at an appropriate ratio. The infection time was 1 hour. After that, the virus solution was replaced with RPMI-1640 medium containing 10 v / v% fetal bovine serum, and the culture was continued for 12 hours before the samples were collected.

[0081] 2. Experimental Results To further investigate the mechanism by which co-infection with PEDV and PoRVA exacerbates clinical symptoms in piglets, this study selected the porcine small intestinal epithelial cell line (IPEC-J2) as a cell infection model to establish cell models infected with PEDV, PoRVA, and both. After culturing IPEC-J2 cells to a monolayer confluence state, they were infected with PEDV, PoRVA, and a mixture of both viruses. The viral infection concentration (MOI=0.1) and infection time (12 h) were determined through prior optimization. Subsequently, the cell infection efficiency was determined by observing the cytopathic effect (CPE) and detecting viral load using RT-qPCR.

[0082] After establishing an IPEC-J2 cell model co-infected with PEDV and PoRVA, Western blotting was used to detect the levels of p-RIPK3 and p-MLKL, confirming that co-infection with PEDV and PoRVA exacerbates clinical symptoms by promoting necrosis and apoptosis. Necrophage-induced apoptosis is a form of programmed cell death, characterized most notably by its high inflammatory nature compared to traditional apoptosis. Necrophage-induced apoptosis can be mediated by viral infection and other mechanisms, activating the key molecule RIPK3, thereby activating the necroptosis pathway, causing changes in membrane permeability, ultimately leading to cell lysis and the release of intracellular contents, resulting in host tissue damage and death. Compared to single viral infection, co-infection with PEDV and PoRVA can further enhance RIPK3 activity. Figure 9 The activation of RIPK3 (a key molecule in the necroptosis signaling pathway), particularly in A and B, induces higher levels of necroptosis, amplifies the inflammatory response, damages the intestinal mucosal barrier, causes more severe diarrhea, and increases piglet mortality. Therefore, precisely regulating the activation level of RIPK3, a key molecule in the necroptosis signaling pathway, holds promise as a new strategy for controlling co-infectious diseases of diarrhea in pigs.

[0083] Example 5: Inhibition of necrotic apoptosis can reduce clinical symptoms and mortality in co-infected piglets. 1. Effects of necrosis-apoptosis inhibitors on PEDV and PoRVA co-infected cells. IPEC-J2 cells were cultured in DMEM medium containing 10% FBS and incubated at 37°C with 5% CO2 until cell confluence reached 80%. A stock solution of the necrosis-apoptosis pathway inhibitor UH15-38 was prepared by dissolving it in DMSO to a concentration of 20 mg / mL. The stock solution was diluted to the desired concentration using a mixture of 5% DMSO, 30% PEG300, 5% Tween 80, and 60% sterile water. Cells were infected with a viral solution containing PEDV and PoRVA (MOI = 0.1) for 1 hour. After adsorption, the viral solution was removed, and the cells were treated with the inhibitor (diluted to 150 mM / mL). The control group was treated with an equal volume of solute, and the cells were cultured for another 12 hours. Cell supernatants and cell lysates were collected, and Western blot analysis was performed to analyze the expression of viral proteins and related genes.

[0084] 2. Animal experiment on the protective effect of necrotizing and apoptosis inhibitors against 7-day-old piglets co-infected with PEDV and PoRVA. To investigate the ameliorative effect of inhibiting the necroptosis pathway on PEDV and PoRVA co-infection in 7-day-old piglets, healthy 7-day-old piglets were randomly selected and divided into four experimental groups: (1) control group (Mock group, administered DMEM only); (2) co-infection group (Co-infection group, administered PEDV and PoRVA orally simultaneously); (3) co-infection treatment group (Co-infection + UH15-38 group, administered PEDV and PoRVA orally simultaneously, and treated with the necroptosis pathway inhibitor UH15-38); (4) control group treatment (Mock group, treated with the necroptosis pathway inhibitor UH15-38). UH15-38 drug preparation and administration: Drug preparation: UH15-38 was dissolved in DMSO to prepare a stock solution of 20 mg / mL. The stock solution was diluted to the required concentration using a mixture of 5 v / v DMSO, 30 v / v PEG300, 5 v / v THREE 80 and 60 v / v % sterile water.

[0085] Administration: In the co-infection + treatment group and the DMEM treatment group, UH15-38 was initiated within 1 hour of viral infection. The dosage was 5 mg per kilogram of body weight, administered intravenously once daily for 3 consecutive days.

[0086] During the experiment, piglets in both the co-infection and co-infection treatment groups were simultaneously orally infected with strain XY (virus dose 1×10⁻⁶) on day 0. 5 TCID 50 / mL, 1mL per piglet) and PoRVA / CHN / GD / 2024 (virus dose 1×106 TCID 50 / mL, 1mL per piglet), piglets in the DMEM group and piglets in the DMEM-treated group were orally administered the same amount of DMEM as a negative control.

[0087] For 7 consecutive days after viral challenge, clinical symptoms in piglets were observed and recorded. Fecal swabs were collected for viral load detection, and piglet survival was monitored. On day 7, all surviving piglets were euthanized, and jejunal tissue was collected for viral load RT-qPCR detection, histopathological (H&E) staining, and immunohistochemical (IHC) detection to assess intestinal pathological damage and viral antigen distribution. Furthermore, immunohistochemical methods were used to detect IL-1β and p-MLKL protein expression to assess changes in the necroptosis pathway and verify the inhibitory effect of UH15-38 on the necroptosis pathway.

[0088] 3. Experimental Results UH15-38 is a novel receptor-interacting protein kinase 3 (RIPK3) inhibitor characterized by high efficacy and selectivity. RIPK3 plays a crucial role in programmed necroptosis, and overactivation of this pathway can lead to severe inflammation and tissue damage. UH15-38 reduces inflammation and tissue damage by inhibiting RIPK3 activity, thus blocking the necroptosis signaling pathway.

[0089] The effect of co-infection of IPEC-J2 cells with PEDV and PoRVA using the necrosis-apoptosis pathway inhibitor UH15-38 was verified. Results showed that UH15-38 effectively reduced the viral load of PEDV and PoRVA in IPEC-J2 cells co-infected with PEDV and PoRVA. UH15-38 also reduced the phosphorylation levels of RIPK3 and MLKL in IPEC-J2 cells co-infected with PEDV and PoRVA, thereby inhibiting viral replication. Figure 10 (A, B)

[0090] This embodiment further explores the therapeutic effect of the necroptosis pathway inhibitor UH15-38 on piglets co-infected with PEDV and PoRVA. Piglets were randomly divided into a PEDV and PoRVA co-infection group (Co-infection group), a co-infection drug treatment group (Co-infection + UH15-38 group), a treatment control group (DMEM + UH15-38 group), and a DMEM control group. Piglets in the treatment group were administered the drug intravenously for 3 days after challenge at a dose of 5 mg / kg body weight, once daily. Subsequently, a systematic analysis was performed on the fecal viral load, clinical manifestations, survival rate, and jejunal histopathological changes in different groups. Figure 11 (A)

[0091] The results showed that compared with the co-infection group, the viral load in stool swabs was significantly lower in the drug treatment group, with both PoRVA and PEDV viral copy numbers showing a significant decrease. Figure 11 (D, E). The clinical scores of the treatment group were significantly lower than those of the untreated co-infection group, indicating that UH15-38 significantly alleviated the clinical diarrhea symptoms caused by co-infection. Figure 11 (C). Meanwhile, the survival rate of piglets in the treatment group (66.6%) was significantly higher than that in the co-infected group (33.3%). Figure 11 (B), further confirming the protective effect of UH15-38.

[0092] Further pathological analysis of jejunal tissue revealed severe intestinal lesions in the co-infected group, characterized by villus atrophy and extensive inflammatory cell infiltration, while the intestinal inflammatory response was significantly reduced in the treatment group. Furthermore, immunohistochemical results showed that the positive signal intensity of PEDV N protein and PoRVA VP6 protein in the treatment group was significantly lower than that in the untreated co-infected group, indicating that the use of UH15-38 effectively inhibited viral replication in the intestine. Figure 12 ).

[0093] To further clarify the mechanism of action of UH15-38, immunohistochemistry was used to detect the expression levels of key inflammatory cytokine IL-1β and p-MLKL, a core molecule in the necroptosis pathway. The results showed that the expression levels of both IL-1β and p-MLKL in the jejunal tissue of the treatment group were significantly reduced, indicating that UH15-38 alleviated intestinal damage and inflammatory response caused by viral infection by inhibiting the necroptosis pathway. Figure 13 ).

[0094] In summary, this experiment is the first to demonstrate that inhibiting the necroptosis pathway can effectively reduce enterovirus replication, histopathological damage, and clinical symptoms caused by co-infection with PEDV and PoRVA, providing a new treatment approach and theoretical basis for the clinical prevention and control of porcine viral diarrhea.

[0095] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The use of UH15-38 or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and treatment of porcine viral diarrhea; The chemical formula of UH15-38 is C 26 H 27 N5O2; The structural formula is shown in equation (Ⅰ): Equation (I); The porcine viral diarrhea is caused by infection with any of the following groups of viruses: 1) to 3). 1) Porcine epidemic diarrhea virus; 2) Porcine rotavirus A; 3) Porcine epidemic diarrhea virus and porcine rotavirus A.

2. The application according to claim 1, characterized in that: The aforementioned swine viral diarrhea is caused by co-infection with porcine epidemic diarrhea virus and porcine rotavirus A.

3. The application according to claim 1, characterized in that: The drug includes pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that: Pharmaceutically acceptable excipients include carriers.

5. The application according to claim 3, characterized in that: The dosage form of the drug includes one of the following: powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

6. The application according to claim 3, characterized in that: The drug can be administered via the gastrointestinal tract or non-gastrointestinal route.

Images