Medicament against blue ear virus

Abstract

The application belongs to the field of biological medicine, and particularly relates to a medicine for blue ear virus. The application mainly provides a medicine combination based on polyether long-chain antibiotics, which is used for treating and / or preventing a disease caused by a porcine reproductive and respiratory syndrome virus (PRRSV). The medicine of the application can efficiently inhibit the replication of the PRRSV, helps to maintain the health state and production performance of pigs, has a simple preparation method, is suitable for large-scale production, and provides a new and effective solution for the animal husbandry.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a drug targeting porcine reproductive and respiratory syndrome virus (PRRSV). Background Technology

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as blue ear disease, is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV belongs to the Arteriviridae family and is a single-stranded, positive-sense RNA virus that primarily affects the reproductive and respiratory systems of pigs. Since its initial discovery in North America and Europe in the late 1980s, PRRS has rapidly spread globally, causing significant economic losses to the pig industry. The clinical manifestations of PRRS vary depending on the age of the pig, the strain of the virus, and environmental factors. Infected sows often exhibit abortion, premature birth, stillbirth, and decreased fertility. Infected piglets and growing pigs typically present with respiratory distress, fever, coughing, slow weight gain, and high mortality. Infected finishing pigs primarily exhibit respiratory diseases, leading to stunted growth and decreased feed conversion ratio. Statistics show that porcine reproductive and respiratory syndrome (PRRS) can cause a 20% decrease in growth rate, a 7% decrease in feed intake, a 15% decrease in feed utilization, and a 15-day extension of the fattening period in growing and finishing pigs. The economic loss per pig due to PRRS can exceed 80 yuan, making it a major problem urgently needing to be solved in the pig farming industry. Currently, the prevention and control of PRRS mainly relies on biosecurity measures, vaccination, and management methods. Biosecurity measures include restricting the entry and exit of personnel and equipment, strict disinfection procedures, and isolating newly introduced pigs. While these measures can reduce the risk of virus transmission, they are difficult to completely prevent the introduction and spread of the virus, especially in high-density farming environments. Regarding vaccination, various vaccines are available on the market, including live attenuated vaccines and inactivated vaccines. However, the high variability of PRRSV results in limited vaccine protection, often leading to immune escape. Furthermore, vaccination may cause side effects, affecting the health and production performance of pigs. Management methods for PRRSV include health monitoring of the pig herd, early diagnosis of the disease, and isolation of infected pigs. These methods require significant human and material resources and are difficult to implement completely effectively in practice.

[0003] Polyether long-chain antibiotics are a class of antibiotics widely used in animal husbandry, exhibiting good antibacterial and antiprotozoal activities. Lasalocid, methyl salinomycin, and salinomycin are three major representatives. Lasalocid is a polycyclic lactone ionotropic antibiotic, primarily used for the prevention and treatment of coccidiosis in poultry and livestock. It inhibits the growth and reproduction of pathogens by forming ion channels on the cell membrane, disrupting the ion gradient. Studies have shown that Lasalocid exhibits good anticoccidial activity in poultry feed, particularly compared to other ionotropic antibiotics such as monensin and salinomycin (Owles, 1984). Furthermore, in immunosuppressed rats, Lasalocid showed activity against Cryptosporidium, indicating its potential in combating parasitic infections (Rehg, 1993). Salinomycin is an antibiotic widely used in animal husbandry, primarily for the prevention and treatment of coccidiosis in poultry. Salinomycin exhibits good antibacterial and antiprotozoal activities by affecting the ion balance inside and outside cells. Salinomycin has shown good anticoccal activity against a variety of coccidia (e.g., E. acervulina, E. mivati, E. necatrix, and E. tenella) (Migaki et al., 1979). In treating spontaneously infected rabbits, salinomycin showed significant inhibitory effects, increasing the rabbits' body weight (Pakandl, 1986). These polyether long-chain antibiotics exhibit broad antimicrobial activity by altering the ion balance of pathogens and show significant potential in inhibiting PRRSV, overcoming the shortcomings of existing vaccines and biosecurity measures, and providing a highly efficient and low-risk solution for the swine industry. Summary of the Invention

[0004] This invention aims to develop a new treatment and / or prevention regimen for porcine reproductive and respiratory syndrome (PRRS), and provides the application of an antibiotic in the preparation of a drug for the prevention and / or treatment of PRRS, wherein the antibiotic is a polyether long-chain antibiotic.

[0005] Polyether long-chain antibiotics have a unique mechanism for interfering with viral replication. They can disrupt the electrolyte balance of the virus, thereby inhibiting the viral replication process within the host cell.

[0006] The polyether-based long-chain antibiotics described in this invention are ion-carrier antibiotics; as ion carriers, these compounds primarily function by forming ion channels on the cell membrane. They can affect the ion balance inside and outside the cell, especially sodium and potassium ions, thereby disrupting the ion gradient and membrane potential of pathogens. This mechanism of action leads to the inhibition of pathogen growth and reproduction, particularly against bacteria, protozoa, and certain viruses.

[0007] Preferably, the ionotropic antibiotic is selected from one or more of Lasalocid, methylsalicylic acid, or salicylic acid.

[0008] The structure of salinomycin is shown below:

[0009]

[0010] Salinomycin is a widely used antibiotic in animal husbandry, particularly as an anticoccidial agent. Salinomycin exhibits its antimicrobial activity by altering electrolyte and ion permeability on cell membranes. In the case of PRRSV, salinomycin can reduce the virus's infectivity or interfere with its life cycle.

[0011] The structure of methylsalicylic acid is shown below:

[0012]

[0013] Methylsalicylic acid is a derivative of salicylic acid and possesses similar antibiotic properties. This compound works by disrupting the ion balance of bacteria and potentially virus-infected cells, thereby helping to inhibit viral infection and replication. In the context of PRRSV, methylsalicylic acid may exhibit its antiviral effects by affecting the virus's ability to invade host cells or by interfering with the viral replication cycle.

[0014] The Lasalocid structure is shown below:

[0015]

[0016] Lasalocid is a polyether compound composed of a polycyclic ester structure, which allows it to effectively form complexes with ions, especially sodium ions. Lasalocid primarily works by forming ion channels on the cell membrane, altering the concentration of ions, particularly sodium ions, inside and outside the cell. This mechanism disrupts the intracellular environment, affecting cell survival and proliferation. Lasalocid is mainly used in poultry (e.g., chickens) and livestock (e.g., cattle and sheep) for the prevention and treatment of protozoan diseases, such as coccidiosis. As a feed additive, it helps control parasitic infections, improving animal growth efficiency and overall health. In scientific research, Lasalocid has also been used to study the transport and regulation of intracellular sodium ions, as well as related cellular physiological processes. Overall, Lasalocid is an important antiprotozoal drug widely used in animal husbandry. Its ion-carrier properties enable it to effectively control the physiological processes of various organisms, and these properties also open up possibilities for its further application in medicine and scientific research.

[0017] The structure of monensin is shown below:

[0018]

[0019] Monensin is an ionotropic antibiotic primarily used in poultry and livestock for the prevention and treatment of certain parasitic diseases. In antiviral applications, although monensin is mainly targeted at protozoa, its mechanism—altering ion channels and membrane potential in host cells—may adversely affect the viral replication environment.

[0020] The drug described in this invention is used for the prevention and / or treatment of porcine reproductive and respiratory syndrome caused by PRRSV virus;

[0021] Preferably, the PRRSV virus is selected from PRRSV-1 or PRRSV-2;

[0022] Preferably, the PRRSV-1 or PRRSV-2 includes its different geographically distributed subtypes and lineages;

[0023] Preferably, the PRRSV-1 includes lineages common in Europe, and the PRRSV-2 includes lineages common in North America and / or Asia;

[0024] Preferably, the lineage of the PRRSV-2 includes lineages with high pathogenicity and / or viral variants;

[0025] Preferably, the viral variant is selected from one or more of PRRSV Ch-1a strain, WH3 strain, or SD16 strain; more preferably, the viral variant is PRRSV WH3 strain.

[0026] The drug of this invention prevents and / or treats porcine reproductive and respiratory syndrome by blocking the expression and / or replication of the PRRSV virus.

[0027] Another aspect of the present invention provides a drug for combating PRRSV virus or for preventing and / or treating porcine reproductive and respiratory syndrome, wherein the drug contains one or more of the above-mentioned polyether long-chain antibiotics.

[0028] In one embodiment, the present invention evaluated the inhibitory effect of methylsalicylic acid on porcine reproductive and respiratory syndrome virus (PRRSV). Experimental results showed that methylsalicylic acid significantly inhibited PRRSV replication at different concentrations, especially at a high concentration of 5 μM, where the inhibitory effect was most significant, manifested as a significantly increased CT value, indicating a significant reduction in viral replication. These results provide strong experimental support for the potential of methylsalicylic acid as an anti-PRRSV drug.

[0029] In one embodiment, the present invention evaluated the inhibitory effect of salinomycin against porcine reproductive and respiratory syndrome virus (PRRSV). Experiments with different concentrations of salinomycin showed that even at lower concentrations, salinomycin significantly increased the CT value of viral replication, demonstrating its ability to inhibit viral replication. At a high concentration of 5 μM, salinomycin exhibited a stronger viral inhibitory effect. This finding highlights the effectiveness and application potential of salinomycin as an anti-PRRSV drug.

[0030] In one embodiment, the present invention evaluated the inhibitory effect of Lasalocid on porcine reproductive and respiratory syndrome virus (PRRSV). Experimental data showed that Lasalocid significantly increased the CT value of PRRSV at all tested concentrations, particularly at a high concentration of 5 μM, exhibiting the strongest viral inhibition effect. This result indicates that Lasalocid has a significant effect in inhibiting PRRSV replication, supporting its feasibility as an anti-PRRSV treatment option.

[0031] In one embodiment, this invention evaluated the inhibitory effects of different polyether long-chain antibiotics—methylsalicylic acid, salicylic acid, lasalocid, and monensin—on porcine reproductive and respiratory syndrome virus (PRRSV) at the same concentration (5 μM). The results showed that methylsalicylic acid, salicylic acid, and lasalocid all exhibited significant inhibitory effects, especially methylsalicylic acid, whose significant increase in CT value indicated potent inhibition of PRRSV replication. In contrast, monensin showed a weaker effect at this concentration, suggesting that the specific mechanism of action of the drug and its performance under different conditions should be considered when selecting anti-PRRSV treatment regimens.

[0032] In one embodiment, the present invention evaluated the effects of different target compounds—methylsalicylic acid, salicylic acid, and lasalocid—on the survival of PAM cells at different concentrations to confirm their safety at effective antiviral concentrations. Experimental results showed that changes in cell viability at different concentrations of methylsalicylic acid, salicylic acid, and lasalocid indicated that these compounds had low cytotoxicity at low to moderate concentrations. In particular, under experimental conditions above the concentration threshold, these compounds did not significantly affect cell survival, demonstrating good cell compatibility.

[0033] In one embodiment, the concentration of the polyether long-chain antibiotic in the drug is selected from 0.1 to 50 μM;

[0034] Preferably, the concentration is selected from 0.1 to 5 μM;

[0035] Preferably, the concentration is selected from 1 μM, 2.5 μM or 5 μM; more preferably, the concentration is 5 μM.

[0036] In selecting the antibiotics and their concentrations, this invention comprehensively considers the inhibitory effects of different drugs on the virus and the results of cytotoxicity experiments. Methylsalinomycin, salinomycin, and Lasalocid all demonstrated significant inhibitory effects on PRRSV at specific concentrations, significantly increasing the viral CT value, indicating effective inhibition of viral replication. Considering the balance between antiviral activity and cytotoxicity, the antibiotics and their concentration ranges not only ensure the antiviral effect of the drugs but also minimize cytotoxicity.

[0037] In one embodiment, the dosage form of the drug is selected from one or more of topical preparations, spray preparations, injectable preparations, or oral preparations;

[0038] Preferably, the topical preparation is selected from one or more of creams, gels, lotions, or medicinal sprays;

[0039] Preferably, the spray formulation is selected from one or more of solution spray, suspension spray, or powder spray;

[0040] Preferably, the injectable formulation is selected from one or more of solution injections, suspension injections, or emulsion injections;

[0041] Preferably, the oral formulation is selected from one or more of tablets, capsules, suspensions or granules.

[0042] The drug of the present invention further includes an excipient, wherein the excipient is selected from one or more of excipients, sustained-release agents, stabilizers, antioxidants, preservatives, solvents and solubilizers;

[0043] In one embodiment, the excipient is selected from one or more of starch, lactose, cellulose, or derivatives thereof;

[0044] In one embodiment, the sustained-release agent is selected from one or more of hydroxypropyl methylcellulose, polyvinyl alcohol, or copolymers thereof;

[0045] In one embodiment, the stabilizer is selected from one or more of ethylenediaminetetraacetic acid (EDTA) or its sodium salt;

[0046] In one embodiment, the antioxidant is selected from one or more of vitamin E, vitamin C, or derivatives thereof;

[0047] In one embodiment, the preservative is selected from one or more of sodium benzoate and potassium sorbate;

[0048] In one embodiment, the solvent is selected from one or more of water, ethanol, or propylene glycol;

[0049] In one embodiment, the solubilizer is selected from one or more of polysorbate 80 (Tween 80) or polyoxyethylene castor oil.

[0050] In one embodiment, the drug of the present invention is applicable to dolphin animals;

[0051] Preferably, the pig-like animals include, but are not limited to, wild and domesticated species;

[0052] Preferably, the domesticated breed is a breed of pig for consumption, including but not limited to Large White, Duroc, and Hampshire pigs; more preferably, the breed of pig for consumption is PRRSV-sensitive.

[0053] The polyether long-chain antibiotics of this invention have demonstrated significant progress and advantages in the application against PRRSV (Porcine Reproductive and Respiratory Syndrome Virus), especially in the veterinary field, where their unique pharmacological properties and versatility make them stand out in the market.

[0054] 1. Highly Effective Inhibition of Viral Replication: This invention provides the application of polyether long-chain antibiotics in the preparation of antiviral drugs, particularly showing a significant inhibitory effect on PRRSV replication. These antibiotics inhibit the growth and reproduction of pathogens by forming ion channels on the cell membrane and disrupting the ion gradient of the pathogen. Experiments have shown that salinomycin, for example, has a significant inhibitory effect on PRRSV replication at different concentrations, effectively reducing viral load and thus controlling the spread and impact of the disease.

[0055] 2. Low side effects: These polyether long-chain antibiotics exhibit low toxicity and fewer side effects at therapeutic doses. This helps maintain the health and production performance of pigs and reduces adverse drug reactions. For example, methylsalicylic acid has been shown to effectively inhibit PRRSV at low concentrations without significant toxicity to host cells, making it advantageous for long-term treatment.

[0056] 3. Multifunctionality: In addition to antiviral activity, the antibiotics of this invention also possess broad-spectrum antibacterial and antiprotozoal activities. For example, Lasalocid and salinomycin not only demonstrate advantages in antiviral activity but also effectively prevent and treat coccidiosis in poultry and livestock. This multifunctionality allows these antibiotics to address multiple infection problems in a single pharmaceutical formulation, improving farming efficiency.

[0057] In summary, the polyether long-chain antibiotics of this invention have broad application prospects in the development of anti-PRRSV drugs. By combining their advanced pharmacological properties with treatment strategies, they can significantly improve the efficacy of veterinary medication and meet the needs of the livestock industry for efficient and low-risk treatment solutions. Attached Figure Description

[0058] Figure 1 The changes in viral replication CT values ​​are shown in the control group and in samples with different concentrations of methylsalicylic acid.

[0059] Figure 2 The changes in viral replication CT values ​​are shown in the control group and in samples with different concentrations of salinomycin.

[0060] Figure 3 The changes in viral replication CT values ​​are shown in the control group and in samples with different concentrations of Lasalocid.

[0061] Figure 4 The changes in viral replication CT values ​​are shown in the control group and different target compound sample groups.

[0062] Figure 5 The changes in viral replication CT values ​​are shown in the control group and in samples with different concentrations of monensin.

[0063] Figure 6 The cell viability results after methylsalicylic acid treatment are shown.

[0064] Figure 7 The cell viability results after salinomycin treatment are shown.

[0065] Figure 8 The results show the cell viability after Lasalocid treatment. Detailed Implementation

[0066] Definitions and Explanations

[0067] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. It should be understood that this invention is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. It should also be understood that the terminology used in this invention is for describing specific embodiments only and is not intended to be limiting.

[0068] As used in this article, the term "Porcine Reproductive and Respiratory Syndrome (PRRS)" refers to an infectious disease caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which primarily affects the reproductive and respiratory systems of pigs. It is a serious viral disease that severely impacts pigs, mainly manifesting as respiratory and reproductive dysfunction. This disease poses a significant challenge to the global pig industry, particularly in causing sow abortions and high piglet mortality rates, leading to severe economic losses.

[0069] As used in this article, the term "PRRSV" refers to porcine reproductive and respiratory syndrome virus, an arteritis virus with two main types: PRRSV-1 and PRRSV-2, which are prevalent in Europe, North America, and Asia, respectively. This virus exhibits high genetic diversity and variability, leading to the emergence of multiple strains.

[0070] As used in this article, the term "polyether long-chain antibiotics" refers to a class of antibiotics whose structure contains polyether chains. These antibiotics function as ion carriers and can interfere with the ion balance of pathogens by forming ion channels on the cell membrane.

[0071] As used herein, the term "dosage form" refers to the physical form or configuration of a drug, which determines its release, distribution, and administration. This includes topical formulations, spray formulations, injectable formulations, and oral formulations. Drug forms are designed to accommodate different routes of administration and enhance efficacy. Different dosage forms can affect drug absorption, distribution, metabolism, and excretion, thus influencing efficacy and safety. For example, topical formulations are suitable for treating skin diseases, spray formulations for respiratory diseases, injectable formulations for rapid and precise administration, and oral formulations are typically used for long-term treatment.

[0072] As used herein, the term "topical preparation" refers to a pharmaceutical preparation applied directly to the skin or specific areas of the skin. Topical preparations are primarily intended to act directly on the skin or mucous membranes. Through local application, these preparations can reduce systemic side effects and increase drug concentration at the site of infection. Common forms of topical preparations include creams, gels, lotions, and medicated sprays. Each form has its specific applications and advantages; for example, creams are suitable for dry or cracked skin, gels are suitable for cleansing or moisturizing, lotions are used to clean large areas of skin, and medicated sprays are convenient for covering wide areas.

[0073] As used herein, the term "aerosol formulation" refers to a drug that is formulated into fine particles and released through a nebulizer for application to the body surface or interior. This is achieved by converting the drug into fine droplets or powder, which can be administered via inhalation or spraying. Such formulations are particularly suitable for conditions requiring rapid action, such as asthma or allergic reactions. Aerosol formulations can be categorized into solution sprays, suspension sprays, and powder sprays based on their carrier, with solution sprays being widely used due to their uniformity and easily controllable dosage.

[0074] As used herein, the term "injectable formulation" refers to a drug formulation injected into the body via a syringe, which can act directly on the bloodstream or specific tissues. Injectable formulations can be solutions, suspensions, or emulsions, each with its own advantages and specific uses. Injectable solutions are widely used because of their homogeneous composition and immediate release of the drug, while injection suspensions and emulsions are used for treatments requiring sustained release because they can prolong the duration of drug action.

[0075] As used in this article, the term "oral preparation" refers to a drug preparation ingested orally, which is one of the most common methods of drug administration. These preparations include tablets, capsules, suspensions, and granules.

[0076] As used herein, the term "external ingredient" refers to any other component, besides the active pharmaceutical ingredient, added to a pharmaceutical formulation to improve the physical, chemical, or biological usability of the drug. These ingredients include excipients, sustained-release agents, stabilizers, antioxidants, preservatives, solvents, and solubilizers. The selection and use of these ingredients depend on the desired drug release rate, stability, patient acceptability, and other pharmaceutical requirements. For example, excipients such as starch and lactose provide the drug in a solid form and for palatability; sustained-release agents such as hydroxypropyl methylcellulose prolong the release time of the drug in vivo; stabilizers such as ethylenediaminetetraacetic acid (EDTA) prevent the degradation of the drug component during storage; antioxidants such as vitamin E and vitamin C prevent drug oxidation; preservatives such as sodium benzoate and potassium sorbate prevent the growth of microorganisms in the formulation; solvents such as water and ethanol adjust the solubility of the drug; and solubilizers such as polysorbate 80 (Tween 80) and polyoxyethylene castor oil increase the absorption efficiency of the drug in the body to improve its physicochemical properties, stability, or acceptability. This includes excipients, sustained-release agents, stabilizers, antioxidants, preservatives, solvents, and solubilizers.

[0077] As used in this article, the term "PAM cells" refers to porcine macrophages, a macrophage line derived from the alveoli of pigs, which are commonly used in virology research, particularly to study the host cell response and viral replication mechanisms of porcine reproductive and respiratory syndrome virus (PRRSV).

[0078] As used in this article, the term "PRRSV WH3 strain" refers to a specific strain of porcine reproductive and respiratory syndrome virus that is widely used in scientific research to help understand the transmission and infection mechanisms of the virus, as well as to test the effectiveness of new treatments.

[0079] As used herein, the term "quantitative fluorescent PCR" (qPCR) refers to a molecular biology technique used to simultaneously amplify and quantify a target DNA template, commonly used to detect and measure viral load or gene expression levels. This method determines the initial quantity of a specific DNA sequence by monitoring the increase in fluorescence signal in real time.

[0080] As used in this article, the term "CT value" (threshold cycle value) refers to the number of cycles required to reach the detection threshold during quantitative real-time PCR. The CT value reflects the initial amount of target DNA in the sample; a lower CT value indicates a higher initial amount of target DNA, and conversely, a higher CT value indicates a lower initial amount of DNA. This value is a key indicator for assessing viral replication efficiency and the antiviral activity of drugs.

[0081] As used in this article, the term "cytotoxicity" refers to the harmful effects of a compound or physical factor on cells, leading to impaired cell function, growth inhibition, or cell death. Cytotoxicity can be achieved through mechanisms such as directly damaging cell structure, interfering with cell metabolism, or affecting cell growth signaling.

[0082] As used in this article, the term "cytotoxicity assay" refers to a series of experimental methods used to assess the effects of compounds or conditions on cell viability and health. These assays typically involve measuring parameters such as cell viability, cellular metabolic activity, and cell membrane integrity. Common cytotoxicity assays include the MTT assay, LDH release assay, and flow cytometry.

[0083] As used in this article, the term "CC50" (Concentration for 50% of maximal cytotoxic effect) refers to the concentration at which a compound reduces cell viability by 50% under certain conditions. This value is commonly used to measure the effect of a compound on cell viability and is an important indicator for judging the cytotoxicity of a compound. A higher CC50 indicates lower cytotoxicity of the compound, meaning that a significant toxic effect on cells is only produced at a higher concentration.

[0084] Example

[0085] Materials and Methods

[0086] Cells: PAM cells were isolated from pig lungs, and the isolation method was based on Ait-Ali, Tahar, et al. "Innate immune responses to replication of porcine reproductive and respiratory syndrome virus in isolated Swine alveolar macrophages." Viral Immunology 20.1(2007):105-118.

[0087] Virus: PRRSV WH3 strain, isolated from pig lungs, with the isolation method described in the above literature.

[0088] Reagents: TRIzol (for RNA extraction), reverse transcription kit (for cDNA synthesis), and porcine reproductive and respiratory syndrome virus universal (PRRSV-U) nucleic acid detection kit (fluorescent PCR method) (for virus copy number detection), purchased from Wuhan Keqian Biotechnology Co., Ltd.

[0089] The target compounds, methylsalicylic acid, salicylic acid, lasalocid, and monensin, were all purchased from commercial reagent companies. Methylsalicylic acid was purchased from Yuanye Biotechnology (catalog number B28443), salicylic acid was purchased from Yuanye Biotechnology (catalog number S80781), lasalocid was purchased from TargetMol (catalog number T15717), and monensin was purchased from Yuanye Biotechnology (catalog number S17047).

[0090] Example 1: Inhibitory effect of methylsalicylic acid on PRRSV

[0091] This embodiment aims to evaluate the inhibitory effect of methylsalicylic acid on porcine reproductive and respiratory syndrome virus (PRRSV) and determine its effect on viral replication at different concentrations, in order to provide experimental evidence for the development of highly effective and low-toxicity anti-PRRSV drugs.

[0092] Grouping method:

[0093] Control group: PAM cells were treated with DMEM medium containing 8% FBS, with only DMSO solvent without methylsalicylic acid added, and then infected with PRRSV.

[0094] Sample groups: PAM cells were treated with methylsalicylic acid at concentrations of 0.1 μM, 0.5 μM, 1 μM, and 5 μM, respectively. Each concentration was pre-diluted in DMEM medium containing 8% FBS and added to the cells as needed, followed by PRRSV infection.

[0095] Experimental methods:

[0096] Cell culture: PAM cells were cultured at a rate of 2 × 10⁶ cells / year. 6 The concentration of cells was seeded into 6-well plates containing glass slides and incubated at 37°C in a 5% CO2 incubator until the cells adhered to the plate and formed a monolayer (approximately 12 hours).

[0097] Drug treatment: Using analytical grade DMSO as a solvent, methylsalicylic acid powder was dissolved in an appropriate amount of DMSO to prepare a high-concentration stock solution. This stock solution was then serially diluted using sterile 8% FBS DMEM medium to ultimately prepare the required working concentrations (0.1 μM, 0.5 μM, 1 μM, 5 μM). Following the grouping method, different concentrations of methylsalicylic acid (0.1 μM, 0.5 μM, 1 μM, 5 μM) were added to the cell culture medium to prepare sample groups of different concentrations, as well as a control group containing only DMSO solvent. PAM cells were treated at 37℃.

[0098] Viral infection: After drug treatment, PRRSV WH3 strain (MOI=0.1) was inoculated and co-cultured in a 37°C, 5% CO2 incubator for 36 hours.

[0099] RNA extraction and reverse transcription: TRIzol was added to extract RNA from cells, and the RNA was reverse transcribed to synthesize cDNA.

[0100] Quantitative real-time PCR: Using reverse-transcribed cDNA as a template and PRRSV-NF and PRRSV-NR as primers, quantitative real-time PCR was performed to detect the PRRSV copy number in the sample. The primers used for quantitative real-time PCR were:

[0101] PRRSV-NF: 5'-AATAACAACGGCAAGCAGCAG-3'

[0102] PRRSV-NR: 5'-CCTCTGGACTGGTTTTGTTGG-3'

[0103] Fluorescence data are collected after each cycle using a real-time PCR instrument, and the CT value for each sample is automatically calculated. The CT value is the number of PCR cycles required to reach a preset fluorescence threshold. This value reflects the number of cycles required to reach the detection threshold. A lower CT value indicates a higher viral load, i.e., active viral replication; an increased CT value indicates a lower viral load, i.e., suppressed viral replication.

[0104] Data analysis: The CT values ​​of the sample group were compared with those of the control group to analyze and evaluate the effect of methylsalicylic acid on PRRSV replication.

[0105] Results analysis and discussion:

[0106] This experiment used RT-PCR to detect the replication of PRRSV in PAM cells. The results showed that methylsalicylic acid at different concentrations significantly inhibited the replication of PRRSV. Figure 1The figure shows a bar chart illustrating the changes in viral replication CT values ​​in the control group and different concentrations of methylsalicylic acid (MSA). The mean MS value in the control group was 14.69, indicating normal viral replication in PAM cells. In the 0.5 μM MSA group, the mean MS value increased to 21.40, showing a significant difference compared to the control group (P < 0.05), indicating that MSA significantly inhibited viral replication. The mean MS value in the 1 μM MSA group further increased to 26.49, showing a significant difference compared to the control group (P < 0.05), demonstrating a stronger inhibitory effect. The mean MS value in the 5 μM MSA group reached 29.45, showing a significant difference compared to the control group (P < 0.05), demonstrating the highest inhibitory effect. These data indicate that MSA significantly reduces the PRRSV load in PAM cells by interfering with viral replication, and the inhibitory effect becomes more significant with increasing drug concentration.

[0107] Experimental results show that methylsalicylic acid exhibits remarkable efficacy in inhibiting PRRSV replication. As a polyether-based long-chain antibiotic, methylsalicylic acid inhibits viral replication within host cells by forming ion channels on the cell membrane, disrupting the virus's ion balance. This mechanism allows methylsalicylic acid to exhibit potent antiviral activity at low concentrations while maintaining low toxicity. By significantly inhibiting PRRSV replication, methylsalicylic acid demonstrates great potential as an anti-PRRSV drug, providing a solid scientific foundation for the development of highly effective and low-toxicity anti-PRRSV drugs. Through systematic experimental research and comparative analysis, methylsalicylic acid shows promise as an effective new drug for the treatment and prevention of PRRSV infection, offering an innovative and effective solution for the swine industry.

[0108] Example 2: Inhibitory effect of salinomycin on PRRSV

[0109] This embodiment aims to evaluate the inhibitory effect of salinomycin on porcine reproductive and respiratory syndrome virus (PRRSV), determine its effect on viral replication at different concentrations, and explore its effectiveness as a potential anti-PRRSV drug.

[0110] Grouping method:

[0111] Control group: PAM cells were treated with DMEM medium containing 8% FBS, with only DMSO solvent without salinomycin added, and then infected with PRRSV.

[0112] Sample groups: PAM cells were treated with 0.1 μM, 0.5 μM, 1 μM, and 5 μM salinomycin, respectively. Each concentration was pre-diluted with DMEM medium containing 8% FBS and added to the cells as needed, followed by PRRSV infection.

[0113] Experimental methods:

[0114] Same as Example 1.

[0115] Results analysis and discussion:

[0116] Figure 2 The figure shows a bar chart illustrating the changes in viral replication CT values ​​in the control group and samples with different concentrations of salinomycin. As shown in the figure, the average CT value of the virus in the control group was 14.69, indicating normal viral replication in PAM cells. At a concentration of 0.5 μM, salinomycin increased the average CT value of the virus from 14.69 to 15.74 compared to the control group, indicating that salinomycin can inhibit the replication of PRRSV in host cells. At higher drug concentrations, the average CT value of the virus at a concentration of 1 μM was 22.01, and the average CT value at a concentration of 5 μM was 26.34, which were significantly different from the control group (P<0.05), indicating a significant reduction in viral replication capacity.

[0117] Experimental results clearly demonstrate that salinomycin exhibits a significant effect in inhibiting PRRSV replication. Furthermore, salinomycin possesses broad-spectrum antimicrobial activity, not only inhibiting viral replication but also effectively preventing and controlling infections from various bacteria and protozoa. This versatility makes salinomycin a promising candidate for widespread application in the livestock industry, capable of addressing various pathogen infections and improving farming efficiency. In conclusion, salinomycin's significant effect in inhibiting PRRSV, its low toxicity, and its versatility make it a highly promising new antiviral drug, providing a highly efficient and low-risk solution for the pig farming industry.

[0118] Example 3: Inhibitory effect of Lasalocid on PRRSV

[0119] This embodiment aims to evaluate the inhibitory effect of Lasalocid on porcine reproductive and respiratory syndrome virus (PRRSV), determine its effect on viral replication at different concentrations, and explore its effectiveness as a potential anti-PRRSV drug.

[0120] Grouping method:

[0121] Control group: PAM cells were treated with DMEM medium containing 8% FBS and DMSO solvent without Lasalocid was added, followed by PRRSV infection.

[0122] Sample groups: PAM cells were treated with Lasalocid at concentrations of 0.1 μM, 0.5 μM, 1 μM, and 5 μM, respectively. Each concentration was pre-diluted with DMEM medium containing 8% FBS and added to the cells as needed, followed by PRRSV infection.

[0123] Experimental methods:

[0124] Same as Example 1.

[0125] Results analysis and discussion:

[0126] This embodiment uses RT-PCR to detect PRRSV replication in PAM cells, revealing the inhibitory effect of Lasalocid on PRRSV replication at different concentrations. Figure 3 The figure shows a bar chart illustrating the changes in viral replication CT values ​​in the control group and samples treated with different concentrations of Lasalocid. As shown, the average CT value of PRRSV in the control group was 14.69, indicating normal viral replication in PAM cells. In samples treated with 0.1 μM Lasalocid, the average CT value increased to 15.67, indicating that Lasalocid began to exert an inhibitory effect at lower concentrations. With increasing concentration, the average CT values ​​in the 0.5 μM and 1 μM treatment groups increased to 13.53 and 17.90, respectively, showing more significant viral replication inhibition. At a high concentration of 5 μM, the average CT value reached 28.07, showing a significant difference compared to the control group (P < 0.05), demonstrating the potent inhibition of PRRSV replication by Lasalocid. These data indicate that Lasalocid effectively reduces viral load by interfering with the replication process of PRRSV in host cells, and its inhibitory effect increases with increasing drug concentration.

[0127] Lasalocid, a polyether-based long-chain antibiotic, inhibits PRRSV replication by adjusting the ion balance on the cell membrane. This mechanism not only exhibits good antiviral activity at low concentrations but also low cytotoxicity. Compared to traditional control measures, Lasalocid demonstrates higher efficiency and fewer side effects, significantly improving its safety in clinical applications. Furthermore, Lasalocid's broad-spectrum antimicrobial activity makes it a potential candidate for preventing and controlling various bacterial and protozoan infections, providing an effective solution for pathogen infection control in the livestock industry and contributing to improved farming efficiency and animal health. Therefore, Lasalocid is not only an effective anti-PRRSV drug but also a highly versatile treatment option, bringing new treatment strategies to the swine industry.

[0128] Example 4: Inhibitory effects of different target compounds on PRRSV

[0129] This embodiment aims to evaluate the inhibitory effects of different target compounds—methylsalicycin, salicycin, lasalocid, and monensin—on porcine reproductive and respiratory syndrome virus (PRRSV) replication at the same concentration (5 μM) to determine which compound is optimal in controlling the virus.

[0130] Grouping method:

[0131] Control group: PAM cells were treated with DMEM medium containing 8% FBS, with only DMSO solvent without salinomycin added, and then infected with PRRSV.

[0132] Sample groups: PAM cells were treated with 5 μM methylsalicylic acid, salicylic acid, lasalocid, and monensin, respectively. Each concentration was pre-diluted in DMEM medium containing 8% FBS and added to the cells as needed, followed by PRRSV infection.

[0133] Experimental methods:

[0134] Same as Example 1.

[0135] Results analysis and discussion:

[0136] Figure 4 The changes in viral replication CT values ​​in the control group and different target compound sample groups are shown, where:

[0137] The mean CT value for viral replication in the methylsalicylic acid (MSA) treatment group was 29.45, demonstrating the strongest inhibitory effect. Compared to the control group, the CT value was significantly higher, indicating that MSA is highly effective in blocking PRRSV replication. This result suggests that MSA may significantly reduce viral load by interfering with a key stage of viral replication. This highly efficient inhibitory ability of MSA makes it a promising candidate for anti-PRRSV treatment. Its potent antiviral activity at low concentrations, coupled with fewer side effects, makes MSA a very attractive candidate drug.

[0138] The mean CT value in the salinomycin treatment group was 26.34, also demonstrating a significant viral suppression effect. Although slightly lower than that of methylsalinomycin, it still effectively inhibited viral replication. The mechanism of action of salinomycin may be similar to that of methylsalinomycin, disrupting the viral ion balance by forming ion channels on the cell membrane and preventing viral replication within the host cell. This antiviral ability of salinomycin makes it another effective candidate drug for PRRSV treatment, especially in cases requiring combination therapy, where its potent antiviral activity will help improve treatment outcomes.

[0139] The average CT value of the Lasalocid-treated group was 28.07, close to that of methylsalicylate, demonstrating a very strong viral inhibitory effect. The mechanism of action of Lasalocid may also involve altering cell membrane ion permeability, thereby inhibiting viral replication. Lasalocid's significant effect in inhibiting PRRSV makes it an important antiviral drug, particularly in the prevention and treatment of viral infections. Its broad-spectrum antimicrobial activity not only inhibits viral replication but also effectively prevents and controls various bacterial and protozoan infections, further increasing the application prospects of Lasalocid in aquaculture.

[0140] Unlike the other three antibiotics, the mean CT value of the monensin treatment group was 13.72, indicating that its inhibitory effect on PRRSV was not significant under these experimental conditions, and it may even have promoted viral replication. This result may be related to the mechanism of action of monensin, suggesting that further research is needed to clarify its effects under different conditions. Figure 5 The bar chart shows the changes in the mean CT value of viral replication in the control group and the sample groups with different concentrations of monensin. In the monensin sample groups at different concentrations, the mean CT value was lower than that in the control group, especially at higher concentrations (1 μM and 5 μM), where the CT value did not show an increasing trend. This performance of monensin reminds us that when selecting anti-PRRSV drugs, it is necessary to fully consider the specific mechanism of action of the drug and experimental conditions to ensure the selection of the most effective treatment regimen.

[0141] In summary, methylsalicylic acid, salicylic acid, and lasalocid have demonstrated significant effects in inhibiting PRRSV, with methylsalicylic acid being particularly outstanding in terms of low toxicity and high efficacy. These antibiotics effectively reduce viral load by interfering with viral replication, providing new ideas and methods for PRRSV treatment. The results of monensin indicate that research and optimization are necessary to ensure the safety and efficacy of all candidate drugs. These findings not only provide a scientific basis for developing new PRRSV treatments but also bring new strategies to the swine industry.

[0142] Example 5: Cytotoxicity Experiment

[0143] This embodiment aims to evaluate the effects of different target compounds—methylsalicycin, salicycin, and lasalocid—on the survival rate of PAM cells at different concentrations, in order to confirm their safety at effective antiviral concentrations.

[0144] Materials and methods

[0145] Cell line: PAM cells were used as the experimental model.

[0146] Cells were cultured in DMEM medium. Different target compounds were first dissolved in DMSO and then diluted with medium to the required experimental concentration. PAM cells were seeded and incubated to allow cell adhesion. Medium containing different concentrations of the target compounds was added to treat the cells. Cell viability was tested using the CCK-8 assay kit. 10 μL of CCK-8 solution was added to each well, and after incubation for 2–4 hours, the absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability at each concentration was calculated and compared with the control group.

[0147] Calculate cell viability using the following formula:

[0148] Cell viability (%) = (Experimental group OD - Blank control group OD) / (Control group OD - Blank control group OD) x 100% Results Analysis and Discussion:

[0149] Figure 6 The image shows the cell viability results after methylsalicylate treatment. Figure 7 The image shows the cell viability results after salinomycin treatment. Figure 8 The image shows the cell viability results after Lasalocid treatment. The experimental results show that, using the compound concentration that causes 50% of PAM cells to lose normal metabolic function or growth capacity as a baseline, at the highest concentration of methylsalicylic acid (22.75 μM), PAM cell viability was greater than 50%, meaning that the CC50 (concentration for 50% of maximal cytotoxic effect) of methylsalicylic acid on PAM cells was greater than 22.75 μM. At lower concentrations (below 5.60 μM), cell viability gradually recovered to nearly 100%, indicating that methylsalicylic acid had low cytotoxicity at these concentrations. The effect of salicylic acid on PAM cells was relatively gradual; under salicylic acid treatment, at the highest concentration of 10.000 μM, PAM cell viability remained above approximately 60%. This indicates that the CC50 value of salicylic acid on PAM cells was higher than 10.000 μM. For Lasalocid, experimental data showed that even at a high concentration of 25.46 μM, the viability of PAM cells remained above 50%, and when further reduced to 0.79 μM, cell viability significantly increased, approaching 100%. This indicates that the CC50 value of Lasalocid is greater than 25.46 μM, suggesting that Lasalocid has low toxicity to PAM cells at these concentrations.

[0150] The foregoing detailed description of the present invention should not be construed as limiting its specific implementation. All documents mentioned herein are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that simple deductions or substitutions made by those skilled in the art without departing from the inventive concept are within the scope of protection of the present invention.

Claims

1. The use of an antibiotic in the preparation of a drug for the prevention and / or treatment of porcine reproductive and respiratory syndrome caused by PRRSV virus, wherein, The antibiotic is a polyether long-chain antibiotic; The polyether-based long-chain antibiotic is an ion carrier antibiotic; The ion carrier antibiotic is selected from one or more of Lasalocid, methylsalicylic acid, or salicylic acid. When the ion carrier antibiotic is Lasalocid, the concentration of the polyether long-chain antibiotic in the drug is selected from 1 to 5 μM; When the ion carrier antibiotic is methylsalicylic acid, the concentration of the polyether long-chain antibiotic in the drug is selected from 0.5 to 5 μM; When the ion carrier antibiotic is salinomycin, the concentration of the polyether long-chain antibiotic in the drug is selected from 1 to 5 μM; The PRRSV virus in question is the variant strain PRRSV WH3.

2. The application according to claim 1, wherein, The drug prevents and / or treats porcine reproductive and respiratory syndrome by blocking the expression and / or replication of the PRRSV virus.

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