Novel drug against porcine reproductive and respiratory syndrome virus

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a novel drug against the porcine reproductive and respiratory syndrome virus. The present invention mainly provides a polyether long-chain antibiotic-based pharmaceutical combination, which is used for treating and / or preventing diseases caused by the porcine reproductive and respiratory syndrome virus (PRRSV). The drug of the present invention can efficiently inhibit the replication of PRRSV, and helps maintain the health state and reproduction performance of pigs. The preparation method is simple and convenient, is suitable for large-scale production, and provides a new and effective solution for the livestock industry.

Description

Novel drug against blue-ear virus

[0001] This application claims priority to Chinese Patent Application No. 2024108250507, filed on June 25, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the field of biological medicine, and specifically relates to a novel drug against blue-ear virus. BACKGROUND

[0003] 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 mainly affects the reproductive and respiratory systems of pigs. Since its first 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 pigs, the strain of the infection, and environmental factors. After being infected with PRRSV, sows often show signs of abortion, premature birth, stillbirth, and decreased reproductive rate. Piglets and growing pigs infected with PRRSV usually exhibit respiratory distress, fever, cough, slow weight gain, and high mortality. Finishing pigs mainly show respiratory symptoms after infection, leading to growth retardation and decreased feed conversion rate. According to statistics, blue-ear disease 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 finishing period. The economic loss caused by blue-ear disease to a single finishing pig is as high as 80 yuan, which has become a major problem in the pig breeding industry that needs to be addressed. Currently, the prevention and control of PRRS mainly relies on biological safety measures, vaccination, and management methods. Biological safety measures include limiting 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 cannot completely prevent the introduction and spread of the virus, especially in high-density breeding environments. In terms of vaccination, there are various vaccines on the market, including attenuated live vaccines and inactivated vaccines. However, the high variability of PRRSV limits the protective effect of vaccines, often resulting in immune escape. In addition, vaccination may cause side effects, affecting the health and production performance of pigs. Management methods for PRRSV include health monitoring of pig herds, early diagnosis of diseases, and isolation of infected pigs. These methods require a large amount of manpower and resources, and are difficult to be completely effective in actual operation.

[0004] Polyether long-chain antibiotics are a class of antibiotics widely used in animal husbandry, with good antibacterial and antiprotozoal activity. Lasalocid, methyl salinomycin and salinomycin are three main representatives among them. Lasalocid is a polycyclic lactone ionophore antibiotic, mainly used for the prevention and treatment of coccidiosis in poultry and livestock. It forms ion channels in the cell membrane, disrupts the ion gradient of the pathogen, and thus inhibits its growth and reproduction. Studies have shown that Lasalocid shows good anticoccidial activity in poultry feed, especially when compared with other ionophore antibiotics such as monensin and salinomycin (Owles, 1984). In addition, Lasalocid shows activity against Cryptosporidium in immunosuppressed rats, indicating its potential in anti-parasitic infections (Rehg, 1993). Salinomycin is an antibiotic widely used in animal husbandry, mainly used for the prevention and treatment of coccidiosis in poultry. Salinomycin shows good antibacterial and antiprotozoal activity by affecting the ion balance inside and outside the cell. Salinomycin shows good anticoccidial activity against various coccidia such as E. acervulina, E. mivati, E. necatrix, and E. tenella (Migaki et al., 1979). When treating naturally infected coccidia in rabbits, salinomycin shows significant inhibitory effect, increasing the body weight of rabbits (Pakandl, 1986). These polyether long-chain antibiotics show extensive antimicrobial activity by changing the ion balance of the pathogen, and have significant potential in inhibiting PRRSV, which can overcome the shortcomings of existing vaccines and biosecurity measures, and provide an efficient and low-risk solution for pig farming. SUMMARY

[0005] The present application aims to develop a new treatment and / or prevention scheme for porcine reproductive and respiratory syndrome, and provides a use of an antibiotic in the preparation of a medicine for preventing and / or treating porcine reproductive and respiratory syndrome, wherein the antibiotic is a polyether long-chain antibiotic.

[0006] Polyether long-chain antibiotics have a unique mechanism of interfering with viral replication, which can interfere with the electrolyte balance of the virus, thereby inhibiting the replication process of the virus in the host cell.

[0007] The polyether-based long-chain antibiotics described herein are ionophore antibiotics; as ionophores, these compounds work primarily by forming ion channels in the cell membrane. They are able to affect the ion balance, particularly sodium and potassium ions, inside and outside the cell, thereby disrupting the ion gradient and membrane potential of the pathogen. This mechanism of action results in the inhibition of growth and reproduction of the pathogen, particularly against bacteria, protozoa, and certain viruses.

[0008] In one embodiment, the ionophore antibiotic is selected from one or more of Lasalocid, Methylsalinomycin, or Salinomycin.

[0009] The structure of Salinomycin is shown below:

[0010] Salinomycin is an antibiotic widely used in the livestock industry, particularly as an anticoccidial drug. Salinomycin exhibits its antimicrobial activity by altering the electrolyte and ion permeability of the cell membrane. For PRRSV, salinomycin can reduce the ability of the virus to infect or interfere with the life cycle of the virus.

[0011] The structure of Methylsalinomycin is shown below:

[0012] Methylsalinomycin is a derivative of salinomycin with similar antibiotic properties. This compound works by disrupting the ion balance of bacteria and possibly virus-infected cells, thereby helping to inhibit viral infection and replication. In the context of PRRSV, methylsalinomycin can exhibit its antiviral effect by affecting the ability of the virus to invade host cells or interfering with the virus replication cycle.

[0013] The structure of Lasalocid is shown below:

[0014] Lasalocid is a polyether compound composed of a polycyclic polyester structure, which allows it to effectively form complexes with ions, particularly sodium ions. Lasalocid primarily functions by forming ion channels in cell membranes, altering the concentration of ions, especially sodium ions, between the inside and outside of cells. This mechanism can lead to disturbances in the cellular environment, affecting cell survival and proliferation. Lasalocid is mainly used in poultry (such as chickens) and livestock (such as cows and sheep) to prevent and treat protozoan diseases, such as coccidiosis. As a feed additive, it helps control parasitic infections, improves the growth efficiency and overall health of animals. In scientific research, Lasalocid is also used to study the transport and regulation of sodium ions in cells and related cellular physiological processes. In summary, 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, which also provides the possibility for further application in medicine and scientific research.

[0015] Monensin has the following structure:

[0016] Monensin is an ionophore antibiotic mainly used in poultry and livestock to prevent and treat certain parasitic diseases. In antiviral applications, although Monensin is mainly directed against protozoa, its mechanism of changing the ion channels and membrane potential of host cells can have adverse effects on the viral replication environment.

[0017] The drug described in the present application is used for preventing and / or treating porcine reproductive and respiratory syndrome caused by PRRSV.

[0018] In an embodiment, the PRRSV is selected from PRRSV-1 or PRRSV-2.

[0019] In an embodiment, the PRRSV-1 or PRRSV-2 includes different geographical subtypes and lineages of its subordinates.

[0020] In an embodiment, the PRRSV-1 includes a commonly seen lineage in Europe, and the PRRSV-2 includes a commonly seen lineage in North America and / or Asia.

[0021] In an embodiment, the lineage of the PRRSV-2 includes a lineage with high pathogenicity and / or a viral variant strain.

[0022] In an embodiment, the viral variant strain is selected from one or more of PRRSV Ch-1a strain, WH3 strain, or SD16 strain; more preferably, the viral variant strain is PRRSV WH3 strain.

[0023] The drug of the present application prevents and / or treats porcine reproductive and respiratory syndrome by blocking the expression and / or replication of the PRRSV.

[0024] In another aspect, the present application provides an anti-PRRSV or a drug 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.

[0025] In one embodiment, the present application evaluates the inhibitory effect of methylsalinomycin on PRRSV. The experimental results show that methylsalinomycin has a significant inhibitory effect on the replication of PRRSV at different concentrations, especially at a high concentration of 5 μM, which shows a significant increase in CT value, i.e., a significant decrease in viral replication. These results provide strong experimental support for the potential of methylsalinomycin as an anti-PRRSV drug.

[0026] In one embodiment, the present application evaluates the inhibitory effect of salinomycin on PRRSV. By testing different concentrations of salinomycin, the results show that even at a lower concentration, salinomycin can significantly increase the CT value of viral replication, indicating its ability to inhibit viral replication. At a high concentration of 5 μM, salinomycin exhibits a stronger viral inhibitory effect. This finding highlights the effectiveness and application potential of salinomycin as an anti-PRRSV drug.

[0027] In one embodiment, the present application evaluates the inhibitory effect of Lasalocid on PRRSV. Experimental data show that Lasalocid can significantly increase the CT value of PRRSV at each tested concentration, especially at a high concentration of 5 μM, showing the strongest viral inhibitory effect. This result indicates that Lasalocid has a significant effect on inhibiting PRRSV replication, supporting its feasibility as an anti-PRRSV treatment option.

[0028] In one embodiment, the present application evaluates the inhibitory effect of different polyether long-chain antibiotics, methylsalinomycin, salinomycin, Lasalocid, and monensin, on PRRSV at the same concentration (5 μM). The experimental results show that methylsalinomycin, salinomycin, and Lasalocid all exhibit significant inhibitory effects, especially methylsalinomycin, which shows a significant increase in CT value, indicating a strong inhibition of PRRSV replication. In contrast, monensin has a weaker effect at this concentration, which suggests that we need to consider the specific mechanism of action of the drug and its performance under different conditions when choosing an anti-PRRSV treatment option.

[0029] In one embodiment, the present application evaluates the effect of different target compounds, methylsalinomycin, salinomycin and Lasalocid on PAM cell viability at different concentrations to confirm their safety at effective antiviral concentrations. The experimental results show that the change of cell viability of methylsalinomycin, salinomycin and Lasalocid at different concentrations indicates that these compounds have low toxicity to cells at low to moderate concentrations. In particular, under experimental conditions above the concentration threshold, these compounds do not significantly affect the survival rate of cells, i.e. show good cell compatibility.

[0030] In one embodiment, the concentration of the polyether long-chain antibiotic in the medicament is selected from 0.1 to 50 μM.

[0031] In one embodiment, the concentration of the polyether long-chain antibiotic in the medicament is selected from 0.1 to 5 μM.

[0032] In one embodiment, the concentration of the polyether long-chain antibiotic in the medicament is selected from 1 μM, 2.5 μM or 5 μM.

[0033] In one embodiment, the concentration of the polyether long-chain antibiotic in the medicament is 5 μM.

[0034] The present application takes into account the results of viral inhibition and cytotoxicity experiments in selecting the antibiotic and its concentration in the medicament. Methylsalinomycin, salinomycin and Lasalocid all exhibit significant inhibition of PRRSV at specific concentrations, significantly increasing the CT value of the virus, indicating that viral replication is effectively inhibited. Considering the balance between antiviral activity and cytotoxicity, the antibiotic and its concentration range not only ensure the antiviral effect of the medicament, but also minimize cytotoxicity.

[0035] In one embodiment, the dosage form of the medicament is selected from one or more of a topical preparation, a spray preparation, an injection preparation or an oral preparation.

[0036] In one embodiment, the topical preparation is selected from one or more of a cream, a gel, a lotion or a medicinal spray.

[0037] In one embodiment, the spray preparation is selected from one or more of a solution spray, a suspension spray or a powder spray.

[0038] In one embodiment, the injection preparation is selected from one or more of a solution injection, a suspension injection or an emulsion injection.

[0039] In one embodiment, the oral preparation is selected from one or more of a tablet, a capsule, a suspension or a granule.

[0040] The medicament of the present application further comprises an auxiliary ingredient, wherein the auxiliary ingredient is selected from one or more of excipient, sustained release agent, stabilizer, antioxidant, preservative, solvent and solubilizer.

[0041] In one embodiment, the excipient is selected from one or more of starch, lactose, cellulose or its derivatives.

[0042] In one embodiment, the sustained release agent is selected from one or more of hypromellose, polyvinyl alcohol or its copolymer.

[0043] In one embodiment, the stabilizer is selected from one or more of ethylenediaminetetraacetic acid (EDTA) or its sodium salt.

[0044] In one embodiment, the antioxidant is selected from one or more of vitamin E, vitamin C or its derivatives.

[0045] In one embodiment, the preservative is selected from one or more of sodium benzoate, potassium sorbate.

[0046] In one embodiment, the solvent is selected from one or more of water, ethanol or propylene glycol.

[0047] In one embodiment, the solubilizer is selected from one or more of polysorbate 80 (Tween 80) or polyoxyethylene castor oil.

[0048] In another aspect of the present application, there is provided use of the aforementioned medicament in the preparation of a medicament for preventing and / or treating PRRSV-mediated disease.

[0049] In another aspect of the present application, there is provided use of the aforementioned medicament in the preparation of a medicament for preventing and / or treating porcine reproductive and respiratory syndrome.

[0050] In another aspect of the present application, there is provided use of the aforementioned medicament in the preparation of a PRRSV inhibitor.

[0051] In another aspect of the present application, there is provided a PRRSV inhibitor, wherein the inhibitor comprises the aforementioned medicament.

[0052] In another aspect of the present application, there is provided a method of preventing and / or treating porcine reproductive and respiratory syndrome, wherein the method comprises administering the aforementioned medicament to a subject in need thereof.

[0053] In one embodiment, the medicament prevents and / or treats porcine reproductive and respiratory syndrome by blocking the expression and / or replication of the PRRSV.

[0054] In one embodiment, the subject to which the medicament of the present application is applicable is an artiodactyl.

[0055] In one embodiment, the Artiodactyla animals include, but are not limited to, wild species and domestic species.

[0056] In one embodiment, the domestic species is a food pig species, including but not limited to, Large White, Duroc, and Hampshire, etc. More preferably, the food pig species is a PRRSV-susceptible type.

[0057] In one embodiment, the effective application concentration of the medicine for preventing and / or treating porcine reproductive and respiratory syndrome is 0.1-5 μM, such as 0.1 μM, 0.5 μM, 1 μM, or 5 μM.

[0058] In one embodiment, the effective concentration is a concentration acting on the cellular level. The polyether long-chain antibiotics in the present application have shown significant progress and advantages in the application of anti-PRRSV, especially in the field of veterinary medicine, and their unique pharmacological properties and multifunctionality make them stand out in the market.

[0059] 1. High efficiency in inhibiting viral replication: The present application provides the use of polyether long-chain antibiotics in the preparation of antiviral drugs, especially in the significant inhibition of the replication of PRRSV. These antibiotics form ion channels on the cell membrane, disrupt the ion gradient of the pathogen, and thus inhibit its growth and reproduction. Experiments have shown that, for example, salinomycin has a significant inhibitory effect on the replication of PRRSV at different concentrations, and can effectively reduce the viral load, thereby controlling the spread and impact of the disease.

[0060] 2. Low side effects: These polyether long-chain antibiotics show low toxicity and fewer side effects at therapeutic doses. This helps to maintain the health and production performance of pigs and reduce adverse reactions caused by drugs. For example, methyl salinomycin has been shown to effectively inhibit PRRSV at lower concentrations without significant toxicity to host cells, which makes it advantageous in long-term treatment.

[0061] 3. Multifunctionality: In addition to the antiviral effect, the antibiotics of the present application also have broad antibacterial and antiprotozoal activity. For example, Lasalocid and salinomycin not only show advantages in antiviral aspects, but also can effectively prevent and treat coccidiosis in poultry and livestock. This multifunctionality allows these antibiotics to solve multiple infection problems in one drug preparation, improving breeding efficiency.

[0062] In summary, the polyether long-chain antibiotics in the present application have broad application prospects in the development of anti-PRRSV drugs. By combining their advanced pharmacological properties and treatment strategies, the effect of veterinary drugs can be significantly improved to meet the needs of the livestock industry for efficient and low-risk treatment programs. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 shows the change of viral replication CT value in the control group and different concentrations of the sample group of methyl salinomycin.

[0064] Figure 2 shows the change of viral replication CT value in the control group and different concentrations of the sample group of salinomycin.

[0065] Figure 3 shows the change of viral replication CT value in the control group and different concentrations of the sample group of Lasalocid.

[0066] Figure 4 shows the change of viral replication CT value in the control group and different target compound sample groups.

[0067] Figure 5 shows the change of viral replication CT value in the control group and different concentrations of the sample group of monensin.

[0068] Figure 6 shows the cell viability results after methyl salinomycin treatment.

[0069] Figure 7 shows the cell viability results after salinomycin treatment.

[0070] Figure 8 shows the cell viability results after Lasalocid treatment. DETAILED DESCRIPTION

[0071] I. Definitions and Explanation

[0072] For the purposes of the present invention, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used herein have the meanings that are commonly understood by one of ordinary skill in the art to which this invention belongs. It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, as such can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0073] As used herein, the term “porcine reproductive and respiratory syndrome (PRRS)” refers to an infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which mainly affects the reproductive and respiratory systems of pigs, and is a viral disease that seriously affects pigs, mainly manifested as respiratory dysfunction and reproductive dysfunction. This disease poses a great challenge to the global pig industry, especially in terms of causing problems such as sow abortion, high mortality of piglets, etc., resulting in serious economic losses.

[0074] As used herein, the term “PRRSV” refers to porcine reproductive and respiratory syndrome virus, which is an arterivirus, divided into two main types of PRRSV-1 and PRRSV-2, prevalent in Europe and North America, Asia, etc. This virus has high genetic diversity and variability, leading to the emergence of multiple strains.

[0075] As used herein, the term "PRRSV inhibitor" is capable of reducing, preventing the activity, replication process of PRRSV, or modulating the associated physiopathological responses it triggers, at the cellular level, by interacting with PRRSV or specific targets within the cells affected by this virus, at effective concentrations of 0.1 μΜ, 0.5 μΜ, 1 μΜ or 5 μΜ, among others, thus preventing and / or treating porcine reproductive and respiratory syndrome.

[0076] As used herein, the term "polyether-based long-chain antibiotic" refers to a class of antibiotics that contain a polyether chain in their structure, which have ionophore function and can interfere with the ion balance of pathogens by forming ion channels in the cell membrane.

[0077] As used herein, the term "dosage form" refers to the physical form or configuration of a drug that determines its release, distribution, and application. It includes topical preparations, spray preparations, injection preparations, or oral preparations, among others. The physical form of a drug is designed to accommodate the method of drug administration and improve efficacy. Different dosage forms can affect the absorption, distribution, metabolism, and excretion of drugs, thus affecting efficacy and safety. For example, topical preparations are suitable for the treatment of skin diseases, spray preparations are suitable for respiratory diseases, injection preparations are suitable for rapid and precise administration, and oral preparations are usually used for long-term treatment.

[0078] As used herein, the term "topical preparation" refers to a drug preparation that is applied directly to the body surface or a specific body surface site. Topical preparations are mainly used for direct action on the skin or mucosal surface of the body surface, and such preparations can reduce systemic side effects and increase drug concentration at the lesion site by topical application. Common forms of topical preparations include creams, gels, lotions, and medicinal sprays, each with its own specific application scenarios and advantages, such as creams for dry or cracked skin, gels for cleaning or moisturizing, lotions for cleaning large areas of skin, and medicinal sprays for easy coverage of extensive areas.

[0079] As used herein, the term "spray preparation" refers to a drug preparation that is made into fine particles and released through a sprayer for application to the body surface or body. By making the drug into fine droplets or powder, it can be administered by inhalation or spraying. This preparation is particularly suitable for conditions that require rapid action, such as asthma or allergic reactions. Spray preparations can be divided into solution sprays, suspension sprays, and powder sprays according to their carriers, among which solution sprays are widely used due to their uniformity and easy-to-control dosage.

[0080] As used herein, the term "injectable formulation" refers to a pharmaceutical formulation that is injected into the body through a syringe, which can act directly on the blood system or specific tissues. The injectable formulation can be a solution, a suspension or an emulsion, each with its own advantages and specific use occasions. Solution injection is widely used because of its uniform composition and immediate release of drug efficacy, while suspension injection and emulsion are used for treatments that require sustained release because they can prolong the action time of the drug.

[0081] As used herein, the term "oral formulation" refers to a pharmaceutical formulation that is taken through the mouth and is one of the most commonly used methods of drug administration. Such formulations include tablets, capsules, suspensions and granules, etc.

[0082] As used herein, the term "auxiliary ingredient" refers to other ingredients added to the pharmaceutical formulation in addition to the active pharmaceutical ingredient, which are substances added to the formulation to improve the physical, chemical properties or bioavailability of the drug. These ingredients include excipients, sustained-release agents, stabilizers, antioxidants, preservatives, solvents and solubilizers. The choice and use of these ingredients depend on the required drug release rate, stability, patient acceptance and other pharmaceutical requirements. For example, excipients such as starch and lactose are used to provide solid form and palatability of the drug; sustained-release agents such as hydroxypropyl methylcellulose are used to prolong the release time of the drug in the body; stabilizers such as ethylenediaminetetraacetic acid (EDTA) are used to prevent degradation of drug ingredients during storage; antioxidants such as vitamin E and vitamin C are used to prevent oxidation of the drug; preservatives such as sodium benzoate and potassium sorbate are used to prevent the growth of microorganisms in the formulation; solvents such as water and ethanol are used to adjust the solubility of the drug; solubilizers such as polysorbate 80 (Tween 80) and polyoxyethylene castor oil are used to increase the absorption efficiency of the drug in the body. To improve the physical and chemical properties, stability or acceptance of the drug. Including excipients, sustained-release agents, stabilizers, antioxidants, preservatives, solvents and solubilizers, etc.

[0083] As used herein, the term "PAM cell" refers to a porcine macrophage, which is a macrophage cell line from porcine alveoli, commonly used in virology research, especially in the study of host cell response and viral replication mechanism of porcine reproductive and respiratory syndrome virus (PRRSV).

[0084] As used herein, the term "blue ear virus PRRSV WH3 strain" refers to a specific strain of porcine reproductive and respiratory syndrome virus, which 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 treatment methods.

[0085] As used herein, the term "Fluorogenic Quantitative Polymerase Chain Reaction" (qPCR) refers to a molecular biology technique used to amplify and quantify target DNA templates simultaneously, commonly used to detect and measure viral load or gene expression levels. This method measures the initial amount of a specific DNA sequence by monitoring the increase in fluorescence signal in real-time.

[0086] As used herein, the term "threshold cycle value" (CT value) refers to the number of cycles required to reach the detection threshold during a fluorogenic quantitative PCR process. The CT value reflects the initial amount of target DNA in the sample; a lower CT value means a higher initial amount of target DNA, conversely, a higher CT value indicates a lower initial DNA amount. This value is a key indicator for assessing viral replication efficiency and drug antiviral activity.

[0087] As used herein, 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 direct damage to cell structures, interference with cell metabolism, or influence on cell growth signals.

[0088] As used herein, the term "cytotoxicity test" refers to a series of experimental methods used to evaluate the effects of compounds or conditions on cell viability and health. These tests are usually performed by measuring parameters such as cell survival rate, cell metabolic activity, cell membrane integrity, etc. Common cytotoxicity test methods include MTT assay, LDH release assay, flow cytometry, etc.

[0089] As used herein, the term "CC50" (Concentration for 50% of maximal cytotoxic effect) refers to the concentration of a compound required to reduce cell survival rate by 50% under certain conditions. This value is commonly used to measure the impact of a compound on cell viability and is an important indicator of the strength of a compound's cytotoxicity. A higher CC50 indicates lower cytotoxicity of the compound, i.e., it will only have a significant toxic effect on cells at higher concentrations.

[0090] As used herein, the term "Artiodactyla" refers to an order of the class Mammalia. There are 10 families, 75 genera, and 184 species in the order, hence the name Artiodactyla, meaning even-toed. Most of these animals have horns on their heads; the thoracolumbar vertebrae are fewer than in the Perissodactyla; the femur has no third trochanter; the third and fourth toes are equally developed for weight support, and the stomach is mostly multilocular, with a short, small cecum. The order includes pigs, badgers, hippos, camels, deer, antelope, giraffes, musk deer, and tufted deer. These animals walk on the tips of their toes, usually with the heavy third and fourth toes balancing the body weight, and the second and fifth toes small and pedunculate, or absent. The premolars and molars are large and well developed, and the canines are often reduced. The incisors are only the lower ones, which are large, and the upper ones are small or absent, so that the food is taken in by the hard upper lip. The premolars and molars are high-crowned in structure, with a masticating surface adapted for grinding. The cheek teeth of a pig, for example, have numerous small tuberculate projections, and are of the lophodont type.

[0091] II. Examples

[0092] Materials and Methods

[0093] Cells: PAM cells were isolated from pig lung, and the isolation method was referred to 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.

[0094] Virus: PRRSV WH3 strain was isolated from pig lung, and the isolation method was referred to the above literature.

[0095] Reagents: TRIzol (for RNA extraction), reverse transcription kit (for cDNA synthesis), PRRSV-U nucleic acid detection kit (fluorescent PCR method) (for virus copy number detection) were purchased from Wuhan Keygen Biotech Co., Ltd.

[0096] Target compounds: Methylmycin, salinomycin, Lasalocid and monensin were purchased from commercial reagent companies. Methylmycin was purchased from Sigma-Aldrich (catalog number B28443); salinomycin was purchased from Sigma-Aldrich (catalog number S80781); Lasalocid was purchased from TargetMol (catalog number T15717); monensin was purchased from Sigma-Aldrich (catalog number S17047).

[0097] Example 1: Inhibition effect of methylmycin on PRRSV

[0098] This example aims to evaluate the inhibitory effect of methyl salinomycin on PRRSV, determine its effect on viral replication at different concentrations, and provide experimental basis for the development of efficient and low-toxicity anti-PRRSV drugs.

[0099] Grouping method:

[0100] Control group: PAM cells were treated with DMEM medium containing 8% FBS, only DMSO solvent without methyl salinomycin was added, followed by PRRSV infection.

[0101] Sample group: PAM cells were treated with methyl salinomycin 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.

[0102] Experimental method:

[0103] Cell culture: PAM cells were seeded in 6-well plates containing glass slides at a concentration of 2×10 6 / well, and incubated in a 37°C, 5% CO2 incubator. After the cells adhered to form a monolayer (about 12 hours), they were treated with methyl salinomycin.

[0104] Drug treatment: Analytical pure DMSO was prepared as a solvent, methyl salinomycin powder was dissolved in an appropriate amount of DMSO to prepare a high-concentration stock solution, and sterile 8% FBS DMEM medium was used for stepwise dilution to prepare the required working concentrations (0.1 μM, 0.5 μM, 1 μM, and 5 μM). According to the grouping method, different concentrations of methyl salinomycin (0.1 μM, 0.5 μM, 1 μM, and 5 μM) were added to the cell culture medium to prepare different concentrations of the sample group, and the control group was only added with DMSO solvent. PAM cells were treated at 37°C.

[0105] Virus infection: After drug treatment, PRRSV WH3 strain (MOI = 0.1) was inoculated, and the cells were co-cultured in a 37°C, 5% CO2 incubator for 36 hours.

[0106] RNA extraction and reverse transcription: RNA was extracted from the cells using TRIzol, and the RNA was reverse transcribed to synthesize cDNA.

[0107] Real-time fluorescence quantitative PCR: The cDNA after reverse transcription was used as the template, PRRSV-N-F and PRRSV-N-R were used as primers, and real-time fluorescence quantitative PCR was performed to detect the copy number of PRRSV in the sample. The real-time fluorescence quantitative PCR primers used were:

[0108] PRRSV-N-F: 5'-AATAACAACGGCAAGCAGCAG-3'

[0109] PRRSV-N-R: 5'-CCTCTGGACTGGTTTTGTTGG-3'

[0110] The fluorescence data was collected after each cycle using a fluorescence quantitative PCR instrument, and the CT value of each sample was automatically calculated. The CT value is the number of PCR cycles required to reach the 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 increase in CT value indicates a decrease in viral load, i.e., inhibition of viral replication.

[0111] Data analysis: Compare the CT values of the sample group with the control group to analyze and evaluate the effect of methyl salinomycin on PRRSV replication.

[0112] Results analysis and discussion:

[0113] In this experiment, the replication of PRRSV in PAM cells was detected by RT-PCR, and the results showed that methyl salinomycin at different concentrations had a significant inhibitory effect on the replication of PRRSV. Figure 1 is a bar chart of the change in viral replication CT value in the control group and different concentrations of methyl salinomycin sample groups. As shown in the figure, the average CT value of the virus in the control group was 14.69, indicating normal replication of the virus in PAM cells; the average CT value of the virus in the 0.5 μM methyl salinomycin sample group rose to 21.40, indicating that at this concentration, there was a significant difference compared with the control group (P<0.05), indicating that methyl salinomycin significantly inhibited the replication of the virus; the average CT value of the virus in the 1 μM methyl salinomycin sample group further rose to 26.49, and there was a significant difference compared with the control group (P<0.05), showing a stronger inhibitory effect; the average CT value of the virus in the 5 μM methyl salinomycin sample group reached 29.45, and there was a significant difference compared with the control group (P<0.05), showing the highest inhibitory effect. These data show that methyl salinomycin significantly reduces the viral load in PAM cells by interfering with the viral replication process, and the inhibitory effect is more significant as the drug concentration increases.

[0114] The experimental results show that methyl salinomycin has excellent effect in inhibiting PRRSV replication. As a polyether long-chain antibiotic, methyl salinomycin forms ion channels on the cell membrane, disrupts the ion balance of the virus, and thus inhibits its replication in host cells. This mechanism of action enables methyl salinomycin to exhibit strong antiviral activity at a lower concentration, while ensuring lower toxicity. By significantly inhibiting the replication of PRRSV, methyl salinomycin shows great potential as an anti-PRRSV drug, providing a solid scientific basis for the development of an efficient and low-toxicity anti-PRRSV drug. Through systematic experimental research and comparative analysis, methyl salinomycin is expected to become an effective new drug for the treatment and prevention of PRRSV infection, providing an innovative and effective solution for the pig industry.

[0115] Example 2: Inhibition effect of salinomycin on PRRSV

[0116] This example aims to evaluate the inhibition effect of salinomycin on PRRSV, determine its impact on viral replication at different concentrations, and explore its effectiveness as a potential anti-PRRSV drug.

[0117] Grouping method:

[0118] Control group: PAM cells were treated with DMEM medium containing 8% FBS, only adding DMSO solvent without salinomycin, followed by PRRSV infection.

[0119] Sample group: PAM cells were treated with salinomycin 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.

[0120] Experimental method:

[0121] The same as Example 1.

[0122] Results analysis and discussion:

[0123] Figure 2 is a column chart of the CT value changes of viral replication in the control group and different concentrations of salinomycin sample groups. As shown in the figure, the average CT value of the virus in the control group is 14.69, indicating that the virus is normally replicated in PAM cells; salinomycin at a concentration of 0.5 μM can increase the average CT value of the virus from 14.69 to 15.74 compared with the control group, indicating that salinomycin can inhibit the replication of blue ear virus in host cells; at higher drug concentrations, the average CT value of the virus at a concentration of 1 μM is 22.01, and the average CT value of the virus at a concentration of 5 μM is 26.34, which has a significant difference (P<0.05) compared with the control group, indicating that the ability of viral replication is significantly weakened.

[0124] The experimental results clearly show that salinomycin has a significant effect on inhibiting PRRSV replication. In addition, salinomycin also has broad-spectrum antimicrobial activity, not only can inhibit the replication of viruses, but also can effectively prevent and control a variety of bacterial and protozoan infections. This multifunctionality makes salinomycin have a wide application prospect in the breeding industry, which can solve the problem of multiple pathogen infections and improve the breeding efficiency. In summary, the significant effect of salinomycin on inhibiting PRRSV, low toxicity and its multifunctionality make it a potential new antiviral drug, providing an efficient and low-risk solution for the pig industry.

[0125] Example 3: Inhibition effect of Lasalocid on PRRSV

[0126] This example aims to evaluate the inhibition effect of Lasalocid on PRRSV and determine its impact on viral replication at different concentrations, in order to explore its effectiveness as a potential anti-PRRSV drug.

[0127] Grouping method:

[0128] Control group: PAM cells were treated with DMEM medium containing 8% FBS, only adding DMSO solvent without Lasalocid, followed by PRRSV infection.

[0129] Sample group: 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.

[0130] Experimental method:

[0131] The same as Example 1.

[0132] Results analysis and discussion:

[0133] The present embodiment detects the replication of PRRSV in PAM cells by RT-PCR, and reveals the inhibitory effect of Lasalocid on PRRSV replication at different concentrations. FIG. 3 is a column chart of the CT value changes of virus replication in the control group and different concentrations of Lasalocid sample groups. As shown in the figure, the average CT value of PRRSV in the control group is 14.69, indicating that the virus is normally replicated in PAM cells. In the sample treated with 0.1 μM Lasalocid, the average CT value rises to 15.67, indicating that Lasalocid has begun to exert an inhibitory effect at a lower concentration. With the increase of concentration, the average CT value of the 1 μM treatment group rises to 17.90, showing more obvious inhibition of virus replication. At a high concentration of 5 μM, the average CT value reaches 28.07, which is significantly different from the control group (P<0.05), showing that Lasalocid has a strong inhibitory effect on PRRSV replication. These data show that Lasalocid effectively reduces viral load by interfering with the replication process of PRRSV in host cells, and the inhibitory effect is enhanced with the increase of drug concentration.

[0134] The experimental results show that Lasalocid, as a polyether long-chain antibiotic, inhibits the replication of PRRSV by adjusting the ion balance on the cell membrane. This mechanism not only exhibits good antiviral activity at a lower concentration, but also has lower cytotoxicity. Compared with traditional prevention and control measures, Lasalocid shows higher efficiency and lower side effects, significantly improving its safety in clinical application. In addition, the broad-spectrum antimicrobial activity of Lasalocid also makes it have potential application value in preventing and controlling various bacterial and protozoan infections, which provides an effective solution to pathogen infection for the breeding industry and helps to improve breeding efficiency and animal health. Therefore, Lasalocid is not only an effective anti-PRRSV drug, but also a highly functional therapeutic option, bringing a new treatment strategy to the pig industry.

[0135] Example 4: Inhibitory effect of different target compounds on PRRSV

[0136] The present embodiment aims to evaluate the inhibitory effect of different target compounds, i.e. methyl salinomycin, salinomycin, Lasalocid and monensin, which are four polyether long-chain antibiotics, on PRRSV replication at the same concentration (5 μM), in order to determine which compound performs best in controlling the virus.

[0137] Grouping method:

[0138] Control group: PAM cells were treated with DMEM medium containing 8% FBS, only adding DMSO solvent without salinomycin, followed by PRRSV infection.

[0139] Sample groups: PAM cells were treated with methylsalinomycin, salinomycin, lasalocid and monensin at a concentration of 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.

[0140] Experimental method:

[0141] The same as Example 1.

[0142] Results analysis and discussion:

[0143] Figure 4 shows the changes in viral replication CT values in the control group and different target compound sample groups, where:

[0144] The average CT value of the methylsalinomycin treatment group was 29.45, showing the strongest inhibitory effect. Compared with the control group, the CT value was significantly increased, indicating that methylsalinomycin was very effective in blocking PRRSV replication. This result suggests that methylsalinomycin may significantly reduce viral load by interfering with a key replication stage of the virus. The high inhibitory capacity of methylsalinomycin makes it have great application potential in anti-PRRSV therapy. Because it can exhibit strong antiviral activity at a lower concentration, with fewer side effects, methylsalinomycin is an extremely attractive candidate drug.

[0145] The average CT value of the salinomycin treatment group was 26.34, also showing significant viral inhibition effect. Although slightly lower than methylsalinomycin, it still effectively inhibited viral replication. The mechanism of action of salinomycin may be similar to that of methylsalinomycin, by forming ion channels on the cell membrane, disrupting the ion balance of the virus, and preventing the replication process of the virus in the host cell. The antiviral ability of salinomycin makes it another effective PRRSV treatment candidate drug, especially in cases where multiple drugs need to be used in combination, its strong antiviral activity will help improve treatment effectiveness.

[0146] The average CT value of the lasalocid treatment group was 28.07, close to methylsalinomycin, showing very strong viral inhibition effect. The mechanism of action of lasalocid may also change the ion permeability of the cell membrane, thereby preventing viral replication. The significant effect of lasalocid in inhibiting PRRSV makes it an important antiviral drug, especially in preventing and treating 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 the farming industry.

[0147] Unlike the other three antibiotics, the average CT value for the monensin-treated group was 13.72, indicating that it did not significantly inhibit PRRSV replication under these experimental conditions and may even have promoted viral replication. This result may be related to the mechanism of action of monensin, suggesting the need for further research to clarify its effects under different conditions. Figure 5 shows a bar chart of the changes in the average CT values of viral replication in the control group and different concentrations of monensin sample groups. In different concentrations of monensin sample groups, the average CT value was lower than the control group, especially at higher concentrations (1 μM and 5 μM), the CT value did not show an upward trend. The performance of monensin reminds us that when choosing anti-PRRSV drugs, the specific mechanism of action of the drug and the experimental conditions need to be fully considered to ensure the selection of the most effective treatment plan.

[0148] In summary, methylsalinomycin, salinomycin, and Lasalocid have shown significant effects in inhibiting PRRSV, especially methylsalinomycin, which has outstanding performance in low toxicity and high efficiency. These antibiotics effectively reduce viral load by interfering with the replication process of the virus, providing new ideas and methods for the treatment of PRRSV. The results of monensin indicate that research and optimization are necessary to ensure the safety and effectiveness of all candidate drugs. These findings not only provide scientific basis for the development of new PRRSV treatment options, but also bring new strategies for the pig industry.

[0149] Example 5: Cytotoxicity experiment

[0150] This example aims to evaluate the effects of different target compounds—methylsalinomycin, salinomycin, and Lasalocid—at different concentrations on the survival rate of PAM cells to confirm their safety at effective antiviral concentrations.

[0151] Materials and methods

[0152] Cell line: PAM cells were used as the experimental model.

[0153] Cells were cultured in DMEM medium, and different target compounds were first dissolved in DMSO and then diluted with culture medium to the desired experimental concentration. PAM cells were inoculated and incubated to adhere to the cells. The culture medium containing different concentrations of target compounds was added for cell treatment. The CCK-8 kit (Cell Counting Kit-8) was used for cell viability testing. Add 10 μL of CCK-8 solution to each well, incubate for 2-4 hours, and then use a microplate reader to measure the absorbance (OD value) at 450 nm. Calculate the cell viability at each concentration and compare it with the control group.

[0154] The cell viability was calculated using the following formula:

[0155] Cell viability (%) = (OD of experimental group - OD of blank control group) / (OD of control group - OD of blank control group) x 100%

[0156] Results analysis and discussion:

[0157] Figure 6 shows the cell viability results after methylsalinomycin treatment, Figure 7 shows the cell viability results after salinomycin treatment, and Figure 8 shows the cell viability results after Lasalocid treatment. The experimental results show that, with the compound concentration that makes 50% of PAM cells lose normal metabolic function or growth ability as the line, at the highest concentration of 22.75 μM of methylsalinomycin, the viability of PAM cells is more than 50%, which means that the toxicity CC50 (Concentration for 50% of maximal cytotoxic effect) of methylsalinomycin to PAM cells is greater than 22.75 μM. At low concentrations (5.60 μM or less), the cell activity gradually recovers to nearly 100%, indicating that the cytotoxicity of methylsalinomycin is low at these concentrations. The effect of salinomycin on PAM cells is relatively gentle, and at the highest concentration of 10.000 μM, the viability of PAM cells is maintained at about 60% or more. This indicates that the toxicity CC50 value of salinomycin to PAM cells is higher than 10.000 μM. For Lasalocid, the experimental data show that even at a higher concentration of 25.46 μM, the viability of PAM cells is still higher than 50%, and when it is further reduced to 0.79 μM, the viability of the cells is significantly improved, close to 100%. This indicates that the CC50 value of Lasalocid is greater than 25.46 μM, indicating that the toxicity of Lasalocid to PAM cells is low at these concentrations.

[0158] The above is a further detailed description of the present application, which cannot be considered as a limitation to the specific implementation of the present application. All the documents mentioned in the present application are cited in the present application as references, just as each document is cited as a reference. In addition, it should be understood that for those skilled in the art, simple deductions or replacements without departing from the concept of the present application are within the scope of protection of the present application.

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, wherein, The antibiotic is a polyether-based long-chain antibiotic.

2. The application according to claim 1, wherein, The polyether-based long-chain antibiotic is an ion carrier antibiotic; Preferably, the ionotropic antibiotic is selected from one or more of Lasalocid, methylsalicylic acid, or salicylic acid.

3. The application according to claim 1 or 2, wherein, The drug is used to prevent and / or treat porcine reproductive and respiratory syndrome caused by PRRSV; Preferably, the PRRSV is selected from PRRSV-1 or PRRSV-2; Preferably, the PRRSV-1 or PRRSV-2 includes its different geographically distributed subtypes and lineages; Preferably, the PRRSV-1 includes lineages common in Europe, and the PRRSV-2 includes lineages common in North America and / or Asia; Preferably, the lineage of the PRRSV-2 includes lineages with high pathogenicity and / or viral variants; 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.

4. The application according to any one of claims 1 to 3, wherein, The drug prevents and / or treats porcine reproductive and respiratory syndrome by blocking the expression and / or replication of PRRSV.

5. A drug for the prevention and / or treatment of porcine reproductive and respiratory syndrome (PRRSV), wherein, The drug contains one or more of the polyether long-chain antibiotics as described in claim 1 or 2.

6. The drug according to claim 5, wherein, The concentration of the polyether long-chain antibiotic in the drug is selected from 0.1 to 50 μM; Preferably, the concentration is selected from 0.1 to 5 μM; Preferably, the concentration is selected from 1 μM, 2.5 μM or 5 μM; more preferably, the concentration is 5 μM.

7. The drug according to claim 5 or 6, wherein, The dosage form of the drug is selected from one or more of the following: topical preparations, spray preparations, injectable preparations, or oral preparations; Preferably, the topical preparation is selected from one or more of creams, gels, lotions, or medicinal sprays; Preferably, the spray formulation is selected from one or more of solution spray, suspension spray, or powder spray; Preferably, the injectable formulation is selected from one or more of solution injections, suspension injections, or emulsion injections; Preferably, the oral formulation is selected from one or more of tablets, capsules, suspensions or granules.

8. The medicament according to any one of claims 5 to 7, wherein, The drug also includes excipients.

9. The medicament according to claim 8, wherein, The auxiliary ingredients are selected from one or more of excipients, sustained-release agents, stabilizers, antioxidants, preservatives, solvents, and solubilizers; The excipient is selected from one or more of starch, lactose, cellulose or their derivatives; The sustained-release agent is selected from one or more of hydroxypropyl methylcellulose, polyvinyl alcohol, or copolymers thereof; The stabilizer is selected from one or more of ethylenediaminetetraacetic acid (EDTA) or its sodium salt; The antioxidant is selected from one or more of vitamin E, vitamin C, or their derivatives; The preservative is selected from one or more of sodium benzoate and potassium sorbate; The solvent is selected from one or more of water, ethanol, or propylene glycol; The solubilizer is selected from one or more of polysorbate 80 (Tween 80) or polyoxyethylene castor oil.

10. The medicament according to any one of claims 5 to 9, wherein, The drug is applicable to even-toed ungulates; Preferably, the even-toed ungulates include, but are not limited to, wild and domesticated species; 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.

11. A method for preventing and / or treating porcine reproductive and respiratory syndrome, wherein, The method includes administering the drug as described in any one of claims 5 to 9 to a person in need; Preferably, the drug prevents and / or treats porcine reproductive and respiratory syndrome by blocking the expression and / or replication of PRRSV; Preferably, the object is an even-toed ungulate; Preferably, the even-toed ungulates include, but are not limited to, wild and domesticated species; 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.

12. Use of the medicament according to any one of claims 5 to 9 in the preparation of a medicament for the prevention and / or treatment of PRRSV-mediated diseases.

13. Use of the medicament according to any one of claims 5 to 9 in the preparation of a PRRSV inhibitor.

14. Use of the medicament according to any one of claims 5 to 9 in the preparation of a medicament for the prevention and / or treatment of porcine reproductive and respiratory syndrome.

Images