A lipid composition for resisting porcine cystic virus infection and use thereof

Abstract

The application discloses an oil and fat composition for resisting porcine enterovirus infection and application thereof. The oil and fat composition comprises at least two of glyceryl monopentanoate, glyceryl monolinoleate and glyceryl monomyristate. The oil and fat composition can further comprise anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil, Tween 80, lactose, mannitol, soluble starch, water and other auxiliary materials for preparing oil and fat composition preparations. The oil and fat composition can inhibit enterovirus through multiple channels and multiple levels, and improve the antiviral effect. Adding the oil and fat composition in the feed of pigs can not only improve the antiviral ability, but also reduce the inflammatory reaction caused by pathogenic infection through the anti-inflammatory and immune regulation effects, effectively prevent and treat the infection of porcine enterovirus diarrhea, and thus reduce the incidence of porcine enterovirus diarrhea and the mortality of pigs with the disease. The oil and fat composition can be used for preparing a medicine or a feed additive for treating and preventing porcine enterovirus diarrhea.

Description

Technical Field

[0001] This application relates to the field of porcine enveloped virus infection prevention and control technology, specifically to an oil composition for preventing porcine enveloped virus infection and its application. Background Technology

[0002] Porcine enveloped virus infection is a major challenge in pig farming. Due to the presence of an outer envelope, enveloped viruses are more prone to mutation and have a stronger ability to evade the immune system, making prevention and control more difficult. Common porcine enveloped viruses include porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), and African swine fever virus (ASFV).

[0003] Porcine Epidemic Diarrhea (PED) caused by Porcine Epidemic Virus (PEV) is a highly contagious enteric disease, a serious threat to the global pig industry. Typical symptoms of PED include diarrhea, vomiting, and dehydration, with the greatest impact on newborn piglets. Mortality rates in piglets under 7 days old can reach 100%, resulting in significant losses. Epidemiological data shows that PEDV has become a major pathogen of porcine viral diarrhea in my country, with its variant strains being the predominant pathogens in epidemics. These variants often exhibit stronger pathogenicity and transmissibility, rendering existing vaccines ineffective and further increasing the difficulty of disease control. PEDV infection is primarily transmitted through the digestive tract, but can also spread through contaminated environments, objects, and the excrement of infected pigs. After entering the pig's body, the virus mainly infects the small intestinal epithelial cells, causing damage to intestinal villi and malabsorption, leading to severe diarrhea and dehydration, resulting in rapid weight loss, weakened immunity, and even death in piglets. Although adult pigs may exhibit milder symptoms, they may still experience decreased appetite and reduced litter size, significantly impacting their production performance.

[0004] Porcine reproductive and respiratory syndrome (PRRS) is a highly dangerous enveloped virus belonging to the Arteriviridae family. It is a single-stranded, positive-sense RNA virus. PRRS infection leads to reproductive disorders and respiratory symptoms, affecting all stages of pig growth. PRRSV is transmitted through various routes, including airborne transmission, direct contact, and transmission via contaminated feed and the environment, and is particularly prone to outbreaks in intensive farming conditions. In sows, PRRSV infection causes abortion, stillbirth, mummified fetuses, and decreased farrowing rates, significantly impacting herd reproductive performance. In piglets and finishing pigs, PRRSV infection manifests as rapid breathing, fever, decreased appetite, and weight loss. Infected herds are also more susceptible to secondary infections, such as bacterial pneumonia, which exacerbate the condition and increase mortality. The presence of PRRSV not only reduces farm productivity but also significantly increases farming costs, especially the economic losses due to increased morbidity and mortality. PRRSV's high variability makes it prone to immunization failure, posing a significant challenge to vaccine control. Therefore, controlling PRRSV infection is challenging, and currently relies primarily on comprehensive prevention and control measures, including biosecurity protocols, sound management strategies, and vaccination. Nevertheless, PRRSV remains one of the major threats to the global swine industry, significantly impacting economic efficiency and food security.

[0005] African swine fever virus (ASFV) is a double-stranded DNA virus belonging to the African swine fever virus family. ASFV is highly contagious and can survive for extended periods in the natural environment, making it one of the major infectious diseases in the pig industry. ASFV spreads widely, including through direct contact, feed and water contamination, and vector-borne transmission (such as soft ticks), and can spread rapidly once an infected herd is formed. ASFV can survive for months or even years under low temperatures, making it difficult to eradicate from pig farms. Pigs infected with ASFV exhibit symptoms such as high fever, red skin, rapid breathing, and decreased appetite; in severe cases, vomiting, diarrhea, and blood loss may occur, with a mortality rate reaching 100%. ASFV infection is fatal to pigs of all ages, especially piglets and herds with weakened immune systems. ASFV infection causes African swine fever (ASF), primarily characterized by acute hemorrhagic disease; it is highly pathogenic, causing death in infected pigs within a short period. The lethality of ASFV and the lack of an effective vaccine often necessitate the culling of all infected pigs and their penmates during an outbreak, resulting in significant economic losses. Control measures primarily rely on strict biosecurity protocols, including isolation, disinfection, and monitoring, to prevent further spread of the virus. However, the widespread transmission of ASF still poses a significant threat to the global swine industry and greatly impacts the stability of the food supply chain.

[0006] Enveloped viruses such as PEDV, PRRSV, and ASFV exhibit strong variability in their hosts, limiting the effectiveness of traditional vaccines. In the face of these viral infectious diseases, developing safe and highly effective feed additives or oral medications that can broadly inhibit enveloped viruses is of great significance for the health management of swine herds. Summary of the Invention

[0007] In view of this, the purpose of this application is to provide an oil composition for resisting porcine enveloped virus infection and its application. This oil composition comprises at least two of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristate, and has the advantages of high safety and convenient application. It significantly inhibits the replication of enveloped viruses such as PEDV, PRRSV, and ASFV, and reduces the virulence of porcine enveloped viruses. It can be used as a feed additive or oral medication, overcoming the disadvantages of possible immunization failure due to vaccination and viral mutations caused by the use of antiviral drugs. It has no toxic side effects on animals and is safe to use.

[0008] To achieve, or at least partially achieve, the above objectives, this application provides the following technical solution:

[0009] In a first aspect, this application provides an oil composition for resisting porcine enveloped virus infection, said oil composition comprising at least two of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristicate.

[0010] Secondly, this application provides the use of the aforementioned oil composition in the preparation of a drug or feed additive for preventing porcine enveloped virus infection.

[0011] The technical solution provided in this application has at least the following advantages compared with the prior art:

[0012] 1. The oil composition provided in this application, consisting of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristicate, has high safety and will not lead to the development of drug-resistant strains or other toxic side effects with long-term use. This is an advantage that other antiviral drugs do not possess.

[0013] 2. The oil composition provided in this application, consisting of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristicate, can inhibit enveloped viruses through multiple pathways and at multiple levels, thereby improving antiviral efficacy. Adding these three monoesters to pig feed not only enhances antiviral capabilities but also reduces inflammatory responses caused by pathogen infection through their anti-inflammatory and immunomodulatory effects, effectively preventing and treating porcine enveloped viral diarrhea (PED), thus reducing the incidence of PED and the mortality rate of affected pigs. This oil composition can be used to prepare drugs or feed additives for the treatment and prevention of PED.

[0014] 3. In vitro experimental results of this application show that the oil composition has a significant inhibitory effect on the in vitro proliferation of PEDV, PRRSV, and ASFV. Adding the oil composition to the cell culture medium at a concentration of 100 μg / mL can reduce the TCID levels of PEDV, PRRSV, and ASFV on the corresponding cells. 50 Each titer was reduced by approximately 5 to 10 points.

[0015] 4. The in vivo experimental results of this application show that the oil composition enhances natural immune function and has antiviral activity, and can effectively prevent and treat PEDV, PRRSV and ASFV infection. The oil composition can significantly improve the infection protection rate against PEDV, PRRSV and ASFV when taken orally or added to piglet feed. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0017] Those skilled in the art will understand that, unless otherwise stated, the terms "the," "the," and "the foregoing" used in this application may also include plural forms. It should be further understood that the word "comprising" as used in the specification of this application means the presence of the stated features, steps, or operations, but does not exclude the presence or addition of one or more other features, integers, or steps.

[0018] Those skilled in the art will understand that, where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field; and where the manufacturers of the raw materials or instruments and equipment used are not specified, they are all conventional products that can be obtained commercially.

[0019] Those skilled in the art will understand that, unless otherwise stated in this application, when numerical ranges are given in the embodiments, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application, as well as the prior art known to those skilled in the art and the descriptions in this application, can be implemented using any prior art methods, devices, and materials similar to or equivalent to those described, used, or made by means of methods, devices, and materials in the embodiments of this application.

[0020] Glyceryl pentanoate (GP) is a monoester formed by the esterification of glycerol and valerate. Its structure contains a five-carbon fatty acid group and a glycerol molecule. Glyceryl pentanoate belongs to the medium-chain fatty acid glycerides and has good lipophilicity and some hydrophilicity, allowing it to penetrate biological membranes and exert its effects within cells. Glyceryl linoleate (GL) is an important unsaturated fatty acid glyceride, mainly composed of glycerol and linoleic acid. GL is one of the more common fatty acid components found in vegetable oils. Glyceryl myristate (GM) is an esterification product of glycerol and myristic acid, mainly found in coconut oil and palm oil. GM is a long-chain saturated fatty acid glyceride with good lipophilicity and some penetrability. Currently, none of these three single glycerides have been used against porcine enveloped viruses.

[0021] This application embodiment mixes three single glycerides—glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristicate—in a specific mass ratio to form an oil composition. In vivo and in vitro experiments have demonstrated that this oil composition can significantly inhibit the proliferation of PEDV, PRRSV, and ASFV, as well as their infection of cells. Therefore, this oil compound can be used to prepare drugs or feed additives against viruses such as PEDV, PRRSV, and ASFV.

[0022] Based on this, embodiments of this application provide an oil composition for resisting porcine enveloped virus infection, the oil composition comprising at least two of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristicate.

[0023] In some embodiments, the oil composition, by weight, comprises 1 part glyceryl monovalerate and 0.25 to 4 parts glyceryl monolinoleate. In some preferred embodiments, the oil composition, by weight, comprises 1 part glyceryl monovalerate and 1 part glyceryl monolinoleate.

[0024] In some embodiments, the oil composition, by weight, comprises 1 part glyceryl monovalerate and 0.5 to 8 parts glyceryl monomyristicate. In some preferred embodiments, the oil composition, by weight, comprises 1 part glyceryl monovalerate and 4 parts glyceryl monomyristicate.

[0025] In some embodiments, the oil composition, by weight, comprises 1 part glyceryl monolinoleate and 0.5 to 8 parts glyceryl monomyristicate. In some preferred embodiments, the oil composition, by weight, comprises 1 part glyceryl monolinoleate and 2 to 4 parts glyceryl monomyristicate.

[0026] In some embodiments, the oil composition comprises, by weight, 1 part glyceryl monovalerate, 1 part glyceryl monolinoleate, and 0.5 to 8 parts glyceryl monomyristicate.

[0027] In some embodiments, the oil composition further includes excipients, which are at least one of anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil, Tween 80, mannitol, soluble starch, and water.

[0028] In some embodiments, the oil composition is a solution formed by mixing glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristate with anhydrous ethanol, Tween-80, and water, wherein the concentration of the three glycerides in the solution is 20-100 mg / mL. This solution can be used orally as an oral liquid formulation or added to feed.

[0029] Based on this, embodiments of this application provide the use of the aforementioned oil composition in the preparation of drugs or feed additives for preventing porcine enveloped virus infection.

[0030] In some embodiments, the porcine enveloped virus includes at least one of porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus.

[0031] In some embodiments, the method of using the oil composition as a drug or feed additive includes at least one of the following methods:

[0032] (1) Add the oil composition to the feed or drinking water at a dosage of 10-120 mg / kg of pig body weight;

[0033] (2) The oil composition was administered orally at a dose of 10-120 mg / kg of pig body weight;

[0034] (3) The oil composition is added to the feed at a dosage of 0.1% to 0.3% of the total weight of the pig feed.

[0035] The technical solution and the technical effects achieved by this application will be described in detail below through more specific embodiments. In this embodiment, glyceryl monovalerate was purchased from Larodan Company with a purity of 97%; glyceryl monolinoleate and glyceryl monomyristicate were both purchased from Sigma Company and were of analytical grade.

[0036] Prepare oil compositions and oral liquid formulations of oil compositions according to the formulas in Table 1 below.

[0037] Table 1

[0038]

[0039] The specific preparation method for oral liquid formulations is as follows:

[0040] Weigh 2-10 g of the glyceride mixture according to the formula mass ratio in Table 1. Add 20 mL of anhydrous ethanol and 30 mL of Tween-80 to each glyceride mixture. Then add water for injection to 100 mL and mix well to obtain oil composition solutions with different glyceride ratios, i.e., oral liquid preparations of oil composition, wherein the concentration of oil composition in the solution is 20-100 mg / mL.

[0041] The above-mentioned oil and fat composition formulation can be used as a drug or feed additive for the prevention or treatment of porcine enveloped virus infection. For routine prevention of swine diseases, the oil and fat composition formulation can be added to the feed at a dosage of 10 mg / kg of pig body weight, used throughout the entire feeding cycle. When used for clinical treatment of swine diseases, the oil and fat composition formulation can be administered orally at a dosage of 40-120 mg / kg of pig body weight; or the oil and fat composition can be dissolved in water and administered orally at a dosage of 40-120 mg / kg of pig body weight, or mixed with the daily diet and added to the feed three times a day (morning, noon, and evening) for one week. The dosage of the oil and fat composition formulation can also be adjusted according to the severity of the swine disease, with the dosage range controlled within the range of 10-120 mg / kg of pig body weight.

[0042] Inhibitory effect of oil-based compositions on in vitro proliferation of enveloped viruses

[0043] This application's embodiments verified the inhibitory effect of the above-mentioned oil compositions in various mass ratios on the in vitro proliferation of three porcine enveloped viruses: PEDV, PRRSV, and ASFV. The verification methods are as follows:

[0044] 1. Materials and Methods:

[0045] 1.1 Viruses: PEDV DR13 strain (recombinant strain DR13-EGFP with fluorescent tag), YN strain, and PRRSVWH1 strain were donated by the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University; ASFV was isolated and preserved by the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0046] 1.2 Cells: African green monkey kidney cells (Vero cells) were used for the proliferation of PEDV DR13 and YN strains; monkey embryonic kidney epithelial cells (Marc145) were used for the proliferation of PRRSV WH1 strain; and porcine alveolar macrophages (primary cells) were used for the proliferation of ASFV. Vero and Marc-145 cells were laboratory-preserved cell lines, and porcine alveolar macrophages were isolated from the lungs of healthy piglets.

[0047] 1.3 Test Drugs: Glyceryl monovalerate (Larodan, 97% purity), glyceryl monolinoleate (Sigma, analytical grade), and glyceryl monomyristate (Sigma, analytical grade) were formulated into oil compositions Y1~Y36 according to the formulations in Table 1.

[0048] 1.4 Reagents: DMEM medium, EDTA, trypsin, fetal bovine serum and penicillin-streptomycin were all purchased from GIBCO.

[0049] 1.5 In vitro proliferation inhibition assay

[0050] (1) PEDV in vitro proliferation inhibition assay: PEDV YN strain virus solution was diluted 10-fold with DMEM medium, and 10 μL of the solution was used. -2 10 -3 10 -4 10 -5 10 -6 10 -7 Six dilutions were inoculated into 96-well plates confluent with Vero cell monolayers at 100 μL / well. PEDV YN strain virus solution was divided into nine experimental groups and one control group. The nine groups were inoculated with DMEM medium containing different formulations of an oil-based composition at a dosage of 20 μg / mL. The control group was inoculated with DMEM medium without the oil-based composition. The plates were incubated at 37 ℃ with 5% CO2 for 36 h, and cytopathic effects were observed. The number of positive wells for each dilution was counted, and the TCID for each oil-based composition concentration was calculated using the Reed-Muench method. 50 Comparing TCIDs of different groups 50 The value was used to determine the inhibitory effect of the oil composition on the in vitro proliferation of PEDV, and three replicates were performed for each group.

[0051] (2) PRRSV in vitro proliferation inhibition assay: PRRSV virus solution was serially diluted 10-fold using DMEM medium to obtain 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7Six dilutions were inoculated into 96-well plates containing pre-cultured Marc-145 cell monolayers at 100 μL / well. The PRRSV virus solution was divided into nine experimental groups and one control group. The nine groups were inoculated with DMEM medium containing different formulations of an oil-based composition at a dosage of 20 μg / mL. The control group was inoculated with DMEM medium without the oil-based composition. The plates were incubated at 37 ℃ with 5% CO2 for 48 h, and cytopathic effects were observed. The number of positive wells for each dilution was counted, and the TCID for each oil-based composition concentration was calculated using the Reed-Muench method. 50 Comparing TCIDs of different groups 50 The value was used to determine the inhibitory effect of the oil composition on PRRSV proliferation in vitro, and three replicates were performed for each group.

[0052] (3) ASFV in vitro proliferation inhibition assay: ASFV virus solution was serially diluted 10-fold using DMEM medium to obtain 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 Six dilutions were inoculated into 96-well plates containing pre-cultured porcine alveolar macrophage monolayers at 100 μL / well. The ASFV virus solution was divided into nine experimental groups and one control group. The nine groups were inoculated with DMEM medium containing different formulations of an oil-based composition at a dosage of 20 μg / mL. The control group was inoculated with DMEM medium without the oil-based composition. The plates were incubated at 37 ℃ with 5% CO2 for 48 h, and cytopathic effects were observed. The number of positive wells for each dilution was counted, and the TCID for each oil-based composition concentration was calculated using the Reed-Muench method. 50 Comparing TCIDs of different groups 50 The value was used to determine the inhibitory effect of the oil composition on ASFV proliferation in vitro, and three replicates were performed for each group.

[0053] 1.6 Data Analysis: Experimental data were analyzed using one-way ANOVA and Duncan's method for multiple comparisons in SPSS 13.0 statistical software. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly significant. Results are expressed as mean ± standard deviation.

[0054] 2. Experimental Results:

[0055] Table 2 shows the TCID values ​​of PEDV, PRRSV, and ASFV for compositions consisting of glyceryl monovalerate and glyceryl monolinoleate in different mass ratios (i.e., oil compositions Y1~Y9). 50 The results are affected by the differences, where a~g are indicators of significance.

[0056] Table 2

[0057]

[0058] Table 2 shows that the addition of compositions consisting of glyceryl monovalerate and glyceryl monolinoleate in different mass ratios (oil compositions Y1~Y9) to the cell culture medium significantly affected the TCID of PEDV in Vero cells, PRRSV in Marc145 cells, and ASFV in porcine alveolar macrophages. 50 The TCID value increases with the increase of the proportion of monolinoleic acid glycerides. 50 The significant decrease indicates that compositions of glyceryl monovalerate and glyceryl monolinoleate at different mass ratios have a certain inhibitory effect on the proliferation of PEDV in cultured Vero cells, PRRSV in cultured Marc145 cells, and ASFV in cultured porcine alveolar macrophages. However, with the increase of the proportion of glyceryl monolinoleate, TCID... 50 The levels rose again. The results in the table show that when the mass ratio of glyceryl monovalerate to glyceryl monolinoleate is 1:1, i.e., the oil composition Y5 exhibits the most significant inhibitory effect on the virus.

[0059] Table 3 shows the TCID values ​​of PEDV, PRRSV, and ASFV for compositions consisting of glyceryl monovalerate and glyceryl monomyristicate in different mass ratios (i.e., oil compositions Y10~Y18). 50 The results are affected by the differences, where a~g are indicators of significance.

[0060] Table 3

[0061]

[0062] Table 3 shows that the addition of compositions consisting of glyceryl monovalerate and glyceryl monolinoleate in different mass ratios (oil compositions Y10~Y18) to the cell culture medium significantly affected the TCID of PEDV in Vero cells, PRRSV in Marc145 cells, and ASFV in porcine alveolar macrophages. 50 The value of TCID increases with the increase of the proportion of glyceryl monomyristicate. 50The initial decrease followed by an increase indicates that compositions of glyceryl monovalerate and glyceryl monomyristicate in different mass ratios have a certain inhibitory effect on the proliferation of PEDV in Vero cells, PRRSV in Marc145 cells, and ASFV in porcine alveolar macrophages. The results in the table show that the inhibitory effect of the oil compositions on the viruses is more significant when the ratio of glyceryl monovalerate to glyceryl monomyristicate is between 1:0.5 and 8.

[0063] Table 4 shows the TCID of PEDV, PRRSV, and ASFV for compositions consisting of different mass ratios of glyceryl monolinoleate and glyceryl monomyristicate (i.e., oil compositions Y19~Y27). 50 The results are affected by the differences, where a~g are indicators of significance.

[0064] Table 4

[0065]

[0066] Table 4 shows that the addition of different mass ratios of monolinoleic acid glycerides and monomyristic acid glycerides (i.e., oil compositions Y19~Y27) to the cell culture medium significantly affected the TCID of PEDV in Vero cells, PRRSV in Marc145 cells, and ASFV in porcine alveolar macrophages. 50 The value of TCID increases with the increase of the proportion of glyceryl monomyristicate. 50 The initial decrease followed by an increase indicates that compositions composed of mono-linoleic acid glycerides and mono-myristate glycerides in different mass ratios have a certain inhibitory effect on the proliferation of PEDV in Vero cells, PRRSV in Marc145 cells, and ASFV in porcine alveolar macrophages. The results in the table show that when the ratio of mono-linoleic acid glycerides to mono-myristate glycerides is between 1:0.5 and 8, the oil composition exhibits a more significant inhibitory effect on the virus.

[0067] Table 5 shows the TCID values ​​of PEDV, PRRSV, and ASFV for compositions consisting of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristicate in different mass ratios (i.e., oil compositions Y28~Y36). 50 The results are affected by the differences, where a~g are indicators of significance.

[0068] Table 5

[0069]

[0070] Table 5 shows that the addition of different mass ratios of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristate (i.e., oil compositions Y28-Y36) to the cell culture medium significantly affected the TCID of PEDV in Vero cells, PRRSV in Marc145 cells, and ASFV in porcine alveolar macrophages. 50 The value of TCID increases with the amount added. 50 The initial decrease followed by an increase indicates that compositions of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristicate in different mass ratios have a certain inhibitory effect on the proliferation of PEDV in Vero cells, PRRSV in Marc145 cells, and ASFV in porcine alveolar macrophages. The table shows that the inhibitory effect is more significant when the ratio of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristicate is between 1:1:0.5 and 8.

[0071] Protective effect of oil-based compositions on PEDV-infected piglets

[0072] This application's embodiments validated the protective effect of the oil-based composition formulation against PEDV-infected piglets, and the validation method is as follows:

[0073] 1. Materials and Methods:

[0074] 1.1 Test virus: PEDV YN strain was donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0075] 1.2 Test drug: Oral liquid preparations of oil compositions Y31~Y35 prepared according to the aforementioned preparation method.

[0076] 1.3 Experimental Animals: Healthy 7-day-old piglets of the same breed and origin, with similar weight and no maternal antibodies, were selected for the challenge experiment. The piglets were divided into a blank group and three treatment groups (low-dose group, medium-dose group, and high-dose group). The oil composition oral liquid preparation was added to milk and diluted to 3 mL before oral administration. The low-dose group was given an oil composition content of 40 mg / kg pig body weight, the medium-dose group was given an oil composition content of 80 mg / kg pig body weight, and the high-dose group was given an oil composition content of 120 mg / kg pig body weight. Each dose group was further divided into 5 subgroups, each subgroup using an oil composition (Y31~Y35) oral liquid preparation with different mass ratios. The blank group was fed the same volume of milk without any oil composition. Each group consisted of 10 piglets.

[0077] 1.4 Challenge Test: After feeding for one week, piglets in each group were challenged with 10... 7 TCID 50Each animal was challenged with PEDV (YN strain) at a dose of / mL and orally administered 5.0 mL of virus solution. A control group was also included in the experiment, which was orally administered the same dose of cell culture medium (DMEM). The animals were then isolated and observed for 7 consecutive days until the end of the experiment.

[0078] 1.5 Observation Indicators: The diarrhea status of experimental piglets was observed daily. At the end of the experimental period, piglets were slaughtered to observe intestinal lesions. The number of dead pigs was also recorded, and the diarrhea rate, mortality rate, and protection rate were calculated. The inhibitory effect of the combination of monovalerate glycerol, monolinoleate glycerol, and monomyristic acid glycerol on PEDV virus was determined by real-time quantitative PCR detection of the expression levels of PEDV structural genes N or M in the jejunal mucosa of piglets.

[0079] 1.6 Data Analysis: Data were analyzed using one-way ANOVA and Duncan's method for multiple comparisons. Diarrhea and mortality rates were expressed as mean, and PCR results were expressed as mean ± standard deviation. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly statistically significant.

[0080] 2. Experimental Results:

[0081] Table 6 shows the morbidity and mortality rates of piglets in each group after adding different doses and mass ratios of oil-oil mixtures to milk, where a~g are indicators of significant differences.

[0082] Table 6

[0083]

[0084] Table 6 shows that adding different doses and mass ratios of oil-based oral solutions composed of monovalerate glyceryl monostearate, monolinoleate glyceryl monostearate, and monomyristate glyceryl monostearate to piglet milk significantly reduced the diarrhea rate and mortality rate of PEDV-infected piglets. Compared with the control group, the diarrhea rate of PEDV-infected piglets decreased to 20%–60%, while the mortality rate of affected piglets decreased to 10%–40%. The experimental results fully demonstrate that adding different doses and proportions of oil-based preparations to piglet milk can significantly improve the protection rate of piglets against PEDV infection.

[0085] The expression levels of PEDV structural genes M and N in the intestinal mucosa of piglets in each group were detected. The results are shown in Table 7 below, where a~k are statistically significant markers.

[0086] Table 7

[0087]

[0088] Table 7 shows that adding different doses and mass ratios of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristicate to piglet milk can significantly reduce the expression levels of the M and N genes of PEDV, indicating that the addition of this glyceryl monovalerate oral solution can significantly reduce the virulence of PEDV and inhibit PEDV replication.

[0089] Protective effect of oil-oil composition on PRRSV-infected piglets

[0090] This application's embodiments validated the protective effect of the oil-based composition formulation against PRRSV-infected piglets, and the validation method is as follows:

[0091] 1. Materials and Methods:

[0092] 1.1 Test virus: The PRRSV strain was provided by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0093] 1.2 Test drug: Oral liquid preparations of oil compositions Y31~Y35 prepared according to the aforementioned preparation method.

[0094] 1.3 Experimental Animals: Healthy 7-day-old piglets of the same breed and origin, with similar weight and no maternal antibodies, were selected for the challenge experiment. The piglets were divided into a blank group and three treatment groups (low-dose group, medium-dose group, and high-dose group). The oil composition oral liquid preparation was added to milk and diluted to 3 mL before oral administration. The low-dose group was given an oil composition content of 40 mg / kg pig body weight, the medium-dose group was given an oil composition content of 80 mg / kg pig body weight, and the high-dose group was given an oil composition content of 120 mg / kg pig body weight. Each dose group was further divided into 5 subgroups, each subgroup using an oil composition (Y31~Y35) oral liquid preparation with different mass ratios. The blank group was fed the same volume of milk without any oil composition. Each group consisted of 10 piglets.

[0095] 1.4 Challenge Test: After feeding for one week, piglets in each group were challenged with 10... 6 TCID 50 Pigs were challenged with PRRSV at a concentration of 3.0 mL / mL, and each pig was orally administered 3.0 mL. A control group was also included in the experiment, which was orally administered the same dose of cell culture medium (DMEM). The pigs were then isolated and observed for 7 consecutive days until the end of the experiment.

[0096] 1.5 Observation Indicators: Diarrhea in experimental piglets was observed daily. At the end of the experiment, piglets were slaughtered for intestinal lesions, and the number of dead pigs was recorded. Diarrhea rate, mortality rate, and protection rate were calculated. The inhibitory effect of the combination of monovalerate, monolinoleate, and monomyristic acid glyceride on PRRSV virus was determined by real-time quantitative PCR detection of the expression level of the PRRSV virus ORF7 gene in the brain and lung tissues of piglets.

[0097] 1.6 Data Analysis:

[0098] Data were analyzed using one-way ANOVA and Duncan's method for multiple comparisons. Diarrhea and mortality rates are expressed as mean, and PCR results are expressed as mean ± standard deviation. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly statistically significant.

[0099] 2. Experimental Results:

[0100] Table 8 shows the morbidity and mortality rates of piglets in each group after adding different doses and mass ratios of oil-oil mixtures to milk, where a~f are indicators of significant differences.

[0101] Table 8

[0102]

[0103] Table 8 shows that adding different doses and mass ratios of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristate to piglet milk can significantly reduce the morbidity and mortality of PRRSV-infected piglets by 0-30%. The experimental results fully demonstrate that adding different doses and proportions of glyceryl monovalerate to piglet milk can significantly improve the protection rate of piglets against PRRSV infection.

[0104] The expression levels of the ORF7 gene of PRRSV virus in the brain and lung tissues of piglets in each group were detected. The results are shown in Table 9 below, where a~k are statistically significant markers.

[0105] Table 9

[0106]

[0107] Table 9 shows that adding different doses and mass ratios of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristicate to piglet milk as an oral solution can significantly reduce the expression level of the ORF7 gene of PRRSV in the brain and lungs of infected pigs. The addition of the combination of glyceryl monovalerate, glyceryl monolinoleate, and glyceryl monomyristicate can significantly reduce the virulence of PRRSV and inhibit PRRSV replication.

[0108] In summary, the oil composition provided in this application has the effect of resisting porcine enveloped viruses such as PEDV, PRRSV, and ASFV, and can be used to prepare drugs and feed additives for treating and preventing porcine diseases caused by porcine enveloped viruses such as PEDV, PRRSV, and ASFV.

[0109] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. An oil and fat composition for resisting infection with a porcine cecum virus, characterized by comprising, The oil and fat composition comprises, by mass, 1 part of glycerol monovalerate, 1 part of glycerol monolinoleate, and 0.5-8 parts of glycerol monomyristate.

2. The oil composition according to claim 1, characterized by, The oil and fat composition further comprises an auxiliary material, which is at least one of anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil, Tween 80, mannitol, soluble starch, and water.

3. The oil composition according to claim 1, characterized by, The oil and fat composition is a solution of glycerol monovalerate, glycerol monolinoleate, and glycerol monomyristate mixed with anhydrous ethanol, Tween-80, and water, and the concentration of the three glycerides in the solution is 20-100 mg / mL.

4. Use of the oil and fat composition of any one of claims 1-3 in the preparation of a medicine or feed additive for resisting porcine cistern membrane virus infection.

5. Use according to claim 4, characterized in that, The porcine cistern membrane virus comprises at least one of porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus.

6. Use according to claim 4, characterized in that, The method for using the oil and fat composition as a medicine or feed additive comprises at least one of (1)-(3): (1) adding the oil and fat composition to feed or drinking water for use at a dose of 10-120 mg / kg of pig body weight in terms of glyceride content; (2) directly orally taking the oil and fat composition at a dose of 10-120 mg / kg of pig body weight in terms of glyceride content; (3) adding the oil and fat composition to feed for use at a dose of 0.1%-0.3% of the total weight of the pig feed in terms of glyceride content.

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