Grease composition for resisting swine envelope virus infection and application of grease composition

Abstract

The invention discloses a grease composition for resisting porcine envelope virus infection and application of the grease composition. The grease composition comprises at least two of glyceryl monovalerate, glyceryl monolinoleate and glyceryl monomyristate. The grease composition can also comprise absolute ethyl alcohol, propylene glycol, dimethyl sulfoxide, edible oil, lactose in Tween 80, mannitol, soluble starch, water and other auxiliary materials, and is used for preparing a grease composition preparation. The grease composition can inhibit envelop viruses in a multi-way and multi-layer mode, and the antiviral effect is improved. By adding the grease composition into pig feed, not only can the antiviral capability be improved, but also inflammatory response caused by pathogenic infection can be reduced through the anti-inflammatory and immune regulation effects of the grease composition, and the infection of swine envelope viral diarrhea can be effectively prevented and treated, so that the incidence rate of the swine envelope viral diarrhea and the death rate of diseased pigs are reduced. The grease composition can be used for preparing a medicine or a feed additive for treating and preventing swine envelope viral diarrhea.

Description

Technical Field

[0001] The present application relates to the technical field of prevention and treatment of porcine envelope virus infection, and in particular to a fat composition for resisting porcine envelope virus infection and its application. Background Art

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

[0003] PEDV is a highly contact enteric infectious pathogen. The porcine epidemic diarrhea (PED) caused by it is one of the diseases that seriously threatens the global pig industry. Typical symptoms of PED include diarrhea, vomiting and dehydration in pigs, especially for piglets that have just been born. After infection, the mortality rate of piglets under 7 days old can be as high as 100%, causing huge losses. According to epidemiological survey data, PEDV has become an important pathogen of porcine viral diarrhea in my country, and its mutant strains are the main pathogens in the epidemic. These mutant strains often have stronger pathogenicity and transmission ability, making the original vaccines ineffective in prevention and control, and the difficulty of epidemic prevention and control is further increased. PEDV infection is mainly transmitted through the digestive tract, and can also be transmitted through contaminated environments, objects and excrement of sick pigs. After the virus enters the pig's body, it mainly infects the epithelial cells of the small intestine, causing damage to the intestinal villi and absorption dysfunction, causing severe diarrhea and dehydration, which leads to rapid weight loss, decreased immunity and even death of piglets. Although adult pigs have milder symptoms, they may also experience problems such as decreased appetite and reduced litter size, which significantly affect production performance.

[0004] PRRSV is an extremely harmful enveloped virus belonging to the family Arteriviridae and is a single-stranded positive RNA virus. PRRSV infection can cause porcine reproductive and respiratory syndrome (PRRS), which is typically characterized by reproductive disorders and respiratory symptoms, affecting all growth stages of pigs. PRRSV has various transmission routes, including airborne transmission, direct contact transmission, transmission through contaminated feed, environmental transmission, etc., and is prone to outbreaks, especially under intensive breeding conditions. PRRSV infection in sows can cause abortion, stillbirth, mummification and reduced litter rate, and has a particularly significant impact on the reproductive performance of pig herds. In piglets and fattening pigs, symptoms such as shortness of breath, fever, decreased appetite and weight loss will occur after infection with PRRSV. Pigs infected with PRRSV are also more likely to develop secondary infections, such as bacterial pneumonia, which aggravates the disease and leads to increased mortality. The presence of PRRSV not only reduces the production performance of pig farms, but also significantly increases breeding costs, especially the economic losses caused by increased morbidity and mortality. PRRSV has a very high variability, which can easily lead to immune failure, making vaccine prevention and control face great challenges. Therefore, PRRSV infection control is difficult, and currently relies mainly on comprehensive prevention and control measures such as biosafety measures, reasonable management strategies, and vaccination. Despite this, PRRSV remains one of the main threats to the global pig industry, with significant impacts on economic benefits and food safety.

[0005] ASFV is a double-stranded DNA virus belonging to the African swine fever virus family. ASFV is highly contagious and can survive for a long time in the natural environment. It is one of the major epidemic diseases in the pig industry. ASFV is widely transmitted, including direct contact, feed, water pollution, and insect vector transmission (such as soft ticks). Once infected, it can spread rapidly in pigs. ASFV can survive for months or even years under low temperature conditions, making it difficult to eliminate in the pig farm environment. Pigs infected with ASFV will show symptoms such as high fever, red skin, rapid breathing, decreased appetite, and in severe cases, vomiting, diarrhea, and blood loss, with a mortality rate of up to 100%. ASFV infection is fatal to pigs of all ages, especially piglets and pigs with low immunity. ASFV infection causes African swine fever (ASF), the main symptom of which is acute hemorrhagic disease. It is highly pathogenic and infected pigs will die in a short time. The lethality of ASFV and the lack of a specific vaccine mean that once an epidemic breaks out, all infected pigs and pigs in the same pen can only be culled, resulting in huge economic losses. Prevention and control mainly rely on strict biosafety measures, including isolation, disinfection, monitoring, etc., to prevent the further spread of the virus. However, the widespread spread of ASF still poses a major threat to the global pig industry and greatly affects the stability of the food supply chain.

[0006] Envelope viruses such as PEDV, PRRSV and ASFV have strong variability in their hosts, which limits the effectiveness of traditional vaccines. In the face of the challenges of these viral infectious diseases, the development of safe and efficient feed additives or oral agents that can broadly inhibit envelope viruses is of great significance to the health management of pig herds. Summary of the invention

[0007] In view of this, the purpose of the present application is to provide a fat composition for resisting porcine envelope virus infection and its application. The fat composition comprises at least two of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl, has the advantages of high safety and convenient application, has the effect of significantly inhibiting the replication of envelope viruses such as PEDV, PRRSV and ASFV, and reducing the virulence of porcine envelope virus, can be used as a feed additive or oral medicine, overcomes the shortcomings of possible immune failure caused by vaccination and virus mutation caused by the use of antiviral drugs, has no toxic side effects on animals, and can be used safely.

[0008] In order to achieve or at least partially achieve the above objectives, the present application provides the following technical solutions:

[0009] In a first aspect, the present application provides a fat composition for resisting porcine envelope virus infection, wherein the fat composition comprises at least two of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl.

[0010] In a second aspect, the present application provides the use of the aforementioned oil composition in the preparation of a drug or feed additive for resisting porcine envelope virus infection.

[0011] Compared with the prior art, the technical solution provided by this application has at least the following beneficial effects:

[0012] 1. The oil composition composed of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristic acid glyceryl provided in the present application is highly safe and will not lead to the production of drug-resistant strains or other toxic side effects even after long-term use, which is an advantage that other antiviral drugs do not have.

[0013] 2. The grease composition composed of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl provided in the present application can inhibit enveloped viruses through multiple pathways and multiple levels, thereby improving the antiviral effect. Adding these three monoesters to pig feed can not only improve the antiviral ability, but also reduce the inflammatory response caused by pathogen infection through its anti-inflammatory and immunomodulatory effects, and can effectively prevent and treat the infection of porcine enveloped viral diarrhea, thereby reducing the incidence of porcine enveloped viral diarrhea and the mortality rate of pigs with the disease. The grease composition can be used to prepare drugs or feed additives for treating and preventing porcine enveloped viral diarrhea.

[0014] 3. The results of in vitro experiments in this application show that the oil composition has the effect of significantly inhibiting the in vitro proliferation of PEDV, PRRSV and ASFV. Adding the oil composition in a cell culture medium at an addition amount of 100 μg / mL can increase the TCID of PEDV, PRRSV and ASFV on the corresponding cells. 50 The titers were reduced by about 5 to 10 respectively.

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

[0016] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to explain the present application and are not used to limit the present application.

[0017] Those skilled in the art will appreciate that, unless otherwise stated, the words "said", "the", and "the aforementioned" used in the text of the present application may also include plural forms. It should be further understood that the wording "including" used in the specification of the present application means the presence of the features, steps, and operations, but does not exclude the presence or addition of one or more other features, integers, and steps.

[0018] Those skilled in the art will appreciate that, for any specific experimental steps or conditions not specified in the examples, the experiments may be carried out according to the conventional experimental steps or conditions described in the literature in the art; any raw materials or instruments used without specifying the manufacturer are conventional products that can be purchased commercially.

[0019] Those skilled in the art will understand that, unless otherwise specified in the present application, when a numerical range is given in an embodiment, 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 are consistent with the prior art mastery of those skilled in the art and the records of this application, and any methods, equipment and materials of the prior art similar or equivalent to the methods, equipment and materials described in the embodiments of this application may also be used to implement this application.

[0020] Glyceryl Pentanoate (GP) is a monoester formed by the esterification of glycerol and valeric acid, and its structure contains a five-carbon fatty acid group and a glycerol molecule. Glyceryl Pentanoate belongs to medium-chain fatty acid glycerides, which has good fat solubility and certain hydrophilicity, enabling it to penetrate biological membranes and play a role in 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 vegetable oil components in fatty acids. Glyceryl Myristate (GM) is the esterification product of glycerol and myristic acid, and is mainly found in coconut oil and palm oil. GM is a long-chain saturated fatty acid glyceride with good lipophilicity and certain permeability. In the prior art, none of the three single glycerides have been used to resist porcine envelope virus.

[0021] In the embodiment of the present application, three single glycerides, monovaleric acid glyceride, monolinoleic acid glyceride and monomyristate glyceride, are mixed in a certain mass ratio to form a grease composition. In vivo and in vitro experiments have shown that the grease composition can significantly inhibit the proliferation of PEDV, PRRSV and ASFV, and inhibit their infection of cells. Furthermore, the grease compound can be used to prepare drugs or feed additives against viruses such as PEDV, PRRSV and ASFV.

[0022] Based on this, an embodiment of the present application provides a fat composition for resisting porcine envelope virus infection, wherein the fat composition comprises at least two of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl.

[0023] In some embodiments, the oil composition comprises, by weight, 1 part of monovaleric acid glyceryl and 0.25 to 4 parts of monolinoleic acid glyceryl. In some preferred embodiments, the oil composition comprises, by weight, 1 part of monovaleric acid glyceryl and 1 part of monolinoleic acid glyceryl.

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

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

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

[0027] In certain embodiments, the oil composition further comprises an auxiliary material, and the auxiliary material is at least one of anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil, lactose in Tween 80, mannitol, soluble starch, and water.

[0028] In certain embodiments, the oil composition is a solution formed by mixing monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl with anhydrous ethanol, Tween-80 and water, and the concentration of the three glycerides in the solution is 20-100 mg / mL. The solution can be taken orally as an oral liquid preparation or added to feed for use.

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

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

[0031] In certain embodiments, the method of using the oil composition as a medicine or feed additive comprises at least one of the following methods:

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

[0033] (2) directly orally administering the fat composition at a dosage of 10 to 120 mg / kg of pig body weight of glyceride;

[0034] (3) The fat 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 scheme of the present application and the technical effects achieved are described in detail below through more specific embodiments, wherein monovaleric acid glyceryl was purchased from Larodan Company with a purity of 97%; monolinoleic acid glyceryl and monomyristate glyceryl were purchased from Sigma Company with analytical grade.

[0036] According to the formula in Table 1 below, the oil composition and the oil composition oral liquid preparation were prepared respectively.

[0037] Table 1

[0038]

[0039]

[0040] The specific preparation method of the oral liquid preparation is as follows:

[0041] According to the mass ratio of the formula in Table 1, 2 to 10 g of the glyceride mixture were weighed respectively, and the glyceride mixture was mixed; 20 mL of anhydrous ethanol and 30 mL of Tween-80 were added to each glyceride mixture respectively; then water for injection was added to 100 mL, and mixed evenly to obtain oil and fat composition solutions with various glyceride ratios, that is, oil and fat composition oral liquid preparations, wherein the concentration of the oil and fat composition in the solution was 20 to 100 mg / mL.

[0042] The above-mentioned oil composition preparation can be used as a medicine or feed additive to prevent or treat porcine envelope virus infection diseases. When used for routine prevention of pig diseases, the oil composition preparation can be added to feed at a dosage of 10 mg / kg pig body weight of the oil composition, and used throughout the entire feeding cycle; when used for clinical treatment of pig diseases, the oil composition preparation can be orally administered at a dosage of 40 to 120 mg / kg pig body weight of the oil composition; or the oil composition can be dissolved in water and administered or mixed with the daily diet at a dosage of 40 to 120 mg / kg pig body weight of the oil composition, and added to feed three times a day in the morning, noon and evening for continuous use for one week; the dosage of the oil composition preparation can also be adjusted according to the severity of the pig disease, and the dosage range is controlled within a range of 10 to 120 mg / kg pig body weight of the oil composition.

[0043] Inhibitory effect of oil and fat composition on proliferation of enveloped viruses in vitro

[0044] The examples of the present application verify the inhibitory effects of the above-mentioned oil compositions of various mass ratios on the in vitro proliferation of three porcine envelope viruses, PEDV, PRRSV and ASFV, and the verification method is as follows:

[0045] 1. Materials and methods:

[0046] 1.1 Viruses: PEDV DR13 strain (recombinant strain DR13-EGFP with fluorescent label), YN strain, and PRRSV WH1 strain were donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University; ASFV was isolated and preserved by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0047] 1.2 Cells: African green monkey kidney cells (Vero cells) are used for the proliferation of PEDV DR13 and YN strains; pig kidney cells (PK-15) are used for the proliferation of PRRSV WH1 strain; porcine alveolar macrophages (primary cells) are used for the proliferation of ASFV. Vero and Marc-145 cells are laboratory preserved cell lines, and porcine alveolar macrophages are isolated from the lungs of healthy piglets.

[0048] 1.3 Experimental drugs: monovaleric acid glyceryl (Larodan Company, purity 97%), monolinoleic acid glyceryl (Sigma Company, analytical grade), monomyristate glyceryl (Sigma Company, analytical grade) were prepared into oil compositions Y1 to Y36 according to the formula in Table 1.

[0049] 1.4 Reagents: DMEM culture medium, EDTA, trypsin, fetal bovine serum and double antibody (penicillin-streptomycin) were purchased from GIBCO.

[0050] 1.5 In vitro proliferation inhibition assay

[0051] (1) PEDV in vitro proliferation inhibition test: PEDV YN strain virus liquid was diluted 10 times with DMEM medium, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated in 96-well plates covered with Vero cell monolayers, 100 μL / well, and the PEDV YN strain virus solution was divided into nine test groups and one blank group. The nine groups were added with DMEM culture medium containing different formula ratios of oil compositions, and the added dosage of the oil composition was 20 μg / mL culture medium. The blank group was added with DMEM culture medium without oil composition, and continued to be cultured in a 37°C incubator containing 5% CO2 for 36 hours to observe cell lesions. The number of lesion-positive wells in the cell wells of each dilution was counted, and the TCID of each concentration of oil composition group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of the oil composition on the proliferation of PEDV in vitro was judged based on the value of , and three replicates were performed for each group.

[0052] (2) PRRSV in vitro proliferation inhibition test: PRRSV virus solution was 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 of cultured Marc-145 cell monolayers, 100 μL / well, and the PRRSV virus solution was divided into nine test groups and one blank group. The nine groups were added with DMEM culture medium containing different formula ratios of oil compositions, and the dosage of the oil composition was 20 μg / mL culture medium. The blank group was added with DMEM culture medium without oil composition, and continued to be cultured in a 37°C incubator containing 5% CO2 for 48 hours to observe cell lesions. The number of lesion-positive wells in the cell wells of each dilution was counted, and the TCID of each concentration of oil composition group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of the oil composition on the proliferation of PRRSV in vitro was judged based on the value of , and three replicates were performed for each group.

[0053] (3) ASFV in vitro proliferation inhibition test: ASFV virus solution was 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 of cultured Marc-145 cell monolayers, 100 μL / well, and the ASFV virus solution was divided into nine test groups and one blank group. The nine groups were added with DMEM culture medium containing different formula ratios of oil compositions, and the added dosage of the oil composition was 20 μg / mL culture medium. The blank group was added with DMEM culture medium without oil composition, and continued to be cultured in a 37°C incubator containing 5% CO2 for 48 hours to observe cell lesions. The number of lesion-positive wells in the cell wells of each dilution was counted, and the TCID of each concentration of oil composition group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of the oil composition on the proliferation of ASFV in vitro was judged based on the value of , and three replicates were performed in each group.

[0054] 1.6 Data analysis: The experimental data were analyzed using one-way ANOVA and Duncan's multiple comparison method in SPSS13.0 statistical software, with P < 0.05 as the standard for significant difference and P < 0.01 as the standard for extremely significant difference. The results are expressed as mean ± standard deviation.

[0055] 2. Experimental results:

[0056] Table 2 shows the TCID of compositions composed of monovaleric acid glyceryl and monolinoleic acid glyceryl (i.e., fat compositions Y1 to Y9) in different mass ratios against PEDV, PRRSV, and ASFV50 Influence results, where a~g are marks of significant difference.

[0057] Table 2

[0058] Trial Drugs Mass ratio of monovaleric acid glyceryl to monolinoleic acid glyceryl PEDV PRRSV ASFV Blank Group / <![CDATA[10 8.68±0.36a ]]> <![CDATA[10 8.34±0.29a ]]> <![CDATA[10 8.10±0.18a ]]> Y1 1:0.0625 <![CDATA[10 5.92±0.34b ]]> <![CDATA[10 6.02±0.21b ]]> <![CDATA[10 5.02±0.34b ]]> Y2 1:0.125 <![CDATA[10 5.77±0.42bc ]]> <![CDATA[10 5.67±0.29c ]]> <![CDATA[10 4.77±0.42bc ]]> Y3 1:0.25 <![CDATA[10 3.72±0.55d ]]> <![CDATA[10 3.42±0.25d ]]> <![CDATA[10 3.89±0.55d ]]> Y4 1:0.5 <![CDATA[10 3.28±0.37e ]]> <![CDATA[10 2.68±0.27e ]]> <![CDATA[10 3.10±0.37e <!-- 6 -->]]> Y5 1:1 <![CDATA[10 1.19±0.21g ]]> <![CDATA[10 1.32±0.11g ]]> <![CDATA[10 1.49±0.36g ]]> Y6 1:2 <![CDATA[10 2.22±0.20f ]]> <![CDATA[10 2.01±0.18f ]]> <![CDATA[10 2.59±0.20f ]]> Y7 1:4 <![CDATA[10 3.58±0.39de ]]> <![CDATA[10 3.38±0.39d ]]> <![CDATA[10 3.59±0.69de ]]> Y8 1:8 <![CDATA[10 4.87±0.28c ]]> <![CDATA[10 5.61±0.38c ]]> <![CDATA[10 4.52±0.28c ]]> Y9 1:16 <![CDATA[10 5.69±0.42b ]]> <![CDATA[10 5.89±0.28bc ]]> <![CDATA[10 4.99±0.42b ]]>

[0059] The results in Table 2 show that the addition of a composition consisting of monovaleric acid glyceryl and monolinoleic acid glyceryl in different mass ratios in the cell culture medium (oil composition Y1-Y9) significantly affects the TCID of PEDV in Vero cells, PRRSV in Marc145 cells and ASFV in porcine alveolar macrophages 50 As the proportion of monolinoleylglycerol increases, the TCID 50 The results showed that the combination of monovaleric acid glyceryl and monolinoleic acid glyceryl with different mass ratios had a certain inhibitory effect on the proliferation of PEDV in in vitro cultured Vero cells, PRRSV in in vitro cultured Marc145 cells and ASFV in in vitro cultured porcine alveolar macrophages. However, as the proportion of monolinoleic acid glyceryl increased, the TCID 50 It can be seen from the results in the table that when the mass ratio of monovaleric acid glyceryl to monolinoleic acid glyceryl is 1:1, that is, the oil composition Y5 has the most significant inhibitory effect on viruses.

[0060] Table 3 shows the TCID of compositions composed of monovaleric acid glyceryl and monomyristate glyceryl (i.e., fat compositions Y10 to Y18) in different mass ratios against PEDV, PRRSV, and ASFV 50 Influence results, where a~g are marks of significant difference.

[0061] Table 3

[0062] Trial Drugs Mass ratio of monovaleric acid glyceryl to monomyristate glyceryl PEDV PRRSV ASFV Blank Group / <![CDATA[10 9.08±0.66a ]]> <![CDATA[10 8.91±0.89a ]]> <![CDATA[10 8.84±0.92a ]]> Y10 1:0.0625 <![CDATA[10 7.83±0.72b ]]> <![CDATA[10 6.72±0.61b ]]> <![CDATA[10 7.08±0.76b ]]> Y11 1:0.125 <![CDATA[10 6.77±0.42c ]]> <![CDATA[10 6.57±0.39c ]]> <![CDATA[10 5.77±0.32c ]]> Y12 1:0.25 <![CDATA[10 6.52±0.59c ]]> <![CDATA[10 6.39±0.35c ]]> <![CDATA[10 4.49±0.42d ]]> Y13 1:0.5 <![CDATA[10 2.03±0.36f ]]> <![CDATA[10 3.68±0.67d ]]> <![CDATA[10 3.50±0.67e ]]> Y14 1:1 <![CDATA[10 2.49±0.15e ]]> <![CDATA[10 2.39±0.42f ]]> <![CDATA[10 2.48±0.56f ]]> Y15 1:2 <![CDATA[10 3.32±0.36d ]]> <![CDATA[10 2.01±0.25g ]]> <![CDATA[10 2.59±0.30f ]]> Y16 1:4 <![CDATA[10 1.98±0.30f ]]> <![CDATA[10 2.12±0.39g ]]> <![CDATA[10 2.41±0.52f ]]> Y17 1:8 <![CDATA[10 2.68±0.53e ]]> <![CDATA[10 3.01±0.48e ]]> <![CDATA[10 3.66±0.68e ]]> Y18 1:16 <![CDATA[10 6.69±0.31c ]]> <![CDATA[10 6.89±0.58b ]]> <![CDATA[10 6.94±0.88b ]]>

[0063] The results in Table 3 show that the addition of a composition consisting of monovaleric acid glyceryl and monolinoleic acid glyceryl in different mass ratios in the cell culture medium (oil composition Y10-Y18) significantly affects the TCID of PEDV in Vero cells, PRRSV in Marc145 cells and ASFV in porcine alveolar macrophages 50 As the proportion of monomyristyl glycerol increases, the TCID 50It first decreases and then increases, indicating that the composition of monovaleric acid glyceryl and monomyristate glyceryl in different mass ratios has a certain inhibitory effect on the proliferation of PEDV in vitro cultured Vero cells, PRRSV in vitro cultured Marc145 cells and ASFV in vitro cultured porcine alveolar macrophages. From the results in the table, it can be seen that when the ratio of monovaleric acid glyceryl and monomyristate glyceryl is 1:0.5-8, the inhibitory effect of the oil composition on the virus is more significant.

[0064] Table 4 shows the TCID of compositions composed of monolinoleyl glycerol and monomyristyl glycerol in different mass ratios (i.e., oil compositions Y19 to Y27) against PEDV, PRRSV, and ASFV 50 Influence results, where a~g are marks of significant difference.

[0065] Table 4

[0066] Trial Drugs Mass ratio of monolinoleylglycerol to monomyristylglycerol PEDV PRRSV ASFV Blank Group / <![CDATA[10 8.38±0.56a ]]> <![CDATA[10 8.16±0.65a ]]> <![CDATA[10 8.39±0.83a ]]> Y19 1:0.0625 <![CDATA[10 6.63±0.63b ]]> <![CDATA[10 6.82±0.76b ]]> <![CDATA[10 7.28±0.56b <!-- 7 -->]]> Y20 1:0.125 <![CDATA[10 5.52±0.32c ]]> <![CDATA[10 6.87±0.66b ]]> <![CDATA[10 5.37±0.52d ]]> Y21 1:0.25 <![CDATA[10 5.37±0.60c ]]> <![CDATA[10 6.02±0.44c ]]> <![CDATA[10 5.29±0.39d ]]> Y22 1:0.5 <![CDATA[10 3.09±0.28e ]]> <![CDATA[10 4.68±0.52d ]]> <![CDATA[10 3.51±0.32f ]]> Y23 1:1 <![CDATA[10 3.49±0.55d ]]> <![CDATA[10 2.49±0.32f ]]> <![CDATA[10 3.48±0.29f ]]> Y24 1:2 <![CDATA[10 3.02±0.36e ]]> <![CDATA[10 2.51±0.28f ]]> <![CDATA[10 2.69±0.23g ]]> Y25 1:4 <![CDATA[10 2.98±0.30e ]]> <![CDATA[10 3.22±0.28e ]]> <![CDATA[10 2.91±0.12g ]]> Y26 1:8 <![CDATA[10 3.62±0.33d ]]> <![CDATA[10 4.81±0.65d ]]> <![CDATA[10 4.67±0.32e ]]> Y27 1:16 <![CDATA[10 6.69±0.31b ]]> <![CDATA[10 6.89±0.77b ]]> <![CDATA[10 5.94±0.57c ]]>

[0067] The results in Table 4 show that the addition of a composition consisting of monolinoleyl glyceride and monomyristyl glyceride in different mass ratios in the cell culture medium (i.e., oil composition Y19-Y27) significantly affects the TCID of PEDV in Vero cells, PRRSV in Marc145 cells, and ASFV in porcine alveolar macrophages. 50 As the proportion of monomyristyl glycerol increases, the TCID 50 It first decreases and then increases, indicating that the composition composed of monolinoleyl glyceride and monomyristyl glyceride in different mass ratios has a certain inhibitory effect on the proliferation of PEDV in vitro cultured Vero cells, PRRSV in vitro cultured Marc145 cells and ASFV in vitro cultured porcine alveolar macrophages. From the results in the table, it can be seen that when the ratio of monolinoleyl glyceride to monomyristyl glyceride is 1:0.5-8, the oil composition has a more significant inhibitory effect on the virus.

[0068] Table 5 shows the TCID of the compositions composed of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl (i.e., oil compositions Y28 to Y36) in different mass ratios against PEDV, PRRSV and ASFV 50 Influence results, where a~g are marks of significant difference.

[0069] Table 5

[0070] Trial Drugs Mass ratio of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl PEDV PRRSV ASFV Blank Group / <![CDATA[10 8.38±0.56a ]]> <![CDATA[10 8.16±0.65a ]]> <![CDATA[10 8.39±0.83a ]]> Y28 1:1:0.0625 <![CDATA[10 6.63±0.63b ]]> <![CDATA[10 6.82±0.76b ]]> <![CDATA[10 7.28±0.56b ]]> Y29 1:1:0.125 <![CDATA[10 5.52±0.32c ]]> <![CDATA[10 6.87±0.66b ]]> <![CDATA[10 5.37±0.52d ]]> Y30 1:1:0.25 <![CDATA[10 5.37±0.60c ]]> <![CDATA[10 6.02±0.44c ]]> <![CDATA[10 5.29±0.39d ]]> Y31 1:1:0.5 <![CDATA[10 3.09±0.28e ]]> <![CDATA[10 4.68±0.52d ]]> <![CDATA[10 3.51±0.32f ]]> Y32 1:1:1 <![CDATA[10 3.49±0.55d ]]> <![CDATA[10 2.49±0.32f ]]> <![CDATA[10 3.48±0.29f ]]> Y33 1:1:2 <![CDATA[10 3.02±0.36e ]]> <![CDATA[10 2.51±0.28f ]]> <![CDATA[10 2.69±0.23g ]]> Y34 1:1:4 <![CDATA[10 2.98±0.30e ]]> <![CDATA[10 3.22±0.28e ]]> <![CDATA[10 2.91±0.12g ]]> Y35 1:1:8 <![CDATA[10 3.62±0.33d ]]> <![CDATA[10 4.81±0.65d ]]> <![CDATA[10 4.67±0.32e ]]> Y36 1:1:16 <![CDATA[10 6.69±0.31b ]]> <![CDATA[10 6.89±0.77b ]]> <![CDATA[10 5.94±0.57c ]]>

[0071] The results in Table 5 show that the addition of a composition consisting of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl in different mass ratios in the cell culture medium (i.e., oil composition Y28-Y36) significantly affects the TCID of PEDV in Vero cells, PRRSV in Marc145 cells, and ASFV in porcine alveolar macrophages. 50 With the increase of the added amount, TCID 50 It first decreases and then increases, indicating that the composition of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl in different mass ratios has a certain inhibitory effect on the proliferation of PEDV in vitro cultured Vero cells, PRRSV in vitro cultured Marc145 cells and ASFV in vitro cultured porcine alveolar macrophages. It can be seen from the table that when the ratio of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl is between 1:1:0.5 and 8, the inhibitory effect is more significant.

[0072] Protective effect of oil composition on piglets infected with PEDV

[0073] The present application example verifies the protective effect of the oil composition preparation on piglets infected with PEDV, and the verification method is as follows:

[0074] 1. Materials and methods:

[0075] 1.1 Experimental virus: PEDV YN strain was donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0076] 1.2 Test drug: an oral liquid preparation of a fat composition prepared from the above-mentioned fat compositions Y31 to Y35 according to the above-mentioned preparation preparation method.

[0077] 1.3 Experimental animals: 7-day-old healthy piglets of the same breed and source, similar weight and without maternal antibodies were selected for the challenge test. The experimental piglets were divided into a blank group and three treatment groups (low-dose group, medium-dose group and high-dose group). The oral liquid preparation of the oil composition was added to milk and the volume was fixed to 3 mL for oral administration. The low-dose group was added with a dosage of 40 mg / kg pig body weight of the oil composition, the medium-dose group was added with a dosage of 80 mg / kg pig body weight of the oil composition, and the high-dose group was added with a dosage of 120 mg / kg pig body weight of the oil composition. Each dosage group included 5 subgroups, each of which corresponded to oral liquid preparations of oil compositions (Y31-Y35) with different mass ratios. The blank group was gavaged with the same volume of milk without any oil composition added, and each group had 10 piglets.

[0078] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 7 TCID 50 / mL of PEDV (YN strain) was used for infection. Each head was orally infused with 5.0mL of virus solution. A control group was set up in the experiment, which was infused with the same dose of cell culture medium (DMEM), and then isolated and raised, and observed for 7 consecutive days until the end of the experiment.

[0079] 1.5 Observation indicators: The diarrhea of ​​the experimental piglets was observed every day. The experimental piglets were slaughtered at the end of the experiment to observe intestinal lesions. The number of dead pigs was counted and the diarrhea rate, mortality rate and protection rate were calculated. The inhibitory effect of the combination of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl on PEDV virus was determined by detecting the expression of PEDV structural gene N or M in the jejunal mucosa of piglets by real-time quantitative PCR.

[0080] 1.6 Data analysis: One-way ANOVA and Duncan's multiple comparison method were used for data analysis. The diarrhea rate and mortality rate were expressed as mean values, and the PCR results were expressed as mean ± standard deviation. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference.

[0081] 2. Experimental results:

[0082] After adding different doses and different mass ratios of the oil composition into milk, the morbidity and mortality of piglets in each group are shown in Table 6, where a~g are marks of significant difference.

[0083] Table 6

[0084]

[0085]

[0086] The results in Table 6 show that adding different doses and different mass ratios of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl to piglet milk can significantly reduce the diarrhea rate and mortality rate of PEDV-infected piglets. Compared with the blank group, the diarrhea rate of PEDV-infected piglets decreased to 20% to 60%; and the mortality rate of diseased pigs decreased to 10% to 40%. The experimental results fully demonstrate that adding different doses and different ratios of oil composition preparations to piglet milk can significantly improve the protection rate of piglets against PEDV infection.

[0087] The expression levels of PEDV structural genes M and N in the intestinal mucosa of each group of piglets were detected. The test results are shown in Table 7 below, where a~k are significant difference marks.

[0088] Table 7

[0089]

[0090]

[0091] The results in Table 7 show that adding different doses and different mass ratios of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl into piglet milk can significantly reduce the expression levels of M and N genes of PEDV, indicating that the oral liquid of the oil composition can significantly reduce the virulence of PEDV and inhibit the replication of PEDV after addition.

[0092] Protective effect of oil composition on piglets infected with PRRSV

[0093] The present application example verifies the protective effect of the oil composition preparation on PRRSV-infected piglets, and the verification method is as follows:

[0094] 1. Materials and methods:

[0095] 1.1 Experimental virus: The PRRSV strain was donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0096] 1.2 Test drug: an oral liquid preparation of a fat composition prepared from the above-mentioned fat compositions Y31 to Y35 according to the above-mentioned preparation preparation method.

[0097] 1.3 Experimental animals: 7-day-old healthy piglets of the same breed and source, similar weight and without maternal antibodies were selected for the challenge test. The experimental piglets were divided into a blank group and three treatment groups (low-dose group, medium-dose group and high-dose group). The oral liquid preparation of the oil composition was added to milk and the volume was fixed to 3 mL for oral administration. The low-dose group was added with a dosage of 40 mg / kg pig body weight of the oil composition, the medium-dose group was added with a dosage of 80 mg / kg pig body weight of the oil composition, and the high-dose group was added with a dosage of 120 mg / kg pig body weight of the oil composition. Each dosage group included 5 subgroups, each of which corresponded to oral liquid preparations of oil compositions (Y31-Y35) with different mass ratios. The blank group was gavaged with the same volume of milk without any oil composition added, and each group had 10 piglets.

[0098] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 6 TCID 50 / mL of PRRSV was used for infection, and each pig was orally infused with 3.0mL. A control group was set up in the experiment, and the control group was infused with the same dose of cell culture medium (DMEM), then raised in isolation and observed for 7 consecutive days until the end of the experiment.

[0099] 1.5 Observation index: The diarrhea of ​​the experimental piglets was observed every day. The experimental piglets were slaughtered at the end of the test period to observe intestinal lesions, and the number of dead pigs was counted, and the diarrhea rate, mortality rate and protection rate were calculated. The inhibitory effect of the composition of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl on PRRSV virus was determined by detecting the expression of ORF7 gene of PRRSV virus in brain and lung tissues of piglets by real-time quantitative PCR.

[0100] 1.6 Data Analysis:

[0101] Data were analyzed by one-way ANOVA and Duncan's multiple comparison method. The diarrhea rate and mortality rate were expressed as mean values, and the PCR results were expressed as mean ± standard deviation. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference.

[0102] 2. Experimental results:

[0103] After adding different doses and different mass ratios of the oil composition into milk, the morbidity and mortality of piglets in each group are shown in Table 8, where a~f are marks of significant difference.

[0104] Table 8

[0105]

[0106] The results in Table 8 show that adding different doses and different mass ratios of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl to piglet milk can significantly reduce the incidence and mortality of piglets infected with PRRSV by 0-30%; the experimental results fully demonstrate that adding different doses and different ratios of oil composition preparations to piglet milk can significantly improve the protection rate of piglets against PRRSV infection.

[0107] The expression levels of ORF7 gene of PRRSV virus in the brain and lung tissues of each group of piglets were detected respectively. The detection results are shown in Table 9 below, wherein a~k are marks of significant difference.

[0108] Table 9

[0109]

[0110] The results in Table 9 show that adding an oral liquid of a fat composition consisting of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl in different doses and mass ratios to piglet milk can significantly reduce the expression level of the ORF7 gene of PRRSV in the brain and lungs of infected pigs. The addition of the composition of monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristate glyceryl can significantly reduce the virulence of PRRSV and inhibit the replication of PRRSV.

[0111] In summary, the oil composition provided in the embodiments of the present application has the effect of resisting porcine envelope viruses such as PEDV, PRRSV and ASFV, and can be used to prepare drugs and feed additives for treating and preventing pig diseases infected by porcine envelope viruses such as PEDV, PRRSV and ASFV.

[0112] The present application is described in detail above. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the present application and its core ideas. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A fat composition for resisting porcine envelope virus infection, characterized in that: The oil composition comprises at least two of glyceryl monovalerate, glyceryl monolinoleate and glyceryl monomyristate.

2. The grease composition according to claim 1, characterized in that The oil composition, calculated by mass, includes 1 part of monovaleric acid glyceryl and 0.25-4 parts of monolinoleic acid glyceryl; preferably, the oil composition, calculated by mass, includes 1 part of monovaleric acid glyceryl and 1 part of monolinoleic acid glyceryl.

3. The grease composition according to claim 1, characterized in that The oil composition, calculated by mass, comprises 1 part of glyceryl monovalerate and 0.5 to 8 parts of glyceryl monomyristate; preferably, the oil composition, calculated by mass, comprises 1 part of glyceryl monovalerate and 4 parts of glyceryl monomyristate.

4. The grease composition according to claim 1, characterized in that The oil composition, calculated by mass, comprises 1 part of monolinolein glyceryl and 0.5-8 parts of monomyristic glyceryl; preferably, the oil composition, calculated by mass, comprises 1 part of monolinolein glyceryl and 2-4 parts of monomyristic glyceryl.

5. The grease composition according to claim 1, characterized in that The oil composition comprises, by weight, 1 part of glyceryl monovalerate, 1 part of glyceryl monolinoleate and 0.5 to 8 parts of glyceryl monomyristate.

6. The grease composition according to claim 5, characterized in that The oil composition further comprises auxiliary materials, which are at least one of anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil, lactose in Tween 80, mannitol, soluble starch and water.

7. The grease composition according to claim 5, characterized in that The oil composition is a solution formed by mixing monovaleric acid glyceryl, monolinoleic acid glyceryl and monomyristic acid glyceryl with anhydrous ethanol, Tween-80 and water, and the concentration of the three glycerides in the solution is 20-100 mg / mL.

8. Use of the oil composition according to any one of claims 1 to 7 in the preparation of a drug or feed additive for resisting porcine envelope virus infection.

9. The use according to claim 8, characterized in that: The porcine enveloped virus includes at least one of porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus.

10. The use according to claim 8, characterized in that: The method for using the oil composition as a medicine or feed additive includes at least one of (1) to (3): (1) adding the fat composition to feed or drinking water at a dosage of 10 to 120 mg / kg of pig body weight; (2) directly orally administering the fat composition at a dosage of 10 to 120 mg / kg of pig body weight of glyceride; (3) The fat composition is added to the feed at a dosage of 0.1% to 0.3% of the total weight of the pig feed.

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