A polysaccharide composition for resisting porcine enveloped virus infection, its preparation method and its application

Abstract

This application discloses a polysaccharide composition, preparation method, and application for combating porcine enveloped virus infection. The polysaccharide composition, by weight, comprises 1 part Poria cocos polysaccharide, 0.5 parts Codonopsis pilosula polysaccharide, and 0.3 parts fucoidan. The polysaccharide composition may also include excipients, such as at least one selected from montmorillonite, vermiculite, zeolite powder, silica, defatted rice bran, corn cob powder, soluble starch, wheat bran, acetic acid, anhydrous ethanol, and Tween-80. This polysaccharide composition enhances the body's antioxidant defense function, improves the pig's natural immune function and antiviral activity, and can effectively prevent and treat infections of porcine viruses such as PEDV, PRRSV, and AFSV, thereby reducing the incidence of porcine diseases and the mortality rate of infected pigs. This polysaccharide composition can be used to prepare drugs or feed additives against porcine viruses such as PEDV, PRRSV, and AFSV, with drug dosage forms including oral liquids, granules, tablets, and powders.

Description

Technical Field

[0001] This application relates to the field of porcine enveloped virus prevention and control technology, specifically to a polysaccharide composition for resisting porcine enveloped virus infection, its preparation method, and its application. Background Technology

[0002] Disease pathogens can be classified into viral and bacterial infections, and viruses can be further divided into enveloped and non-enveloped viruses. Relatively speaking, enveloped viruses, due to the presence of an outer envelope, are more prone to mutation and have a stronger ability to evade the immune system, making their 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 virus (PEDV) is the main cause of porcine epidemic diarrhea (PED). It is a coronavirus that can infect pigs of all ages and breeds, but it has a high mortality rate in piglets, with a mortality rate of up to 100% in piglets under seven days old. PEDV primarily infects and replicates in the villous cells of the small intestine, and clinical symptoms include vomiting, diarrhea, and dehydration. Porcine reproductive and respiratory syndrome (PRRS), also known as "blue ear disease," is a highly contagious disease in pigs, characterized by reproductive disorders in pregnant sows (abortion, stillbirth, mummified fetuses) and respiratory diseases in pigs of all ages, especially piglets. African swine fever (ASF), caused by ASFV infection, is an acute, febrile, and highly contagious disease characterized by high fever, widespread hemorrhage in the skin and internal organs, respiratory disorders, neurological symptoms, and a high mortality rate.

[0004] PEDV, PRRSV, and ASFV caused the deaths of a large number of piglets, resulting in huge economic losses and seriously hindering the development of the pig farming industry.

[0005] Currently, besides inactivated and attenuated vaccines, there are no other effective prevention and control methods against these enveloped viruses. Although many pig farmers choose to prevent viral infection through vaccination, the effectiveness is often unsatisfactory due to factors such as inconsistent vaccine quality. In my country's high-density farming environment, the widespread use of antiviral drugs not only accelerates the mutation rate of virus strains but also hinders vaccine development, leading to frequent outbreaks of epidemics.

[0006] Therefore, providing pigs with safe and effective functional nutrients to resist enveloped virus infection is particularly important, and this measure has broad application prospects. Summary of the Invention

[0007] In view of this, the purpose of this application is to provide a polysaccharide composition for resisting porcine enveloped virus infection, a method for its preparation, and its application. This polysaccharide composition has the advantages of high safety and ease of application, and exhibits a high protective rate against porcine enveloped virus infection. It can be used to prepare drugs or feed additives for resisting porcine enveloped virus infection.

[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 a polysaccharide composition for resisting porcine enveloped virus infection, the polysaccharide composition comprising at least one of Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and fucoidan.

[0010] Secondly, this application provides a method for preparing a polysaccharide composition, comprising the following steps:

[0011] Prepare polysaccharide and excipient components;

[0012] The polysaccharide and excipient components are mixed in a certain proportion to obtain a polysaccharide composition.

[0013] Thirdly, this application provides the use of the above-mentioned polysaccharide composition in the preparation of a drug or feed additive for preventing porcine enveloped virus infection.

[0014] The technical solution provided in this application has at least the following advantages compared with the prior art:

[0015] 1. The polysaccharide composition for preventing porcine enveloped virus infection provided in this application is formulated based on the nutrients Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and Fucoidan polysaccharide in a mass ratio of 1:0.5:0.3. It has a significant effect on inhibiting viral replication and reducing viral virulence. It can prevent and treat porcine enveloped virus infection simply by adding it to feed or by oral administration. It overcomes the disadvantages of possible immunization failure due to vaccination and viral mutation caused by the use of antiviral drugs. It has no toxic side effects on animals and can be used safely.

[0016] 2. The results of in vitro experiments in this application demonstrate that the polysaccharide composition composed of Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and Fucoido has a significant inhibitory effect on the proliferation of PEDV, PRRSV and ASFV.

[0017] 3. The in vivo experimental results of this application show that the polysaccharide composition composed of Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and fucoidan can effectively prevent and treat the infection of porcine epidemic diarrhea virus and porcine reproductive and respiratory syndrome virus, thereby reducing the diarrhea rate and mortality rate of sick pigs and effectively improving the survival rate of pigs. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In this application text, the terms "first" and "second" are used to distinguish identical or similar items with essentially the same function and effect, solely for the purpose of clearly describing the technical solutions of the embodiments of this application, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated.

[0023] The embodiments of this application have shown through in vivo and in vitro experiments that the polysaccharide composition composed of Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and Fucoidan can significantly inhibit the proliferation of PEDV, PRRSV and ASFV, as well as their infection of cells. The inhibitory effect is better than that of each single polysaccharide. The polysaccharide composition can be used to prepare drugs or feed additives against porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus and African swine fever virus.

[0024] Based on this, embodiments of this application provide a polysaccharide composition for resisting porcine enveloped virus infection, the polysaccharide composition comprising at least one of Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and fucoidan.

[0025] In some embodiments, the polysaccharide composition includes Poria cocos polysaccharide, Codonopsis pilosula polysaccharide, and fucoidan.

[0026] In some preferred embodiments, the polysaccharide composition comprises, by weight, 1 part of Poria cocos polysaccharide, 0.5 parts of Codonopsis pilosula polysaccharide and 0.3 parts of fucoidan.

[0027] In some embodiments, the polysaccharide composition further includes excipients, including at least one selected from montmorillonite, vermiculite, zeolite powder, silica, defatted rice bran, corn cob powder, soluble starch, wheat bran, acetic acid, anhydrous ethanol, and Tween-80.

[0028] In some embodiments, the polysaccharide composition is a solid mixture consisting of Poria cocos polysaccharide, Codonopsis pilosula polysaccharide, fucoidan, and montmorillonite, wherein the polysaccharide has a mass fraction of 45% to 55% in the solid mixture.

[0029] In some embodiments, the polysaccharide composition is a mixed solution of Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and fucoidan, acetic acid, anhydrous ethanol, Tween-80 and water for injection, wherein the concentration of the polysaccharide in the mixed solution is 100-150 mg / mL.

[0030] In some embodiments, the polysaccharide composition is a powdered mixture of Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and fucoidan with soluble starch or wheat bran, wherein the polysaccharide has a mass fraction of 45% to 55% in the powdered mixture.

[0031] Based on this, the present application provides a method for preparing a polysaccharide composition, comprising the following steps: preparing Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and fucoidan and excipient components; mixing the polysaccharide and excipient components in a certain proportion to obtain a polysaccharide composition.

[0032] Based on this, embodiments of this application provide the application of the above-mentioned polysaccharide composition in the preparation of drugs or feed additives for preventing porcine enveloped virus infection.

[0033] 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.

[0034] In some embodiments, the formulation type of the drug or feed additive includes at least one of oral liquid, granules, powder, and tablets.

[0035] In some embodiments, the method of using the drug or feed additive includes at least one of the following methods:

[0036] (1) Administer the polysaccharide component to the pig orally at a dose of 5-20 mg / kg of pig body weight;

[0037] (2) Add the feed to the pigs at a dosage of 0.01% to 0.1% of the total weight of the pig feed and feed it to the pigs.

[0038] The technical solution of this application and the technical effects achieved will be described in detail below through more specific embodiments.

[0039] Example 1

[0040] This embodiment provides a method for preparing a polysaccharide composition that resists porcine enveloped virus infection, the specific steps of which are as follows:

[0041] (1) Weigh out 10g of Poria cocos polysaccharide and 18g of montmorillonite, mix them evenly, and obtain the first mixture;

[0042] (2) Weigh 5g of Codonopsis pilosula polysaccharide and 3g of Fucoidan polysaccharide and add them to the first mixture. Mix them evenly to obtain the second mixture, namely the composition T1 for resisting porcine enveloped virus infection.

[0043] Example 2

[0044] This embodiment provides a method for preparing a polysaccharide composition that resists porcine enveloped virus infection, the specific steps of which are as follows:

[0045] (1) Weigh out 20g of Poria cocos polysaccharide, 10g of Codonopsis pilosula polysaccharide and 6g of fucoidan, mix them evenly to obtain a solid mixture;

[0046] (2) Weigh 12g of solid polysaccharide, add 5mL of 5% acetic acid, 15mL of anhydrous ethanol and 30mL of Tween-80 respectively, mix well, and finally add water for injection to 100mL to obtain a mixed solution, namely the polysaccharide composition T2 for anti-swine enveloped virus infection.

[0047] Example 3

[0048] This embodiment provides a method for preparing a polysaccharide composition that resists porcine enveloped virus infection, the specific steps of which are as follows:

[0049] (1) Weigh out 20g of Poria cocos polysaccharide, 10g of Codonopsis pilosula polysaccharide and 6g of fucoidan, mix them evenly to obtain a polysaccharide mixture;

[0050] (2) Weigh 18g of polysaccharide mixture and mix it with 18g of soluble starch. Pass the mixture through a 60-mesh sieve and package it to obtain a powdered mixture, namely the polysaccharide composition T3 for resisting porcine envelope virus infection.

[0051] The formulations and parameters of the various polysaccharide compositions prepared in the above embodiments are shown in Table 1 below:

[0052] Table 1

[0053]

[0054]

[0055] Inhibitory effect of polysaccharide composition on enveloped viruses

[0056] In this application, the polysaccharide composition prepared above was used to verify its inhibitory effect on the in vitro proliferation of three porcine viruses: PEDV, PRRSV, and ASFV. The verification method is as follows:

[0057] 1. Materials and Methods:

[0058] 1.1 Viruses: Porcine epidemic diarrhea virus (PEDV) YN strain, porcine reproductive and respiratory syndrome virus (PRRSV) NADC30-Like strain, and African swine fever virus (ASFV) were isolated, preserved, and donated by the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0059] 1.2 Cells: African green monkey kidney cells (Vero cells) were used for the proliferation of PEDV YN strain; rhesus monkey embryonic kidney cell line (Marc-145 cells) was used for the proliferation of PRRSV NADC30-Like strain; primary porcine alveolar macrophage cells were used for the proliferation of ASFV. All cells were kindly provided by Huazhong Agricultural University.

[0060] 1.3 Test Drug: A polysaccharide composition obtained by mixing Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and fucoidan (Maclean's, 98% purity) in a mass ratio of 1:0.5:0.3.

[0061] 1.4 Reagents: DMEM medium, EDTA, trypsin, fetal bovine serum and penicillin-streptomycin were all purchased from GIBCO.

[0062] 1.5 In vitro proliferation inhibition:

[0063] (1) PEDV in vitro proliferation inhibition assay: PEDV YN strain virus solution was serially diluted 10-fold with DMEM maintenance medium, and 10 -210 -3 10 -4 10 -5 10 -6 10 -7 Six dilutions were seeded into 96-well plates containing pre-cultured Vero cell monolayers at 100 μL / well. DMEM maintenance medium containing different concentrations of polysaccharide compositions was added to each group, and the plates were incubated at 37°C with 5% CO2 for 36 h. Cytopathic effects were observed. The number of wells with positive cytopathic effects for each dilution was counted, and the TCID for each group 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 polysaccharide compound on the in vitro proliferation of PEDV. Each group was performed in triplicate.

[0064] (2) PRRSV in vitro proliferation inhibition assay: The PRRSV NADC30-Like strain virus solution was serially diluted 10-fold with DMEM maintenance medium, and 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 Six dilutions were seeded into 96-well plates containing pre-cultured Marc-145 cell monolayers at 100 μL / well. DMEM maintenance medium containing different concentrations of polysaccharide compositions was added to each group, and the plates were incubated at 37°C with 5% CO2 for 36 h. Cytopathic effects were observed. The number of wells with positive cytopathic effects for each dilution was counted, and the TCID for each group 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 polysaccharide composition on PRRSV proliferation in vitro, and three replicates were performed for each group.

[0065] (3) ASFV in vitro proliferation inhibition assay: The ASFV strain virus solution was serially diluted 10-fold with DMEM maintenance medium, and 10... -2 10 -3 10 -4 10 -5 10 -6 10 -7 Six dilutions were seeded into 96-well plates containing pre-cultured Vero cell monolayers at 100 μL / well. DMEM maintenance medium containing different concentrations of polysaccharide compositions was added to each group, and the plates were incubated at 37°C with 5% CO2 for 36 h. Cytopathic effects were observed. The number of wells with positive cytopathic effects for each dilution was counted, and the TCID for each group was calculated using the Reed-Muench method. 50Comparing TCIDs of different groups 50 The value was used to determine the inhibitory effect of the polysaccharide composition on the in vitro proliferation of ASFV, and three replicates were performed for each group.

[0066] 1.6 Data Analysis: Experimental data were analyzed using one-way ANOVA and Duncan's method for multiple comparisons in SPSS 26.0 statistical software. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly significant. Results are expressed as mean ± standard deviation.

[0067] 2. Test Results

[0068] Table 2 shows the effects of different concentrations of polysaccharides and polysaccharide compositions on different TCID viruses. 50 The results are affected by the differences, where a, b, c, and d are indicators of significant differences.

[0069] Table 2

[0070] Group PEDV PRRSV ASFV Blank group <![CDATA[10 8.13±0.41a ]]> <![CDATA[10 8.38±0.46a ]]> <![CDATA[10 8.17±0.45a ]]> 10 μg / mL Poria cocos polysaccharide group <![CDATA[10 7.35±0.15a ]]> <![CDATA[10 7.67±0.34a ]]> <![CDATA[10 6.94±0.38b ]]> 10 μg / mL Codonopsis pilosula polysaccharide group <![CDATA[10 6.49±0.39b ]]> <![CDATA[10 6.35±0.31b ]]> <![CDATA[10 6.49±0.51b ]]> 10 μg / mL Fucoidan group <![CDATA[10 7.38±0.37ab ]]> <![CDATA[10 6.88±0.58b ]]> <![CDATA[10 7.01±0.26ab ]]> 10 μg / mL polysaccharide composition group <![CDATA[10 5.16±0.25c ]]> <![CDATA[10 4.45±0.31c ]]> <![CDATA[10 4.79±0.53c ]]> 50 μg / mL polysaccharide composition group <![CDATA[10 4.55±0.74bc ]]> <![CDATA[10 3.19±0.23cd ]]> <![CDATA[10 3.87±0.35cd ]]> 100 μg / mL polysaccharide composition group <![CDATA[10 2.28±0.19d ]]> <![CDATA[10 2.35±0.34d ]]> <![CDATA[10 2.14±0.24d ]]>

[0071] Table 2 shows that the toxic titer of PEDV in Vero cells decreased significantly with increasing polysaccharide composition concentration, indicating that the composition has a certain inhibitory effect on the proliferation of PEDV in cultured Vero cells. The TCID of PRDSV in Marc-145 cells also decreased with increasing polysaccharide composition concentration. 50 The significant decrease indicates that the polysaccharide composition has a significant inhibitory effect on the proliferation of PRRSV in Marc-145 cells cultured in vitro. With increasing polysaccharide composition concentration, the toxic titer of ASFV in porcine alveolar macrophages significantly decreased, indicating that the composition has a significant inhibitory effect on the proliferation of ASFV in porcine alveolar macrophages cultured in vitro.

[0072] Protective effect of polysaccharide composition on piglets infected with porcine epidemic diarrhea virus.

[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: polysaccharide composition T2.

[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 three experimental groups (low, medium, and high dose groups) and a blank group. The polysaccharide composition T2 was dissolved in milk and diluted to 3 mL before oral administration. The low-dose group (added at a polysaccharide content of 5 mg / kg pig body weight), the medium-dose group (added at a polysaccharide content of 10 mg / kg pig body weight), the high-dose group (added at a polysaccharide content of 20 mg / kg pig body weight), and the blank group (fed the same dose of milk without any polysaccharide composition) consisted of 10 piglets in each group.

[0077] 1.4 Challenge Test: After feeding for one week, piglets in each group were challenged with 10... 7 TCID 50 Each animal was challenged with PEDV (YN strain) at a dose of / mL and orally administered 5mL of the medium. The control group 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 for intestinal lesions, and the number of dead pigs was recorded. Growth performance, diarrhea rate, mortality rate, and protection rate were calculated. The inhibitory effect of the polysaccharide composition T2 on PEDV virus was determined by real-time quantitative PCR detection of the expression levels of PEDV structural genes N or M in the intestinal mucosa of piglets; the immune function of piglets was determined by detecting the levels of inflammatory factors in the blood.

[0079] 1.6 Data Analysis: Experimental data were analyzed using one-way ANOVA and Duncan's method for multiple comparisons in SPSS 26.0 statistical software. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly significant. Results are expressed as mean ± standard deviation.

[0080] 2. Experimental Results:

[0081] Table 3 shows the average daily weight gain of piglets infected with PEDV after adding different doses of polysaccharide composition T2 to milk and feeding it to them. A, B, C, and D are indicators of significant differences.

[0082] Table 3

[0083] project control group Blank group 5mg / kg group 10mg / kg group 20mg / kg group Average daily weight gain (g / animal) <![CDATA[122.45±22.78 a ]]> <![CDATA[40.46±15.67 d ]]> <![CDATA[62.66±18.62 c ]]> <![CDATA[94.12±25.38 b ]]> <![CDATA[115.49±27.67 a ]]> Material weight ratio 0.63±0.14 0.72±0.18 0.69±0.21 0.68±0.38 0.62±0.14

[0084] As shown in Table 3, adding different doses of polysaccharide composition T2 can significantly increase the average daily weight gain of PEDV-infected piglets, indicating that the polysaccharide composition provided in this application can improve the growth performance of piglets.

[0085] Table 4 shows the morbidity and mortality rates of piglets in each group, where a, b, c, and d are indicators of significant differences.

[0086] Table 4

[0087]

[0088]

[0089] As shown in Table 4, different doses of the polysaccharide composition T2 can significantly reduce the diarrhea rate and mortality rate of PEDV-infected piglets, indicating that the polysaccharide composition provided in this application can play a protective role against PEDV-infected piglets.

[0090] The expression levels of PEDV structural genes N and M in the intestinal mucosa of the jejunum and ileum of piglets were detected. The results are shown in Table 5 below, where a, b, c, and d are statistically significant markers.

[0091] Table 5

[0092]

[0093] As shown in Table 5, the polysaccharide composition T2 can significantly reduce the expression levels of the N and M genes of PEDV, indicating that the polysaccharide composition T2 provided in this application can inhibit PEDV replication in vivo.

[0094] Table 6 shows the levels of IL-8, IL-1β, and IL-6 in the blood of piglets in different groups, where a, b, and c are indicators of significant differences.

[0095] Table 6

[0096] project control group Blank group 5mg / kg group 10mg / kg group 20mg / kg group IL-8 (pg / mL) <![CDATA[29.47±8.79 c ]]> <![CDATA[42.48±11.56 a ]]> <![CDATA[38.22±8.98 a ]]> <![CDATA[31.07±12.36 b ]]> <![CDATA[27.49±10.67 c ]]> IL-1β (pg / mL) <![CDATA[32.36±10.34 c ]]> <![CDATA[53.17±16.65 a ]]> <![CDATA[49.70±12.76 a ]]> <![CDATA[40.27±14.26 b ]]> <![CDATA[35.91±11.89 c ]]> IL-6 (pg / mL) <![CDATA[24.49±7.59 c ]]> <![CDATA[36.78±15.12 a ]]> <![CDATA[31.30±13.45 ab ]]> <![CDATA[28.46±13.35 b ]]> <![CDATA[27.89±12.91 c ]]>

[0097] As shown in Table 6, the polysaccharide composition T2 can significantly reduce the levels of IL-8, IL-1β and IL-6 in the blood of PEDV-infected piglets, indicating that the polysaccharide composition provided in this application can alleviate the inflammatory response of PEDV-infected piglets and play an immune protective role.

[0098] Protective effect of polysaccharide composition on piglets infected with porcine reproductive and respiratory syndrome virus.

[0099] 1. Materials and Methods:

[0100] 1.1 Test virus: PRRSV NADC30-Like strain was donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0101] 2. Test drug: Polysaccharide composition T3.

[0102] 3. Experimental animals: Healthy 25-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 three experimental groups (low, medium, and high dose groups) and a blank group. The polysaccharide composition T3 was dissolved in milk and diluted to 3 mL before oral administration. The low-dose group (added at a polysaccharide content of 5 mg / kg pig body weight), the medium-dose group (added at a polysaccharide content of 10 mg / kg pig body weight), the high-dose group (added at a polysaccharide content of 20 mg / kg pig body weight), and the blank group (fed the same dose of milk without adding any polysaccharide composition) consisted of 10 piglets in each group.

[0103] 1.4 Challenge Test: After feeding for one week, piglets in each group were challenged with 10... 7 TCID 50 The animals were challenged with (PRRSV) NADC30-Like at a dose of 5 mL / mL, and each animal was orally administered 5 mL. The control group was administered the same dose of cell culture medium (DMEM). The animals were then isolated and observed for 10–15 days until the end of the experiment.

[0104] 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. Growth performance, diarrhea rate, mortality rate, and protection rate were calculated. Piglet immune function was determined by detecting the levels of inflammatory factors in the blood. The inhibitory effect of the polysaccharide composition T3 on PRRSV was determined by detecting the expression levels of the PRRSV ORF7 gene in piglets and lungs using quantitative real-time PCR.

[0105] 1.3 Data Analysis: Experimental data were analyzed using one-way ANOVA and Duncan's method for multiple comparisons in SPSS 26.0 statistical software. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly significant. Results are expressed as mean ± standard deviation.

[0106] 2. Experimental Results:

[0107] Table 7 shows the average daily weight gain of piglets infected with PRRSV after adding different doses of polysaccharide composition T3 to milk and feeding it to them.

[0108] Table 7

[0109] project control group Blank group 5mg / kg group 10mg / kg group 20mg / kg group Average daily weight gain (g / animal) <![CDATA[112.24±21.67 a ]]> <![CDATA[39.35±14.69 d ]]> <![CDATA[60.55±17.51 c ]]> <![CDATA[91.01±24.27 b ]]> <![CDATA[109.38±25.56 a ]]> Material weight ratio 0.70±0.14 0.74±0.16 0.71±0.19 0.70±0.27 0.69±0.21

[0110] As shown in Table 7, adding different doses of polysaccharide composition T3 can significantly increase the average daily weight gain of PRRSV-infected piglets, indicating that the polysaccharide composition provided in this application can improve the growth performance of piglets.

[0111] Table 8 shows the morbidity and mortality rates of piglets in each group, where a, b, c, and d are indicators of significant differences.

[0112] Table 8

[0113] Group Diarrhea rate (%) mortality rate(%) Protection rate (%) Blank group <![CDATA[100 a ]]> <![CDATA[100 a ]]> <![CDATA[0 d ]]> 5mg / kg group <![CDATA[75 b ]]> <![CDATA[70 b ]]> <![CDATA[30 c ]]> 10mg / kg group <![CDATA[60 c ]]> <![CDATA[40 c ]]> <![CDATA[60 b ]]> 20mg / kg group <![CDATA[30 d ]]> <![CDATA[15 c ]]> <![CDATA[75 a ]]>

[0114] As shown in Table 8, different doses of the polysaccharide composition T3 can significantly reduce the diarrhea rate and mortality rate of PRRSV-infected piglets, indicating that the polysaccharide composition provided in this application can play a protective role against PRRSV-infected piglets.

[0115] The expression levels of the PRRSV ORF7 gene in the brain and lungs of piglets were detected, and the results are shown in Table 9 below.

[0116] Table 9

[0117]

[0118] As shown in Table 9, different doses of the polysaccharide composition T3 can significantly reduce the expression level of the PRRSV ORF7 gene, which fully demonstrates that the polysaccharide composition provided in this application can significantly reduce the replication of PRRSV in the pig brain and lungs after addition.

[0119] Table 10 shows the levels of IL-8, IL-1β, and IL-6 in the blood of piglets in different groups, where a, b, and c are indicators of significant differences.

[0120] Table 10

[0121] project control group Blank group 5mg / kg group 10mg / kg group 20mg / kg group IL-8 (pg / mL) <![CDATA[28.36±9.68 c ]]> <![CDATA[41.37±12.45 a ]]> <![CDATA[37.12±9.87 a ]]> <![CDATA[30.16±11.25 b ]]> <![CDATA[26.37±11.78 c ]]> IL-1β (pg / mL) <![CDATA[31.25±11.23 c ]]> <![CDATA[52.28±15.54 a ]]> <![CDATA[48.59±13.67 a ]]> <![CDATA[41.18±13.15 b ]]> <![CDATA[34.89±10.78 c ]]> IL-6 (pg / mL) <![CDATA[23.38±7.48 c ]]> <![CDATA[35.67±14.01 a ]]> <![CDATA[31.56±12.34 ab ]]> <![CDATA[29.16±13.15 b ]]> <![CDATA[24.31±8.80 c ]]>

[0122] As shown in Table 10, the polysaccharide composition T3 can significantly reduce the levels of IL-8, IL-1β and IL-6 in the blood of PRRSV-infected piglets, indicating that the polysaccharide composition provided in this application can alleviate the inflammatory response of PRRSV-infected piglets and play an immune protective role.

[0123] In summary, the polysaccharide composition provided in this application has the effect of resisting porcine 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 viruses such as PEDV, PRRSV, and ASFV. The dosage forms of the drugs and feed additives include oral liquids, granules, powders, tablets, etc.

[0124] 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. A polysaccharide composition for resisting porcine enveloped virus infection, characterized in that, The polysaccharide composition comprises, by weight, 1 part of Poria cocos polysaccharide, 0.5 parts of Codonopsis pilosula polysaccharide, and 0.3 parts of fucoidan.

2. The polysaccharide composition according to claim 1, characterized in that, The polysaccharide composition further includes excipients, which include at least one of montmorillonite, vermiculite, zeolite powder, silica, defatted rice bran, corn cob powder, soluble starch, wheat bran, acetic acid, anhydrous ethanol, and Tween-80.

3. The polysaccharide composition according to claim 1, characterized in that, The polysaccharide composition is at least one of the following (1) to (3): (1) a solid mixture composed of Poria cocos polysaccharide, Codonopsis pilosula polysaccharide, fucoidan and montmorillonite, wherein the polysaccharide in the solid mixture has a mass fraction of 45% to 55%; (2) A mixed solution formed by Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and fucoidan, acetic acid, anhydrous ethanol, Tween-80 and water for injection, wherein the concentration of polysaccharide in the mixed solution is 100~150 mg / mL; (3) A powdered mixture composed of Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and fucoidan with soluble starch or wheat bran, wherein the polysaccharide has a mass fraction of 45% to 55% in the powdered mixture.

4. A method for preparing a polysaccharide composition, characterized in that, The method for preparing the polysaccharide mixture according to any one of claims 1-3 comprises the following steps: Prepare Poria cocos polysaccharide, Codonopsis pilosula polysaccharide, and fucoidan, as well as excipients; The polysaccharide and excipient components are mixed in a certain proportion to obtain a polysaccharide composition.

5. The use of the polysaccharide composition of claim 1 in the preparation of a drug or feed additive for preventing porcine enveloped virus infection, wherein the porcine enveloped virus includes at least one of porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus.

6. The application according to claim 5, characterized in that, The formulation types of the drug or feed additive include at least one of oral liquid, granules, powder, and tablets.

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