Polysaccharide composition for resisting porcine envelope virus infection as well as preparation method and application of polysaccharide composition

Abstract

The invention discloses a polysaccharide composition for resisting porcine envelope virus infection as well as a preparation method and application of the polysaccharide composition. The polysaccharide composition comprises the following components in parts by mass: 1 part of pachymaran, 0.5 part of codonopsis pilosula polysaccharide and 0.3 part of fucoidin. The polysaccharide composition can also comprise auxiliary materials, wherein the auxiliary materials comprise at least one of montmorillonite, vermiculite, zeolite powder, white carbon black, defatted rice bran, corncob powder, soluble starch, bran, acetic acid, absolute ethyl alcohol and tween-80. The polysaccharide composition can effectively prevent and treat infection of porcine viruses such as PEDV (porcine epidemic diarrhea virus), PRRSV (porcine reproductive and respiratory syndrome virus) and AFSV (avian influenza virus) by enhancing the antioxidant defense function of a body and improving the natural immune function and antiviral activity of pigs, so that the incidence rate of pig diseases and the death rate of diseased pigs are reduced. The polysaccharide composition can be used for preparing medicines or feed additives for resisting porcine viruses such as PEDV (porcine epidemic diarrhea virus), PRRSV (porcine reproductive and respiratory syndrome virus) and AFSV (acute follicle-state virus), and the dosage forms of the medicines comprise oral liquid, granules, tablets, powder and the like.

Description

Technical Field

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

[0002] Disease pathogens can be divided into viral and bacterial infections, and viruses can be divided into enveloped viruses and non-enveloped viruses. Relatively speaking, enveloped viruses are more prone to mutation and have stronger immune escape ability due to the presence of an outer envelope, making their prevention and control more difficult. Common porcine enveloped viruses include porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), etc.

[0003] Porcine epidemic diarrhea virus (PEDV) is the main cause of porcine epidemic diarrhea (PED). It is a coronavirus that can infect pigs of different ages and breeds, but has a high mortality rate for piglets, with a mortality rate of up to 100% for piglets under seven days of age. PEDV mainly infects and replicates in the villus enterocytes of the small intestine, and clinical symptoms include vomiting, diarrhea, and dehydration in pigs. PRRSV infection can cause porcine reproductive and respiratory syndrome (PRRS), also known as "blue ear disease", a highly contagious disease of 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, extensive bleeding in the skin and internal organs, respiratory disorders, neurological symptoms, and high mortality.

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

[0005] To date, there are no other effective means of prevention and treatment for the above enveloped viruses except inactivated and attenuated vaccines. Although many pig farmers choose to prevent viral infection through vaccination, the vaccination effect is often not ideal due to factors such as inconsistent vaccine quality. In my country's high-density breeding environment, the phenomenon of large-scale use of antiviral drugs is common, which not only accelerates the mutation rate of virus strains, but also makes the progress of vaccine research and development pale in comparison, leading to frequent outbreaks of epidemics.

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

[0007] In view of this, the purpose of the present application is to provide a polysaccharide composition, preparation method and application thereof for resisting porcine envelope virus infection. The polysaccharide composition has the advantages of high safety and convenient application, and has a high protection rate against porcine envelope virus infection, and can be used to prepare drugs or feed additives for resisting porcine envelope virus infection.

[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 polysaccharide composition for resisting porcine envelope virus infection, wherein the polysaccharide composition comprises at least one of tuckahoe polysaccharide, codonopsis polysaccharide and fucoidan.

[0010] In a second aspect, the present application provides a method for preparing a polysaccharide composition, comprising the following steps:

[0011] preparing polysaccharide and excipient components;

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

[0013] In a third aspect, the present application provides the use of the above-mentioned polysaccharide composition in the preparation of a drug or feed additive for resisting porcine envelope virus infection.

[0014] Compared with the prior art, the technical solution provided by this application has at least the following beneficial effects:

[0015] 1. The polysaccharide composition for resistance to piglet envelope virus infection provided in the present application is prepared by compounding the nutrients Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and fucoidan at a mass ratio of 1:0.5:0.3. It has a significant effect of inhibiting viral replication and reducing viral virulence. It can prevent and treat piglet envelope virus infection by simply adding it to feed or taking it orally. It overcomes the shortcomings of possible immune failure caused by vaccination and virus mutation caused by the use of antiviral drugs. It has no toxic side effects on animals and can be used safely.

[0016] 2. The in vitro experimental results of this application show that: the in vitro experimental results prove that the polysaccharide composition composed of Poria polysaccharide, Codonopsis pilosula polysaccharide and Fucus has a significant effect on inhibiting 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 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 diseased pigs and effectively improving the survival rate of pigs. DETAILED DESCRIPTION

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In the text of this application, the terms "first", "second" and the like are used to distinguish between identical or similar items with substantially the same functions and effects. This is only for the purpose of clearly describing the technical solutions of the embodiments of this application, and shall not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.

[0023] The examples of the present application found through in vivo and in vitro experiments that a polysaccharide composition composed of Poria polysaccharide, Codonopsis pilosula polysaccharide, and fucoidan can significantly inhibit the proliferation of PEDV, PRRSV, and ASFV, and inhibit 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, an embodiment of the present application provides a polysaccharide composition for resisting porcine envelope virus infection, wherein the polysaccharide composition includes at least one of Poria cocos polysaccharide, Codonopsis pilosula polysaccharide and fucoidan.

[0025] In certain embodiments, the polysaccharide composition includes pachymaran, codonopsis pilosula polysaccharide and fucoidan.

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

[0027] In certain embodiments, the polysaccharide composition further comprises auxiliary materials, and the auxiliary materials comprise at least one of montmorillonite, vermiculite, zeolite powder, white carbon black, defatted rice bran, corn cob powder, soluble starch, bran, acetic acid, anhydrous ethanol, and Tween-80.

[0028] In certain embodiments, the polysaccharide composition is a solid mixture of tuckahoe polysaccharide, codonopsis pilosula polysaccharide, fucoidan and montmorillonite, wherein the mass fraction of the polysaccharide in the solid mixture is 45% to 55%.

[0029] In certain embodiments, the polysaccharide composition is a mixed solution of Poria 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 certain embodiments, the polysaccharide composition is a powdery mixture of tuckahoe polysaccharide, codonopsis pilosula polysaccharide, fucoidan and soluble starch or bran, wherein the mass fraction of the polysaccharide in the powdery mixture is 45% to 55%.

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

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

[0033] 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.

[0034] In certain embodiments, the drug or feed additive is in the form of an oral solution, a granule, a powder, or a tablet.

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

[0036] (1) Orally administering the polysaccharide component to pigs at a dosage of 5 to 20 mg / kg of pig body weight;

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

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

[0039] Example 1

[0040] This embodiment provides a method for preparing a polysaccharide composition for resisting porcine envelope virus infection, and the specific steps are as follows:

[0041] (1) Weigh 10 g of Poria polysaccharide and 18 g of montmorillonite and mix them evenly to obtain a first mixture;

[0042] (2) Weigh 5 g of Codonopsis pilosula polysaccharide and 3 g of fucoidan, add them to the first mixture, mix them evenly, and obtain a second mixture, i.e., composition T1 for resisting porcine envelope virus infection.

[0043] Example 2

[0044] This embodiment provides a method for preparing a polysaccharide composition for resisting porcine envelope virus infection, and the specific steps are as follows:

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

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

[0047] Example 3

[0048] This embodiment provides a method for preparing a polysaccharide composition for resisting porcine envelope virus infection, and the specific steps are as follows:

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

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

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

[0052] Table 1

[0053]

[0054]

[0055] Inhibitory effect of polysaccharide composition on enveloped viruses

[0056] In the present application, the polysaccharide composition prepared above was used to verify the 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 National 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) were 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 donated by Huazhong Agricultural University.

[0060] 1.3 Experimental drug: A polysaccharide composition was obtained by mixing Poria polysaccharide, Codonopsis pilosula polysaccharide and fucoidan (McLean, purity 98%) in a mass ratio of 1:0.5:0.3.

[0061] 1.4 Reagents: DMEM culture medium, EDTA, trypsin, fetal bovine serum and double antibody (penicillin-streptomycin) were purchased from GIBCO.

[0062] 1.5 In vitro proliferation inhibition:

[0063] (1) PEDV in vitro proliferation inhibition test: PEDV YN strain virus liquid was diluted 10 times in DMEM maintenance medium, and 10 -2, 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated into 96-well plates with cultured Vero cell monolayers, 100 μL / well, and DMEM maintenance solution containing different concentrations of polysaccharide compositions was added to each group. The cells were placed in a 37°C incubator with 5% CO2 for 36 hours and the cell lesions were observed. The number of lesion-positive wells in each dilution was counted, and the TCID of each group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of the polysaccharide on the proliferation of PEDV in vitro was judged by the value of , and three replicates were performed in each group.

[0064] (2) PRRSV in vitro proliferation inhibition test: PRRSV NADC30-Like strain virus liquid was diluted 10 times in DMEM maintenance medium, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated into 96-well plates with cultured Marc-145 cell monolayers, 100 μL / well, and DMEM maintenance solution containing different concentrations of polysaccharide composition was added to each group. The cells were placed in a 37°C incubator with 5% CO2 for 36 hours and the cell lesions were observed. The number of lesion-positive wells in each dilution was counted, and the TCID of each group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of the polysaccharide composition on the proliferation of PRRSV in vitro was judged based on the value of , and three replicates were performed in each group.

[0065] (3) ASFV in vitro proliferation inhibition test: The ASFV virus liquid was diluted 10 times in DMEM maintenance medium, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated into 96-well plates with cultured Vero cell monolayers, 100 μL / well, and DMEM maintenance solution containing different concentrations of polysaccharide compositions was added to each group. The cells were placed in a 37°C incubator with 5% CO2 for 36 hours and the cell lesions were observed. The number of lesion-positive wells in each dilution was counted, and the TCID of each group was calculated using the Reed-Muench method. 50, compare different groups of TCID 50 The inhibitory effect of the polysaccharide composition on the proliferation of ASFV in vitro was judged based on the value of , and three replicates were performed in each group.

[0066] 1.6 Data analysis: The experimental data were analyzed using one-way ANOVA and Duncan's multiple comparison method in SPSS26.0 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.

[0067] 2. Test results

[0068] Table 2 shows the TCID of different viruses by different concentrations of polysaccharides and polysaccharide combinations 50 Influence results, where a, b, c, and d are marks of significant difference.

[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 ]]> 10μg / mL Poria polysaccharide group <![CDATA[10 7.35±0.15a ]]> <![CDATA[10 7.67±0.34a ]]> <![CDATA[10 6.94±0.38b ]]> 10μg / mL Codonopsis pilosula polysaccharide group <![CDATA[10 6.49±0.39b ]]> <![CDATA[10 6.35±0.31b ]]> <![CDATA[10 6.49±0.51b ]]> 10μg / 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] As shown in Table 2, with the increase of the concentration of the polysaccharide composition, the toxicity of PEDV on Vero cells decreased significantly, indicating that the composition has a certain inhibitory effect on the proliferation of PEDV in Vero cells cultured in vitro. 50 The results showed that the polysaccharide composition had a significant inhibitory effect on the proliferation of PRRSV in Marc-145 cells cultured in vitro. As the concentration of the polysaccharide composition increased, the toxicity of ASFV in porcine alveolar macrophages decreased significantly, indicating that the composition had 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 Experimental virus: PEDV YN strain was donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0075] 1.2 Experimental drug: polysaccharide composition T2.

[0076] 1.3 Experimental animals: 7-day-old healthy piglets of the same breed and origin, similar weight and without maternal antibodies were selected for the challenge test. The experimental 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 the volume was fixed to 3 mL for oral administration, including 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 (gavagely fed with an equal dose of milk without adding any polysaccharide composition), with 10 piglets in each group.

[0077] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 7 TCID 50 / mL PEDV (YN strain) was used for infection, and 5 mL was orally infused into each head. The control group was infused with the same dose of cell culture medium (DMEM), and then they were isolated and raised, and observed for 7 consecutive days until the end of the experiment.

[0078] 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 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 detecting the expression of PEDV structural gene N or M in the intestinal mucosa of piglets by real-time quantitative PCR; the immune function of piglets was determined by detecting the content of inflammatory factors in the blood.

[0079] 1.6 Data analysis: The experimental data were analyzed using one-way ANOVA and Duncan's multiple comparison method in SPSS26.0 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.

[0080] 2. Test results:

[0081] After adding different doses of the polysaccharide composition T2 to milk and feeding it to piglets infected with PEDV, the average daily weight gain of the piglets in each group is shown in Table 3, where a, b, c, and d are 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 / head <![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 the polysaccharide composition T2 can significantly increase the average daily weight gain of PEDV-infected piglets, indicating that the polysaccharide composition provided by the present application can improve the growth performance of piglets.

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

[0086] Table 4

[0087]

[0088]

[0089] As can be seen from 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 the present application can protect piglets infected with PEDV.

[0090] The expression levels of PEDV structural gene N or M in the intestinal mucosa of the jejunum and ileum tissues of piglets were detected respectively. The test results are shown in Table 5 below, where a, b, c, and d are significant difference marks.

[0091] Table 5

[0092]

[0093] As can be seen from Table 5, the polysaccharide composition T2 can significantly reduce the expression levels of N and M genes of PEDV, indicating that the polysaccharide composition T2 provided in the present application can inhibit the replication of PEDV 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 marks of significant difference.

[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 can be seen from 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 the present application can alleviate the inflammatory response of PEDV-infected piglets and play an immune protective role.

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

[0099] 1. Materials and methods:

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

[0101] 2. Experimental drug: polysaccharide composition T3.

[0102] 3. Experimental animals: 25-day-old healthy piglets of the same breed and origin, similar weight and without maternal antibodies were selected for the challenge test. The experimental 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 the volume was fixed to 3 mL for oral administration. There were low-dose group (addition of polysaccharide content of 5 mg / kg pig body weight), medium-dose group (addition of polysaccharide content of 10 mg / kg pig body weight), high-dose group (addition of polysaccharide content of 20 mg / kg pig body weight) and blank group (gavage feeding of equal doses of milk without adding any polysaccharide composition), each group had 10 piglets.

[0103] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 7 TCID 50 / mL (PRRSV) NADC30-Like was used for infection, and 5 mL was orally infused into each head. The control group was infused with the same dose of cell culture medium (DMEM), and then they were isolated and raised, and observed continuously for 10 to 15 days until the end of the experiment.

[0104] 1.5 Observation indicators: 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. The number of dead pigs was counted, and the growth performance, diarrhea rate, mortality rate and protection rate were calculated. The immune function of piglets was determined by detecting the content of inflammatory factors in the blood. The inhibitory effect of the polysaccharide composition T3 on PRRSV was determined by detecting the expression of PRRSV ORF7 gene in piglets and lungs by fluorescent quantitative PCR.

[0105] 1.3 Data analysis: The experimental data were analyzed using one-way ANOVA and Duncan's multiple comparison method in SPSS26.0 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.

[0106] 2. Test results:

[0107] After adding different doses of the polysaccharide composition T3 to milk and feeding it to piglets infected with PRRSV, the average daily weight gain of the piglets in each group is shown in Table 7.

[0108] Table 7

[0109] project Control group Blank Group 5mg / kg group 10mg / kg group 20mg / kg group Average daily weight gain g / head <![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 the polysaccharide composition T3 can significantly increase the average daily weight gain of PRRSV-infected piglets, indicating that the polysaccharide composition provided by the present application can improve the growth performance of piglets.

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

[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 the present application can protect PRRSV-infected piglets.

[0115] The expression levels of PRRSV ORF7 gene in the brain and lung of piglets were detected respectively. The detection 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 ORF7 gene of PRRSV. The results fully demonstrate that the polysaccharide composition provided by the present 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 marks of significant difference.

[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 the present application can alleviate the inflammatory response of PRRSV-infected piglets and play an immune protective role.

[0123] In summary, the polysaccharide composition provided in the embodiments of the present 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 pig diseases infected by porcine viruses such as PEDV, PRRSV and ASFV. The dosage forms of the drugs and feed additives include oral liquids, granules, powders, tablets and other dosage forms.

[0124] 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 polysaccharide composition for resisting porcine envelope virus infection, characterized in that: The polysaccharide composition comprises at least one of pachymaran, codonopsis pilosula polysaccharide and fucoidan.

2. The polysaccharide composition according to claim 1, characterized in that The polysaccharide composition, by weight, comprises 1 part of tuckahoe polysaccharide, 0.5 part of codonopsis polysaccharide and 0.3 part of fucoidan.

3. The polysaccharide composition according to claim 1, characterized in that The polysaccharide composition also includes auxiliary materials, which include at least one of montmorillonite, vermiculite, zeolite powder, white carbon black, defatted rice bran, corn cob powder, soluble starch, bran, acetic acid, anhydrous ethanol and Tween-80.

4. 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 consisting of tuckahoe polysaccharide, codonopsis pilosula polysaccharide, fucoidan and montmorillonite, wherein the mass fraction of the polysaccharide in the solid mixture is 45% to 55%; (2) a mixed solution formed by tuckahoe polysaccharide, codonopsis 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 to 150 mg / mL; (3) A powdery mixture consisting of tuckahoe polysaccharide, codonopsis pilosula polysaccharide, fucoidan and soluble starch or bran, wherein the mass fraction of the polysaccharide in the powdery mixture is 45% to 55%.

5. A method for preparing a polysaccharide composition, characterized in that: The following steps are involved: preparing tuckahoe polysaccharide, codonopsis pilosula polysaccharide, fucoidan and auxiliary material components; The polysaccharide and auxiliary material components are mixed in proportion to obtain a polysaccharide composition.

6. Use of the polysaccharide composition according to claim 1 in the preparation of a drug or feed additive for resisting porcine envelope virus infection.

7. The use according to claim 6, 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.

8. The use according to claim 6, characterized in that: The preparation type of the medicine or feed additive includes at least one of oral liquid, granules, powder and tablets.

9. The use according to claim 6, characterized in that: The method of using the drug or feed additive includes at least one of the following (1) and (2): (1) Orally administering the polysaccharide component to pigs at a dosage of 5 to 20 mg / kg of pig body weight; (2) Add the additive to the feed at a dosage of 0.01% to 0.1% of the total weight of the pig feed and feed it to pigs.

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