Application of 25-hydroxyvitamin D3 in preparation of medicine for treating or preventing swine envelope virus, pharmaceutical composition and application of pharmaceutical composition

Abstract

The invention discloses application of 25-hydroxyvitamin D3 in preparation of a medicine for treating or preventing swine envelope virus, a medicine composition and application of the medicine composition. The pharmaceutical composition comprises 25-hydroxyvitamin D3 and auxiliary materials, wherein the auxiliary materials comprise at least one of absolute ethyl alcohol, water, propylene glycol, dimethyl sulfoxide, edible oil, Tween 80, lactose, mannitol, soluble starch, montmorillonite, vermiculite, medical stone, vermiculite, zeolite powder, white carbon black, defatted rice bran, corncob powder, starch and bran. The pharmaceutical composition is high in safety, can be used as a feed additive for a long time to achieve the purpose of preventing epidemic diseases, can effectively prevent and treat infection of porcine envelope viruses such as PEDV (porcine epidemic diarrhea virus), PRRSV (porcine reproductive and respiratory syndrome virus) and AFSV (aflatoxin virus) by enhancing the antioxidant defense function of a body and improving the natural immune function and antiviral activity of pigs, and has a good application prospect. Therefore, the incidence rate of pig diseases and the death rate of diseased pigs are reduced.

Description

Technical Field

[0001] The present application relates to the technical field of porcine virus prevention and treatment, and in particular to the use of 25-hydroxyvitamin D3 in the preparation of drugs for treating or preventing porcine envelope virus, a pharmaceutical composition and its application. Background Art

[0002] The intestine is the main place for the body to digest and absorb nutrients, and it is also the largest immune organ. The intestinal health of piglets is closely related to the production efficiency of the pig industry. Diarrhea is one of the most serious diseases for weaned piglets and poses a major threat to them.

[0003] Weaned piglets have low intestinal immune function and poor gastrointestinal development, which makes them less resistant to pathogens (viruses, bacteria, endotoxins), which is an important cause of intestinal diseases. Existing data show that in farms with poor prevention and control levels, the diarrhea rate of piglets reaches about 75%, and the mortality rate is about 20%, and some are even as high as 60%. Therefore, improving the productivity of piglets by nutritional measures to improve the intestinal environment of piglets has become one of the main research hotspots in the field of animal nutrition.

[0004] So far, PEDV is one of the most important pathogens causing diarrhea in piglets. The main methods used to prevent and control PEDV are vaccination or antiviral drugs. However, due to the variability of PEDV, vaccination or antiviral drugs make the immunoglobulins produced by pigs lose their due effects, resulting in many vaccines or drugs losing their due effects, resulting in unsatisfactory effects of vaccination or antiviral drugs. At the same time, in clinical practice, intestinal health problems in piglets caused by PEDV infection often further cause mixed infections of porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), making it even more difficult to prevent and control viral infectious diseases in piglets. So far, there are still no good prevention and control measures.

[0005] In view of the above situation, the development of therapeutic drugs and methods that can directly inhibit the activity of porcine viruses such as PEDV, PRRSV and ASFV has become a market need. Summary of the invention

[0006] The present application provides the use of 25-hydroxyvitamin D3 in the preparation of drugs for treating or preventing porcine envelope virus, a pharmaceutical composition and its use. 25-hydroxyvitamin D3 can directly inhibit the gene expression of PEDV, PRRSV and AFSV, and improve the pig's own immunity, and can effectively prevent and treat the infection of PEDV, PRRSV and AFSV, thereby reducing the incidence and mortality rate of pigs.

[0007] In a first aspect, the present application provides the use of 25-hydroxyvitamin D3 in the preparation of drugs for treating or preventing porcine envelope virus.

[0008] By adopting the above technical scheme, 25-hydroxyvitamin D3 can be administered alone as a drug for treating or preventing porcine envelope virus; it can also be mixed with excipients to form a pharmaceutical composition for administration as a drug for treating or preventing porcine envelope virus. The drug prepared by 25-hydroxyvitamin D3 can prevent piglets from being infected with porcine envelope virus and treat piglets infected with porcine envelope virus by inhibiting the gene expression of related porcine envelope virus.

[0009] Furthermore, the porcine envelope virus includes at least one of porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus and African swine fever virus.

[0010] By adopting the above technical scheme, 25-hydroxyvitamin D3 can inhibit the gene expression of porcine envelope viruses such as porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (AFSV), so as to reduce the proliferation of PEDV, PRRSV and AFSV, and prepare corresponding preparations for treating or preventing porcine envelope viruses.

[0011] Furthermore, when treating porcine envelope virus, the dosage of 25-hydroxyvitamin D3 is 2 to 3 mg / kg body weight, added 2 to 3 times a day, and the drug is used continuously for 5 to 7 days.

[0012] The above technical solution is used to treat pigs infected with porcine envelope virus. In one embodiment, 25-hydroxyvitamin D3 is added 3 times a day at a dose of 2 mg / kg body weight, or 2 times a day at a dose of 3 mg / kg body weight. The treatment period is 5 to 7 days, which can be 5 days, 6 days or 7 days depending on the recovery of the pig.

[0013] Furthermore, for the prevention of porcine envelope virus, the dosage of 25-hydroxyvitamin D3 is 0.4-0.6 mg / kg body weight, which is used throughout the entire feeding cycle of pigs.

[0014] By adopting the above technical scheme, pigs can be prevented from being infected with porcine envelope virus. In one embodiment, the dosage of 25-hydroxyvitamin D3 is 0.4 mg / kg body weight, 0.5 mg / kg body weight or 0.6 mg / kg body weight, and is used throughout the entire feeding cycle of pigs by directly feeding or mixing into feed.

[0015] In a second aspect, the present application provides a pharmaceutical composition comprising 25-hydroxyvitamin D3 and excipients.

[0016] By adopting the above technical solution, when preparing the pharmaceutical composition, 25-hydroxyvitamin D3 is obtained, and 25-hydroxyvitamin D3 is mixed with auxiliary materials to obtain the pharmaceutical composition.

[0017] Furthermore, the auxiliary material includes at least one of anhydrous ethanol, water, propylene glycol, dimethyl sulfoxide, edible oil, Tween 80, lactose, mannitol, soluble starch, montmorillonite, vermiculite, medical stone, vermiculite, zeolite powder, white carbon black, defatted rice bran, corn cob powder, starch, and bran.

[0018] By adopting the above technical solution, the pharmaceutical composition preparation can be prepared in various forms, specifically configured as at least one of oral liquid, tablets, granules and powders.

[0019] Furthermore, the auxiliary material includes a solution formed by mixing anhydrous ethanol, Tween-80 and water, wherein the concentration of 25-hydroxyvitamin D3 is 5 to 20 mg / ml.

[0020] By adopting the above technical solution, the pharmaceutical composition preparation is prepared in the form of an oral liquid.

[0021] Furthermore, the pharmaceutical composition is configured as a solid mixture of 25-hydroxyvitamin D3, lactose and soluble starch, wherein the mass fraction of 25-hydroxyvitamin D3 in the solid mixture is 20 to 30%.

[0022] By adopting the above technical solution, the pharmaceutical composition preparation is prepared in the form of granules or tablets.

[0023] Furthermore, the pharmaceutical composition is configured as a powdery mixture of 25-hydroxyvitamin D3 and soluble starch or bran, wherein the mass fraction of 25-hydroxyvitamin D3 in the powdery mixture is 20 to 30%.

[0024] By adopting the above technical solution, the pharmaceutical composition preparation is prepared in the form of powder.

[0025] In a third aspect, the present application provides a use of a pharmaceutical composition in the preparation of a drug for treating or preventing porcine envelope virus.

[0026] By adopting the above technical scheme, 25-hydroxyvitamin D3 has the effects of enhancing natural immune function and antiviral activity. The pharmaceutical composition with 25-hydroxyvitamin D3 as the active ingredient can effectively prevent and treat infection by porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus, thereby reducing the mortality rate of diseased pigs and effectively improving the survival rate and production performance of pigs.

[0027] In summary, the present application includes at least one of the following beneficial technical effects:

[0028] 1. The pharmaceutical composition provided in the present application is highly safe and can be used as a feed additive for a long time to prevent epidemic diseases. Long-term use will not lead to the production of drug-resistant strains and will not have other toxic side effects. This is an advantage that other anti-swine vesicular virus disease drugs do not have.

[0029] 2. The pharmaceutical composition provided by the present application can effectively prevent and treat infection by porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus and African swine fever virus by enhancing the body's antioxidant defense function, improving the pig's natural immune function and antiviral activity, thereby reducing the incidence of porcine diarrhea and the death of infected pigs. 25-Hydroxyvitamin D3, as an active ingredient in drugs for the treatment and prevention of porcine envelope virus infection, has significant efficacy.

[0030] 3. The results of in vivo experiments in this application show that 25-hydroxyvitamin D3 has the function of enhancing natural immune function and antiviral activity, and can effectively prevent and treat infection with porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus, thereby reducing the mortality rate of diseased pigs and effectively improving the survival rate and production performance of pigs. Oral administration of 0.5 mg / kg body weight or adding 25-hydroxyvitamin D3 to piglet feed can protect against infection with porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus by about 90%. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] Embodiment 1:

[0036] Application of 25-hydroxyvitamin D3 in preparing drugs for treating or preventing porcine envelope virus

[0037] The preparation of a drug, that is, the preparation of a pharmaceutical composition, comprises the following steps:

[0038] (1) Weigh 0.5 g of 25-hydroxyvitamin D3, 20 mL of anhydrous ethanol, 30 mL of Tween-80, and an appropriate amount of water for injection.

[0039] (2) Dissolve 25-hydroxyvitamin D3 in anhydrous ethanol, then add Tween-80, and finally slowly add water for injection to make up to 100 mL.

[0040] (3) Obtain an oral solution with a 25-hydroxyvitamin D3 concentration of 5 mg / mL, namely, pharmaceutical composition H1.

[0041] During use:

[0042] When used to treat porcine envelope virus, the dose of 25-hydroxyvitamin D3 is 2 mg / kg body weight (ie, the dose of oral solution is 0.4 mL / kg body weight), added 3 times a day, and the drug is used continuously for 7 days.

[0043] As a preventive measure for porcine envelope virus, the dose of 25-hydroxyvitamin D3 is 0.5 mg / kg body weight (i.e. the dose of oral solution is 0.1 mL / kg body weight) and is used throughout the entire feeding cycle of pigs.

[0044] Embodiment 2:

[0045] Application of 25-hydroxyvitamin D3 in preparing drugs for treating or preventing porcine envelope virus

[0046] The preparation of a drug, that is, the preparation of a pharmaceutical composition, comprises the following steps:

[0047] The same as Example 1, except that 2 g of 25-hydroxyvitamin D3 was weighed to obtain an oral solution with a 25-hydroxyvitamin D3 concentration of 20 mg / mL, namely, pharmaceutical composition H2.

[0048] During use:

[0049] When used to treat porcine envelope virus, the dose of 25-hydroxyvitamin D3 is 3 mg / kg body weight (ie, the dose of oral solution is 0.15 mL / kg body weight), added twice a day, and the drug is used continuously for 5 days.

[0050] As a preventive measure for porcine envelope virus, the dose of 25-hydroxyvitamin D3 is 0.5 mg / kg body weight (i.e. the dose of oral solution is 0.025 mL / kg body weight) and is used throughout the entire feeding cycle of pigs.

[0051] Embodiment 3:

[0052] Application of 25-hydroxyvitamin D3 in preparing drugs for treating or preventing porcine envelope virus

[0053] The preparation of a drug, that is, the preparation of a pharmaceutical composition, comprises the following steps:

[0054] (1) Weigh 25 g of 25-hydroxyvitamin D3, 25 g of lactose, and 50 g of soluble starch.

[0055] (2) 25-hydroxyvitamin D3, lactose and soluble starch are mixed and granulated, and then dried and granulated.

[0056] (3) Obtain granules with a mass fraction of 25% of 25-hydroxyvitamin D3, namely, pharmaceutical composition H3.

[0057] During use:

[0058] When used to treat porcine envelope virus, the dose of 25-hydroxyvitamin D3 is 2 mg / kg body weight (ie, the dose of granules is 0.008 g / kg body weight), added 3 times a day, and the drug is used continuously for 6 days.

[0059] When used to prevent porcine envelope virus, the dose of 25-hydroxyvitamin D3 is 0.6 mg / kg body weight (i.e. the dose of granules is 0.0024 g / kg body weight) and is used throughout the entire feeding cycle of pigs.

[0060] Embodiment 4:

[0061] Application of 25-hydroxyvitamin D3 in preparing drugs for treating or preventing porcine envelope virus

[0062] The preparation of a drug, that is, the preparation of a pharmaceutical composition, comprises the following steps:

[0063] The same as Example 3, except that 20 g of 25-hydroxyvitamin D3, 30 g of lactose, and 50 g of soluble starch were weighed, mixed, dried and granulated to obtain granules with a mass fraction of 20% of 25-hydroxyvitamin D3, namely, pharmaceutical composition H4.

[0064] During use:

[0065] When used to treat porcine envelope virus, the dose of 25-hydroxyvitamin D3 is 3 mg / kg body weight (ie, the dose of granules is 0.015 g / kg body weight), added twice a day, and the drug is used continuously for 7 days.

[0066] When used to prevent porcine envelope virus, the dose of 25-hydroxyvitamin D3 is 0.4 mg / kg body weight (i.e. the dose of granules is 0.002 g / kg body weight) and is used throughout the entire feeding cycle of pigs.

[0067] Embodiment 5:

[0068] Application of 25-hydroxyvitamin D3 in preparing drugs for treating or preventing porcine envelope virus

[0069] The preparation of a drug, that is, the preparation of a pharmaceutical composition, comprises the following steps:

[0070] The same as Example 3, except that 30 g of 25-hydroxyvitamin D3, 20 g of lactose, and 50 g of soluble starch were weighed, mixed, dried and granulated to obtain granules with a mass fraction of 25-hydroxyvitamin D3 of 30%, namely, pharmaceutical composition H5.

[0071] During use:

[0072] When used to treat porcine envelope virus, the dose of 25-hydroxyvitamin D3 is 3 mg / kg body weight (ie, the dose of granules is 0.01 g / kg body weight), added twice a day, and the drug is used continuously for 6 days.

[0073] When used to prevent porcine envelope virus, the dose of 25-hydroxyvitamin D3 is 0.6 mg / kg body weight (i.e. the dose of granules is 0.002 g / kg body weight) and is used throughout the entire feeding cycle of pigs.

[0074] Embodiment 6:

[0075] Application of 25-hydroxyvitamin D3 in preparing drugs for treating or preventing porcine envelope virus

[0076] The preparation of a drug, that is, the preparation of a pharmaceutical composition, comprises the following steps:

[0077] The same as Example 3, except that 25-hydroxyvitamin D3, lactose and soluble starch are mixed, compressed and dried using a tablet press to obtain tablets with a mass fraction of 25% of 25-hydroxyvitamin D3, namely, pharmaceutical composition H6.

[0078] Embodiment 7:

[0079] Application of 25-hydroxyvitamin D3 in preparing drugs for treating or preventing porcine envelope virus

[0080] The preparation of a drug, that is, the preparation of a pharmaceutical composition, comprises the following steps:

[0081] (1) Weigh 25 g of 25-hydroxyvitamin D3 and 75 g of soluble starch.

[0082] (2) Mix 25-hydroxyvitamin D3 and soluble starch evenly and pass through a 60-mesh sieve.

[0083] (3) Obtaining a powder with a mass fraction of 25% of 25-hydroxyvitamin D3, namely, pharmaceutical composition H7.

[0084] Embodiment 8:

[0085] Application of 25-hydroxyvitamin D3 in preparing drugs for treating or preventing porcine envelope virus

[0086] The preparation of a drug, that is, the preparation of a pharmaceutical composition, comprises the following steps:

[0087] The same as Example 7, except that 25 g of 25-hydroxyvitamin D3 and 75 g of bran were weighed, mixed and sieved to obtain a powder with a mass fraction of 25% of 25-hydroxyvitamin D3, namely, pharmaceutical composition H8.

[0088] 25-Hydroxyvitamin D3 can directly inhibit the gene expression of porcine envelope virus, improve the pig's own immunity, and further inhibit the gene expression of porcine envelope virus, thereby reducing the incidence and mortality rate of pigs.

[0089] Experiment 1

[0090] This application verifies the inhibitory effect of 25-hydroxyvitamin D3 on the proliferation of three porcine viruses, PEDV, PRRSV and ASFV, and the results show that 25-Hydroxyvitamin D3 can directly inhibit the gene expression of porcine envelope virus. The verification method is as follows:

[0091] 1. Materials and methods

[0092] 1.1 Viruses:

[0093] PEDV DR13 strain, PRRSV, and ASFV were isolated, preserved, and donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0094] 1.2 Cells:

[0095] African green monkey kidney cells (Vero), used for the proliferation of PEDV DR13 strain;

[0096] Monkey embryonic kidney epithelial cells (Marc-145) were used for the proliferation of PRRSV;

[0097] Porcine alveolar macrophages (primary cells) were used for the proliferation of ASFV.

[0098] Vero and Marc-145 cells are laboratory-maintained cell lines, and porcine alveolar macrophages were isolated from the lungs of healthy piglets.

[0099] 1.3 Trial drugs:

[0100] 25-Hydroxyvitamin D3: 25-Hydroxyvitamin D3 was provided by Hubei Haohua Biotechnology Co., Ltd.

[0101] 1.4 Reagents:

[0102] DMEM culture medium, EDTA, trypsin, fetal bovine serum and double antibody (penicillin-streptomycin) were purchased from GIBCO.

[0103] 1.5 In vitro proliferation inhibition:

[0104] (1) PEDV in vitro proliferation inhibition test:

[0105] All in vitro experiments were set up with three experimental groups and a blank control group. Different doses of 25-hydroxyvitamin D3 were added to the culture medium in the experimental groups, including a low-dose group of 25-hydroxyvitamin D3 (added at 5 μg / mL culture medium), a medium-dose group of 25-hydroxyvitamin D3 (added at 10 μg / mL culture medium), a high-dose group of 25-hydroxyvitamin D3 (added at 20 μg / mL culture medium), and a blank control group (no 25-hydroxyvitamin D3 was added to the culture medium). PEDV DR13 strain was used to infect Vero cells (MOI = 0.001), and different concentrations of 25-hydroxyvitamin D3 were added at the same time of infection. After 36 hours after infection, samples were collected and the effect of 25-hydroxyvitamin D3 on the in vitro proliferation of PEDV DR13 strain was determined by qPCR.

[0106] (2) PRRSV in vitro proliferation inhibition test: All in vitro tests were set up with three test groups and one blank control group. Different doses of 25-hydroxyvitamin D3 were added to the culture medium in the test groups, including a low-dose 25-hydroxyvitamin D3 group (added at 20 μg / mL culture medium), a medium-dose 25-hydroxyvitamin D3 group (added at 40 μg / mL culture medium), a high-dose 25-hydroxyvitamin D3 group (added at 80 μg / mL culture medium) and a blank control group (no 25-hydroxyvitamin D3 was added to the culture medium). Marc-145 cells were infected with PRRSV (MOI = 0.001), and different concentrations of 25-hydroxyvitamin D3 were added at the same time of infection (i.e., three test groups and one blank control group). After 36 hours of infection, samples were collected and the effect of 25-hydroxyvitamin D3 on the in vitro proliferation of PRRSV was determined by qPCR.

[0107] (3) ASFV in vitro proliferation inhibition test: All in vitro tests were set up with three test groups and one blank control group. Different doses of 25-hydroxyvitamin D3 were added to the culture medium in the test groups, including a low-dose group of 25-hydroxyvitamin D3 (added at 20 μg / mL culture medium), a medium-dose group of 25-hydroxyvitamin D3 (added at 40 μg / mL culture medium), a high-dose group of 25-hydroxyvitamin D3 (added at 80 μg / mL culture medium) and a blank control group (no 25-hydroxyvitamin D3 was added to the culture medium). ASFV was used to infect porcine alveolar macrophages (MOI = 0.001), and different concentrations of 25-hydroxyvitamin D3 were added at the same time of infection. After 36 hours of infection, samples were collected and the effect of 25-hydroxyvitamin D3 on the in vitro proliferation of ASFV was determined by qPCR.

[0108] 2. Experimental results:

[0109] 2.1 Effect of 25-hydroxyvitamin D3 on the proliferation of PEDV DR13 strain in Vero cells

[0110] The effects of different concentrations of 25-hydroxyvitamin D3 on the expression of N, M and S genes of PEDV are shown in Table 1.

[0111] Table 1

[0112]

[0113] The results in Table 1 show that adding different concentrations of 25-hydroxyvitamin D3 to the culture medium can significantly affect the expression levels of N, M and S genes of PEDV. As the concentration of 25-hydroxyvitamin D3 increases, the expression levels of N, M and S genes of PEDV decrease significantly, indicating that 25-hydroxyvitamin D3 can directly inhibit the proliferation of PEDV.

[0114] It should be noted that in qPCR experiments, the unit of measurement of gene expression is usually expressed as relative expression, and there is no specific unit. The expression of each gene (M, N, S) is expressed by comparing it with the blank control group. The expression of the blank control group is set to 1.000, and the expression of other groups is the multiple change relative to the blank control group to reflect the expression level relative to the blank control group.

[0115] 2.2 Effect of 25-hydroxyvitamin D3 on the proliferation of PRRSV in Marc-145 cells

[0116] The effects of different concentrations of 25-hydroxyvitamin D3 on the expression of ORF5 and ORF7 genes of PRRSV are shown in Table 2.

[0117] Table 2

[0118]

[0119] The results in Table 2 show that adding different concentrations of 25-hydroxyvitamin D3 to the culture medium can significantly affect the gene expression levels of ORF5 and ORF7 of PRRSV. As the concentration of 25-hydroxyvitamin D3 increases, the gene expression levels of ORF5 and ORF7 of PRRSV decrease significantly, indicating that 25-hydroxyvitamin D3 can directly inhibit the proliferation of PRRSV.

[0120] 2.3 Intervention effect of 25-hydroxyvitamin D3 on ASFV proliferation in porcine alveolar macrophages

[0121] The effects of different concentrations of 25-hydroxyvitamin D3 on the expression of P72 and CD2v genes of ASFV are shown in Table 3.

[0122] Table 3

[0123]

[0124] The results in Table 3 show that adding different concentrations of 25-hydroxyvitamin D3 to the culture medium can significantly affect the gene expression levels of P72 and CD2v of ASFV. As the concentration of 25-hydroxyvitamin D3 increases, the gene expression levels of P72 and CD2v of ASFV decrease significantly, indicating that 25-hydroxyvitamin D3 can directly inhibit the proliferation of ASFV.

[0125] Experiment 2

[0126] This application verifies the protective effect of 25-hydroxyvitamin D3 on piglets infected with porcine epidemic diarrhea virus (PEDV). 25-Hydroxyvitamin D3 can directly inhibit theGene expression , and can also improve the pig's Autoimmunity further inhibits the spread of PEDV Gene expression level, verification method is as follows:

[0127] Materials and Methods

[0128] 1. Test virus: PEDV YN strain

[0129] The PEDV YN strain was donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0130] 2.25-Hydroxyvitamin D3:

[0131] 25-Hydroxyvitamin D3 was provided by Hubei Haohua Biotechnology Co., Ltd.

[0132] 3. Experimental Animals:

[0133] 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 of 25-hydroxyvitamin D3) and a blank control group. 25-hydroxyvitamin D3 was dissolved in milk and the volume was made up to 3 mL for oral administration. Among them, there were 25-hydroxyvitamin D3 low-dose group (added at a dose of 0.5 mg / kg body weight), 25-hydroxyvitamin D3 medium-dose group (added at a dose of 1 mg / kg body weight) and 25-hydroxyvitamin D3 high-dose group (added at a dose of 2 mg / kg body weight) and a blank control group (fed with an equal dose of milk without adding 25-hydroxyvitamin D3), with 10 piglets in each group.

[0134] 4. Virus challenge test:

[0135] After one week of feeding, the piglets in each group were fed with 10 7 TCID 50 / mL of PEDV YN strain, 5.0mL was orally infused into each head, and 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.

[0136] 5. Observation indicators:

[0137] The diarrhea of ​​the experimental piglets was observed every day. At the end of the experiment, the experimental piglets were slaughtered to observe intestinal lesions, and the number of dead pigs was counted to calculate the diarrhea rate, mortality rate and protection rate. The inhibitory effect of 25-hydroxyvitamin D3 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.

[0138] 6. Data Analysis:

[0139] 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 of significant difference and P<0.01 as the standard of extremely significant difference. The results are expressed as mean values.

[0140] Experimental results:

[0141] After taking different doses of 25-hydroxyvitamin D3, the morbidity and mortality of piglets in each group are shown in Table 4.

[0142] Table 4

[0143] Group Diarrhea rate (%) mortality rate(%) Protection rate (%) Blank control group 100 100 0.0 Low dose group 80 60 40 Medium dose group 40 30 70 High dose group 20 0 100

[0144] The results in Table 4 show that adding different concentrations of 25-hydroxyvitamin D3 to piglet feed can significantly reduce the diarrhea rate and mortality rate of PEDV-infected piglets. Compared with the blank control group, the diarrhea rate of PEDV-infected piglets decreased by 20% to 80%, and the mortality rate of diseased pigs decreased by 40% to 100%. The experimental results fully demonstrate that the addition of the drug composition can significantly improve the protection rate of piglets against PEDV infection.

[0145] The PEDV structural genes in the intestinal mucosa of the jejunum, ileum, and colon tissues of piglets were detected respectively, and the detection results are shown in Table 5.

[0146] Table 5

[0147]

[0148] The results in Table 5 show that the addition of 25-hydroxyvitamin D3 can significantly reduce the expression levels of N, M and S genes of PEDV. The results fully demonstrate that the addition of 25-hydroxyvitamin D3 can significantly reduce the virulence of PEDV and inhibit the replication of PEDV in the jejunum, ileum and colon.

[0149] In addition, compared with the PEDV in vitro proliferation inhibition test, feeding infected piglets 25-hydroxyvitamin D3 has a better inhibitory effect on PEDV.

[0150] This is because, after entering the jejunum, ileum and colon, 25-hydroxyvitamin D3 can directly inhibit the expression of N, M and S genes of PEDV. When 25-hydroxyvitamin D3 is absorbed and digested by piglets, the content of autologous immunoglobulins in piglets is increased, thereby improving the autoimmunity of piglets and further inhibiting the expression of N, M and S genes of PEDV, thereby reducing the incidence and mortality rate of pigs.

[0151] Experiment 3

[0152] The present invention verifies the protective effect of 25-hydroxyvitamin D3 on piglets infected with porcine reproductive and respiratory syndrome virus (PRRSV). 3. Cocoa can improve the pig's own immunity to inhibit system The gene expression of PRRSV, 25-hydroxyvitamin D3 has a strong protective effect on the intestine, the verification method is as follows:

[0153] Materials and Methods

[0154] 1. Test virus: PRRSV

[0155] PRRSV was gifted by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0156] 2.25-Hydroxyvitamin D3:

[0157] 25-Hydroxyvitamin D3 was provided by Hubei Haohua Biotechnology Co., Ltd.

[0158] 3. Experimental Animals:

[0159] 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 of 25-hydroxyvitamin D3) and a blank control group. 25-hydroxyvitamin D3 was dissolved in milk and the volume was made up to 3 mL for oral administration. Among them, there were 25-hydroxyvitamin D3 low-dose group (added at a dose of 0.5 mg / kg body weight), 25-hydroxyvitamin D3 medium-dose group (added at a dose of 1 mg / kg body weight) and 25-hydroxyvitamin D3 high-dose group (added at a dose of 2 mg / kg body weight) and a blank control group (fed with an equal dose of milk without adding 25-hydroxyvitamin D3), with 10 piglets in each group.

[0160] 4. Virus challenge test:

[0161] After feeding for 1 week, the piglets in each group were fed with 10 5 TCID 50 / mL PRRSV challenge, each head was orally infused with 5.0mL, and the control group was infused with the same dose of normal saline, and then isolated and raised, and observed for 7 consecutive days until the end of the experiment.

[0162] 5. Observation indicators:

[0163] The diarrhea of ​​the experimental piglets was observed every day. At the end of the experiment, the experimental piglets were slaughtered to observe intestinal lesions. At the same time, the number of dead pigs was counted, and the diarrhea rate and mortality rate were calculated.

[0164] 6. Data Analysis:

[0165] 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 of significant difference and P<0.01 as the standard of extremely significant difference. The results are expressed as mean values.

[0166] Experimental Results

[0167] The morbidity and mortality rates of piglets in each group after taking different doses of 25-hydroxyvitamin D3 are shown in Table 6.

[0168] Table 6

[0169] Group Incidence (%) mortality rate(%) Protection rate (%) Blank control group 100.0 70.0 30.0 Low dose group 80.0 40.0 60.0 Medium dose group 30.0 20.0 80.0 High dose group 20.0 10.0 90.0

[0170] The results in Table 6 show that oral administration of different doses of 25-hydroxyvitamin D3 to piglets can significantly reduce the morbidity and mortality of piglets infected with PRRSV. Compared with the blank control group, with the increase of dose, the morbidity of piglets infected with PRRSV decreased by 20% to 80%; and the mortality rate of diseased pigs decreased by 30% to 60%. The experimental results fully demonstrate that the addition of 25-hydroxyvitamin D3 can significantly improve the protection rate of piglets against PRRSV infection.

[0171] The effects of different doses of 25-hydroxyvitamin D3 on the expression levels of ORF5 and ORF7 genes of PRRSV are shown in Table 7.

[0172] Table 7

[0173]

[0174] The results in Table 7 show that the addition of 25-hydroxyvitamin D3 can significantly reduce the expression levels of ORF5 and ORF7 genes of PRRSV. The results fully demonstrate that the addition of 25-hydroxyvitamin D3 can significantly reduce the virulence of PRRSV and inhibit the replication of PEDV in the brain and lungs.

[0175] In addition, compared with the PRRSV in vitro proliferation inhibition test, feeding infected piglets 25-hydroxyvitamin D3 at high doses has a better inhibitory effect on PRRSV. Although the inhibitory effect has improved, the increase is inferior to the increase in the PEDV in vitro proliferation inhibition test compared with the PEDV infected piglet protection test.

[0176] This is because, compared with PEDV, PRRSV mainly causes severe lung damage by invading alveolar macrophages. Piglets infected with PRRSV will have symptoms such as interstitial pneumonia and lymphadenopathy. PRRSV first replicates in local macrophages, then quickly spreads to lymph nodes and lungs, and sometimes to other tissues (heart, brain, kidneys, etc.).

[0177] That is, the main active site of PRRSV is not in the intestine, and the direct inhibitory effect of feeding 25-hydroxyvitamin D3 on PRRSV is inferior to its direct inhibitory effect on PEDV, that is, 25-hydroxyvitamin D3 has a stronger protective effect on the intestine.

[0178] In summary, the pharmaceutical 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.

[0179] 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. Application of 25-hydroxyvitamin D3 in the preparation of drugs for treating or preventing porcine envelope virus.

2. The use according to claim 1, characterized in that: The porcine envelope virus includes at least one of porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus and African swine fever virus.

3. The use according to claim 1, characterized in that: When treating porcine envelope virus, the dosage of 25-hydroxyvitamin D3 is 2 to 3 mg / kg body weight, added 2 to 3 times a day, and the drug is used continuously for 5 to 7 days.

4. The use according to claim 1, characterized in that: For the prevention of porcine envelope virus, the dosage of 25-hydroxyvitamin D3 is 0.4-0.6 mg / kg body weight, which is used throughout the entire breeding cycle of pigs.

5. A pharmaceutical composition, characterized in that Comprising 25-hydroxyvitamin D3 and auxiliary materials.

6. The pharmaceutical composition according to claim 5, characterized in that The auxiliary material comprises at least one of anhydrous ethanol, water, propylene glycol, dimethyl sulfoxide, edible oil, Tween 80, lactose, mannitol, soluble starch, montmorillonite, vermiculite, medical stone, vermiculite, zeolite powder, white carbon black, defatted rice bran, corn cob powder, starch and bran.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is set as a solution formed by mixing 25-hydroxyvitamin D3 with anhydrous ethanol, Tween-80 and water, wherein the concentration of 25-hydroxyvitamin D3 is 5 to 20 mg / ml.

8. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is configured as a solid mixture of 25-hydroxyvitamin D3, lactose and soluble starch, wherein the mass fraction of 25-hydroxyvitamin D3 in the solid mixture is 20-30%.

9. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is configured as a powdery mixture of 25-hydroxyvitamin D3 and soluble starch or bran, wherein the mass fraction of 25-hydroxyvitamin D3 in the powdery mixture is 20-30%.

10. Use of the pharmaceutical composition according to any one of claims 5 to 9 in the preparation of a drug for treating or preventing porcine envelope virus.

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