Application of organic zinc compound in preparation of medicine for inhibiting infection of different types of porcine reproductive and respiratory syndrome viruses

Abstract

The invention discloses application of an organic zinc compound in preparation of drugs for inhibiting infection of different types of porcine reproductive and respiratory syndrome viruses, and belongs to the technical field of veterinary biological products. Researches find that the organic zinc compounds zinc gluconate and zinc acetate are inhibitors capable of remarkably inhibiting infection of high-pathogenicity PRRSV (HP-PRRSV) and classical PRRSV attenuated strains (CH-1R), the PRRSV inhibiting effect of the organic zinc compounds and zinc acetate is superior to that of an inorganic zinc compound zinc sulfate monohydrate, PRRSV infection can be remarkably reduced, and the organic zinc compounds and zinc acetate can be developed into drugs for preventing and treating different types of PRRSV infection and have broad application prospects. Therefore, a brand new thought is provided for prevention and treatment of the PRRS, the research range is expanded, and the method is of great significance to actual production.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, specifically to the application of organozinc compounds in the preparation of drugs that inhibit infection by different types of porcine reproductive and respiratory syndrome virus. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV), also known as "blue ear disease." Its main characteristics include reproductive disorders in pregnant sows (abortion, stillbirth, mummified fetuses) and respiratory diseases in pigs of all ages, especially piglets, causing significant economic losses to the pig farming industry. PRRSV exhibits complex characteristics such as strain diversity, susceptibility to gene recombination, antibody-dependent enhancement (ADE), delayed neutralizing antibody effects, and immunosuppression, making disease control extremely difficult.

[0003] Currently, the prevention and control of porcine reproductive and respiratory syndrome (PRRS) relies primarily on vaccination. However, due to the genetic diversity, variability, and polysaccharide shielding characteristics of PRRSV, vaccine efficacy is limited, failing to provide complete protection. Furthermore, there is currently no specific antiviral drug for PRRSV; treatment is limited to symptomatic relief. Therefore, developing therapeutic drugs capable of inhibiting different types of PRRSV infection is of great significance for the prevention and control of PRRSV. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of organozinc compounds in the preparation of drugs that inhibit infection with different types of porcine reproductive and respiratory syndrome virus.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the invention provides the use of organozinc compounds in the preparation of medicaments that inhibit porcine reproductive and respiratory syndrome virus infection.

[0006] In the above applications, the organozinc compound is one or more of zinc gluconate and zinc acetate.

[0007] Preferably, the organozinc compound is composed of zinc gluconate and zinc acetate in a concentration ratio of 1:1.

[0008] In the above applications, the porcine reproductive and respiratory syndrome virus infection is one or more of the GFP-TA-12 recombinant virus and the CH-1R strain.

[0009] In the above applications, the concentration of organozinc compounds used is 50-200 μM.

[0010] In the above applications, in addition to the aforementioned active ingredients, the drug may also contain one or more pharmaceutically acceptable carriers. Examples include hydroxypropyl methylcellulose, methylcellulose, stearic acid, chitosan, silk fibroin, and liposomes.

[0011] Drugs can be formulated as injectable preparations, such as aqueous suspensions, oil suspensions, emulsions, or solutions; or as oral preparations, such as powders, tablets, capsules, or granules.

[0012] In a second aspect, the present invention provides a medicament for the prevention and / or treatment of porcine reproductive and respiratory syndrome virus infection, said medicament having zinc gluconate and zinc acetate as active ingredients.

[0013] Preferably, the concentration of zinc gluconate and zinc acetate in the drug is 50 μM.

[0014] The beneficial effects of this invention are: Zinc ions exert antiviral effects through mechanisms such as disrupting viral polypeptide synthesis, inhibiting viral genome translation and replication, interfering with viral fusion with host cell membranes, and promoting viral uncoating. Previous reports have identified inorganic zinc compounds (zinc oxide, zinc sulfate, zinc nitrate) for the prevention and treatment of PRRSV. However, organic zinc compounds (such as zinc acetate, zinc protein salts, and zinc gluconate) have higher bioavailability and lower gastrointestinal irritation compared to inorganic zinc compounds (such as zinc sulfate and zinc oxide), resulting in fewer side effects with long-term use. Compared to other organic zinc compounds (such as zinc citrate and zinc glycinate), zinc gluconate and zinc acetate have higher absorption rates and are relatively cheaper. Based on this, the organic zinc compound zinc gluconate was discovered. Compared with the inorganic zinc compound zinc sulfate monohydrate, zinc acetate is an inhibitor that can significantly inhibit the infection of highly pathogenic PRRSV (HP-PRRSV) and the classical attenuated PRRSV strain (CH-1R). It can significantly reduce PRRSV infection. The combination of the two drugs has been found to have better efficacy and effect. It can be developed into a drug for the prevention and treatment of different types of PRRSV infection, thus providing a new approach to the prevention and treatment of PRRS, expanding the research scope, and having extremely important significance for practical production. Attached Figure Description

[0015] Figure 1 Effects of zinc gluconate, zinc acetate, zinc sulfate monohydrate, and the combined use of zinc gluconate and zinc acetate on the activity of Marc-145 cells.

[0016] Effects of zinc sulfate monohydrate on Marc145 cell viability (A), effects of zinc gluconate on Marc145 cell viability (B), effects of zinc acetate on Marc145 cell viability (C), and effects of combined administration of zinc gluconate and zinc acetate on Marc145 cell viability (D).

[0017] Figure 2 Fluorescence screening and inhibition of zinc sulfate monohydrate against PRRSV.

[0018] Fluorescent screening of the antiviral effect of zinc sulfate monohydrate against PRRSV (A), the inhibitory effect of zinc sulfate monohydrate on viral N protein synthesis (B), and the inhibitory effect of zinc sulfate monohydrate on viral mRNA copy number (C).

[0019] Figure 3 Fluorescence screening and inhibition of the antiviral effect of combined zinc gluconate and zinc acetate against PRRSV.

[0020] Fluorescent screening of the antiviral effect of zinc gluconate and zinc acetate combination against PRRSV (A), the inhibitory effect of zinc gluconate and zinc acetate combination on viral N protein synthesis (B), and the inhibitory effect of zinc gluconate and zinc acetate combination on viral mRNA copy number (C).

[0021] Figure 4 : Zinc gluconate and zinc acetate, and TCID in viral supernatant treated with a combination of these drugs 50 .

[0022] Figure 5 Effects of zinc gluconate and zinc acetate on different PRRSV strains.

[0023] Inhibitory effects of zinc gluconate and zinc acetate on PRRSV strain CH-1R N protein synthesis (A), and the effect of zinc gluconate on TCID50 in the supernatant of PRRSV strain CH-1R progeny virus. 50 Effects of zinc acetate on TCID in the supernatant of PRRSV strain CH-1R progeny virus (B) 50 The impact (C). Detailed Implementation

[0024] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0025] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0026] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein: The PRRSV strains used in this invention include the GFP-TA-12 recombinant virus, described in Liu et al. 2015 (Virology Journal), and the CH-1R strain, described in Tian et al. 2009 (Vaccine) + China Veterinary Drug Registration. The public may obtain these strains from the applicant for use in replicating this invention within 20 years from the date of application.

[0027] The maintenance medium used in this invention is DMEM medium containing 2% fetal bovine serum.

[0028] Example 1: Cytotoxicity test 1. Test method: To test the effects of different concentrations of zinc ion compounds on the cell viability of Marc-145 cells, cytotoxicity tests were conducted on zinc sulfate monohydrate, zinc gluconate, zinc acetate, and a combination of zinc gluconate and zinc acetate. The procedures are as follows: (1) Marc145 cells were cultured at a density of 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL in 96-well cells culture plates. The cells were then incubated at 37°C with 5% CO2 for 24 h to allow them to grow into a monolayer.

[0029] (2) Zinc gluconate was diluted in the maintenance solution at concentrations of 0, 10, 50, 100, and 200 μM; zinc acetate at concentrations of 0, 10, 50, 1, and 100 μM; and zinc sulfate monohydrate at concentrations of 0, 10, 50, 100, and 200 μM. Zinc gluconate and zinc acetate were used in combination at a concentration ratio of 1:1, with the sum of their concentrations being 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, and 150 μM, respectively. Double-distilled water was added to the zero-addition group of the three zinc ion compounds. The diluted maintenance solutions containing the drugs were thoroughly mixed on a vortex mixer and set aside for later use.

[0030] (3) Remove the Marc-145 cells that have grown into a monolayer from the cell culture incubator, discard the original culture medium, and wash the cells twice with preheated PBS. Then add 100 μL of the test diluent to each 96-well cell culture plate, and set up control wells and blank wells, with 4 replicates for each group. Incubate the 96-well cell culture plates with the added diluent in a 37°C, 5% CO2 incubator for 36 h.

[0031] (4) The cytotoxicity of different drugs on Marc-145 cells was detected using the CCK-8 kit.

[0032] (5) Calculate the cell survival rate using the formula: Cell viability = (absorbance of test wells - absorbance of blank wells) / (absorbance of control wells - absorbance of blank wells) × 100%.

[0033] 2. Test Results: The results are as follows Figure 1 As shown, the addition of zinc gluconate in the range of 0–100 μM, zinc acetate in the range of 0–100 μM, zinc sulfate monohydrate in the range of 0–100 μM, and the addition of zinc gluconate and zinc acetate in combination in the range of 0–100 μM had no effect on the cell viability of Marc-145 cells and can be used for subsequent experiments.

[0034] Example 2: The effect of zinc sulfate monohydrate on viral replication 1. Test method: (1) Marc145 cells were cultured at a density of 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell / mL in 6-well cells culture plates, with 2 mL of cell suspension added to each well. The cells were then incubated at 37°C with 5% CO2 for 24 h to allow the cells to grow into a monolayer.

[0035] (2) Remove the 6-well cell culture plate from the incubator. Use a pipette to remove the original culture medium from the wells, wash the cells twice with PBS, and infect the PRRSV strain GFP-TA-12 with MOI=0.1. Place the plate in a 37°C incubator with 5% CO2 for 1 hour, and gently shake the cell plate every 15 minutes to ensure that the virus solution is in full contact with the cells for 1 hour.

[0036] (3) Dilute zinc sulfate monohydrate to a concentration of 100µM in the preheated maintenance solution; add an equal amount of H2O as a control; mix thoroughly with a vortex mixer for later use.

[0037] (4) One hour after cell infection with the virus, the cells were replaced with the maintenance solution containing zinc sulfate monohydrate prepared in step (3). Cells were observed under a fluorescence microscope at 0, 12, 24, 36, and 48 hours after virus infection. Cells were collected, and their inhibitory effect on the GFP-TA-12 strain was detected by Western blot. Cells were also collected, RNA was extracted, and reverse transcription was performed for quantitative real-time PCR to verify the viral titer in the cells. (The primers used for quantitative real-time PCR are shown in Table 1.)

[0038] Table 1: Primers used for quantitative fluorescence detection 2. Test Results: The results are as follows Figure 2 As shown, compared with the control group, the fluorescence effect in the experimental group gradually weakened with increasing culture time, and the difference in fluorescence between the experimental group and the control group was most obvious at 36h and 48h. Figure 2 A); Zinc sulfate monohydrate effectively inhibited the replication of GFP-TA-12 strain in Marc-145 cells at 36h and 48h post-infection, and the expression level of PRRSV N protein detected by Western blot showed an inhibitory effect compared with the control group. Figure 2 B).

[0039] With increasing culture time, quantitative real-time PCR showed that the increase in PRRSV viral copy number in cells was inhibited compared to the control group. Figure 2 C).

[0040] Example 3: Antiviral efficacy of zinc gluconate, zinc acetate, and the combination of zinc gluconate and zinc acetate. 1. Test method: (1) The Marc145 cells were cultured according to the method in Example 2 until the cells grew to a monolayer.

[0041] (2) Remove the 6-well cell culture plate from the incubator. Use a pipette to remove the original culture medium from the wells, wash the cells twice with PBS, and infect the PRRSV strain GFP-TA-12 with MOI=0.1. Place the plate in a 37°C incubator with 5% CO2 for 1 hour, and gently shake the cell plate every 15 minutes to ensure that the virus solution is in full contact with the cells for 1 hour.

[0042] (3) Zinc gluconate and zinc acetate were diluted in preheated maintenance solution at a concentration of 100µM and 100µM respectively. Zinc gluconate and zinc acetate were used in combination at a concentration ratio of 1:1, and the sum of their concentrations was 100µM (zinc gluconate 50µM + zinc acetate 50µM). An equal amount of H2O was added as a control. The mixture was thoroughly mixed with a vortex mixer and set aside.

[0043] (4) One hour after cell infection with the virus, the cells were replaced with the maintenance medium containing different organic zincs prepared in step (3). After 36 hours of virus infection, the cells were observed under a fluorescence microscope. Cells were collected and their inhibitory effect on the GFP-TA-12 strain was detected by Western blot. Cells were also collected, RNA was extracted, and reverse transcription was performed for quantitative real-time PCR to verify the viral titer in the cells. The primers used for quantitative real-time PCR were the same as those in Table 1. Cell supernatants from the mock group, zinc gluconate alone, zinc acetate alone, and combined treatment were collected, and the TCID of the virus was calculated using the Reed-Muench method. 50 .

[0044] 2. Test Results: The results are as follows Figure 3 As shown, the combined use of zinc gluconate and zinc acetate showed a significant difference in fluorescence compared to the control group at 36 h, and the effect was superior to that of the inorganic zinc compound zinc sulfate monohydrate under the same treatment conditions. Figure 3 A).

[0045] Western blot results showed that, compared with the control group, the expression levels of PRRSV N protein were significantly reduced in zinc gluconate alone, zinc acetate alone, and the combination of zinc gluconate and zinc acetate; and the combination of zinc gluconate and zinc acetate showed the best effect. Figure 3 B).

[0046] Zinc gluconate alone, zinc acetate alone, and the combination of zinc gluconate and zinc acetate all showed significant inhibitory effects on PRRSV viral replication in cells compared to the control group, as detected by quantitative real-time PCR. Furthermore, under the same treatment concentrations, the combination of zinc gluconate and zinc acetate significantly inhibited viral replication more effectively than either zinc gluconate alone or zinc acetate alone, indicating a synergistic effect between the two treatments on PRRSV viral replication. Figure 3 C).

[0047] The effects of zinc gluconate, zinc acetate, and the combination of zinc gluconate and zinc acetate on the progeny virus titers in cell supernatants are as follows: Figure 4As shown, the results indicate that compared with zinc gluconate treatment alone and zinc acetate treatment alone, the combined use of zinc gluconate and zinc acetate can more significantly reduce the viral titer of PRRSV, achieving a synergistic effect greater than the sum of its parts.

[0048] Example 4: Investigation of the inhibitory effects of zinc gluconate and zinc acetate on different types of PRRSV 1. Test method: (1) Marc145 cells were cultured at a density of 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell / mL in 6-well cells culture plates, with 2 mL of cell suspension added to each well. The cells were then incubated at 37°C with 5% CO2 for 24 h to allow the cells to grow into a monolayer.

[0049] (2) Remove the 6-well cell culture plate from the incubator. Use a pipette to remove the original culture medium from the wells, wash the cells twice with PBS, and infect them with PRRSV strain CH-1R at MOI=0.1. Place the plate at 37°C in a 5% incubator for 1 hour, and gently shake the cell plate every 15 minutes to ensure that the virus solution is in full contact with the cells for 1 hour.

[0050] (3) Prepare drug diluents by maintaining the concentration of zinc gluconate and zinc acetate at 100µM.

[0051] (4) After virus incubation, cell maintenance medium containing 100 µM zinc gluconate was added to the wells of cells infected with PRRSV strain CH-1R, and cell maintenance medium containing 100 µM zinc acetate was added to the wells of cells infected with PRRSV strain CH-1R. A control was set up with maintenance medium containing the same volume of H2O as the drug. The cells were incubated for another 36 h and samples were collected. The inhibitory effect of the drug on the synthesis of N protein of different PRRSV strains and the TCID of progeny virus supernatant were detected. 50 The impact.

[0052] 2. Test Results: The results are as follows Figure 5 As shown, zinc gluconate and zinc acetate at a concentration of 100 µM inhibited the expression of N protein and the progeny virus titer of the attenuated PRRSV strain CH-1R. This demonstrates that zinc gluconate and zinc acetate not only inhibit the activity of strong PRRSV strains but also the attenuated PRRSV strain CH-1R, indicating their broad applicability in antiviral activity against PRRSV.

[0053] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Application of organozinc compounds in the preparation of drugs that inhibit porcine reproductive and respiratory syndrome virus infection.

2. The application according to claim 1, characterized in that, The organozinc compound is one or more of zinc gluconate and zinc acetate.

3. The application according to claim 2, characterized in that, The organozinc compound is composed of zinc gluconate and zinc acetate in a concentration ratio of 1:

1.

4. The application according to claim 1, characterized in that, The porcine reproductive and respiratory syndrome virus infection is one or more of the following: GFP-TA-12 recombinant virus and CH-1R strain.

5. The application according to any one of claims 1-3, characterized in that, The concentration of organozinc compounds used is 50-200 μM.

6. The application according to claim 1, characterized in that, The drug also contains one or more pharmaceutically acceptable carriers.

7. The application according to claim 6, characterized in that, The pharmaceutically acceptable carrier is selected from hydroxypropyl methylcellulose, methylcellulose, stearic acid, chitosan, silk fibroin and / or liposomes.

8. The application according to claim 6, characterized in that, The dosage form of the drug is an aqueous suspension, an oil suspension, an emulsion, a powder, a tablet, a capsule, or a granule.

9. A drug for the prevention and / or treatment of porcine reproductive and respiratory syndrome virus infection, characterized in that, The drug uses zinc gluconate and zinc acetate as its active ingredients.

10. The medicament according to claim 9, characterized in that, The concentration of zinc gluconate in the drug is 50 μM, and the concentration of zinc acetate is 50 μM.

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