Effect of ebseoline in preparation of medicine for preventing and treating porcine virus infection

Abstract

The invention discloses an effect of ebseoline in preparation of a medicine for preventing and treating porcine virus infection. According to the invention, a PRRSV strain HUN4 is used as a representative strain for research, and the result shows that Ebselen has the activity of resisting PRRSV infection. On African green monkey embryonic kidney cells, compared with a control group, the Ebseen treatment can significantly reduce PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) infection. In addition, the Ebseen has an obvious inhibition effect on PRRSV strains of different pedigree and common porcine coronaviruses such as PEDV (porcine epidemic diarrhea virus), PDCoV (PDCoV) and TGEV (transmissible gastroenteritis virus). Therefore, the small molecule compound can be used as a medicine or a feed additive for inhibiting infection of important porcine viruses PRRSV, PEDV, PDCoV and TGEV.

Description

Technical Field

[0001] The invention relates to the technical field of compound application, and in particular to the role of ebselen in preparing medicines for preventing and treating porcine viral infection. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ear disease, is caused by infection with porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV belongs to the genus Arterivirus of the family Arteriviridae of the order Nidovirales. The family also includes equine arteritis virus (EAV), lactate dehydrogenase hyperinfection virus (LDV), and simian hemorrhagic fever virus (SHFV). PRRSV is divided into two genotypes, namely genotype II represented by VR-2332, which was first discovered in North America in the late 1980s, and genotype I represented by LV (Lelystad Virus), which first appeared in Europe in the early 1990s. However, there is only 55-70% nucleotide homology and 50-80% amino acid homology between the two genotypes.

[0003] PRRSV has the characteristics of high variability and immunosuppression, making it difficult for infected pigs to produce sufficient immune protection to fight the virus. Existing protective measures are difficult to effectively eliminate and control PRRSV mutant strains, bringing great difficulties to the pig farming industry.

[0004] Ebselen, also known as ebselen, CAS number: 60940-34-3, has a chemical structure as shown in Formula 1. Ebselen is a new type of chemically synthesized non-steroidal selenium-containing drug with good anti-inflammatory effects. However, there is no relevant report on whether ebselen can resist porcine viral infection.

[0005] (Formula 1) Summary of the invention

[0006] A variety of live vaccines and inactivated vaccines of PRRSV have been widely used. However, due to the high variability and strong recombination ability of PRRSV, the protectiveness of the vaccine is very limited, and the spread and prevalence of PRRSV cannot be effectively controlled. At present, no specific therapeutic drug for PRRSV has been found. Once pigs are infected with PRRSV, there is no specific drug that can completely eliminate the virus or effectively inhibit its replication except for taking some supportive therapies and symptomatic treatments to relieve the symptoms. This situation undoubtedly brings great challenges to the prevention and control of PRRSV. In order to overcome the problems existing in the prior art, the object of the present invention is to provide the effect of ebselen in the preparation of drugs for preventing and treating porcine viral infections.

[0007] The present invention discovers for the first time that ebselen can effectively resist PRRSV infection and significantly reduce the infection rate and morbidity of PRRSV, thereby more effectively preventing and controlling PRRSV and reducing its impact on the breeding industry and agricultural economy.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is: The invention discloses an application of ebselen in the preparation of medicine or feed additive for preventing and / or treating porcine viral diseases, wherein the disease comprises at least one of porcine blue ear disease and porcine epidemic diarrhea.

[0009] Furthermore, the porcine reproductive and respiratory syndrome virus (PRRSV) includes porcine reproductive and respiratory syndrome virus (PRRSV) caused by PRRSV.

[0010] Furthermore, the porcine epidemic diarrhea includes porcine epidemic diarrhea caused by porcine epidemic diarrhea virus PEDV, porcine deltacoronavirus PDCoV and / or porcine transmissible gastroenteritis virus TGEV.

[0011] The invention discloses an application of ebselen in the preparation of a drug for resisting porcine virus infection, wherein the virus comprises at least one of porcine reproductive and respiratory syndrome virus PRRSV, porcine epidemic diarrhea virus PEDV, porcine deltacoronavirus PDCoV and porcine transmissible gastroenteritis virus TGEV.

[0012] Furthermore, the anti-swine virus infection comprises at least one of inhibiting viral gene expression, inhibiting viral protein expression and inhibiting the production of progeny virus particles.

[0013] Furthermore, the viral gene includes the N gene, and the viral protein includes the N protein.

[0014] The invention discloses an application of ebselen in the preparation of anti-swine virus drugs, wherein the viruses include at least one of porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and porcine transmissible gastroenteritis virus (TGEV).

[0015] Furthermore, the anti-swine virus drug is a porcine virus replication inhibitor.

[0016] Furthermore, the porcine reproductive and respiratory syndrome virus PRRSV includes at least one of the HUN4 strain, JX strain, HN07-1 strain, CH-1R strain, BJ4 strain and HNhx strain.

[0017] Furthermore, the porcine deltacoronavirus PDCoV includes the HKU15 strain.

[0018] Furthermore, the porcine epidemic diarrhea virus PEDV includes the CV777 strain.

[0019] Furthermore, the porcine transmissible gastroenteritis virus TGEV includes the H165 strain.

[0020] Furthermore, the effective concentration of ebselen is 2.5-20 μmol / L.

[0021] Furthermore, the ebselen includes ebselen and a pharmaceutically acceptable salt thereof.

[0022] Furthermore, the medicine or feed additive comprises ebselen and at least one pharmaceutically acceptable excipient.

[0023] Furthermore, the dosage form of the drug is a gastrointestinal dosage form or an injection dosage form.

[0024] Furthermore, the gastrointestinal tract administration dosage form is tablets, capsules, granules, pills or pellets.

[0025] Compared with the prior art, the present invention has the following advantages and effects: The present invention explores the new disease treatment field of the existing drug Ebselen. In view of the fact that the safety and effectiveness of Ebselen have been verified to a certain extent, we conduct in-depth research on this basis in order to discover its wider application potential. The present invention preliminarily evaluates the activity of Ebselen against porcine reproductive and respiratory syndrome virus (PRRSV), and selects the highly pathogenic PRRSV (HP-PRRSV) strain HUN4 (GenBank: EF635006.1) as a representative strain for research. The results show that Ebselen has anti-PRRSV infection activity. In African green monkey embryonic kidney cells (Marc145), Ebselen treatment can significantly reduce PRRSV infection compared with the control group. In addition, Ebselen has a significant inhibitory effect on PRRSV strains of different lineages and common porcine coronaviruses such as PEDV, PDCoV and TGEV. Ebselen showed excellent anti-PRRSV activity, providing new ideas and effective response strategies for the prevention and control of PRRSV. This study has important scientific significance for strengthening the prevention and control of PRRSV epidemics. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the results of a study on the effect of Ebselen on the cell viability of Marc145 cells.

[0027] Figure 2This is a graph showing the results of a study on the effect of Ebselen in inhibiting PRRSV RNA levels.

[0028] Figure 3 This is a graph showing the results of a study on the effect of Ebselen in inhibiting the expression of PRRSV proteins.

[0029] Figure 4 This is a graph showing the research results on Ebselen's inhibition of PRRSV progeny virus production.

[0030] Figure 5 This is a graph showing the results of a study on the effect of pre-incubation of Ebselen with PRRSV on the RNA level of PRRSV.

[0031] Figure 6 This is a fluorescence quantitative PCR detection of the inhibitory effect of Ebselen on PRRSV at different treatment times.

[0032] Figure 7 This is a diagram showing the inhibitory effect of Ebselen on different lineages of PRRSV.

[0033] Figure 8 This is a graph showing the results of a study on the effect of Ebselen on the cell viability of Vero E6 cells.

[0034] Fig. 9 This is a graph showing the results of a study on the effect of Ebselen on the cell viability of PK15 cells.

[0035] Fig.10 This is a study result showing that Ebselen inhibits the RNA levels of PEDV, PDCoV and TGEV. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention. Rather than all the embodiments, all other embodiments obtained by ordinary technicians in this field without innovative work based on the embodiments of the present invention are within the scope of protection of the present invention.

[0037] The PRRSV viruses used in the following examples include HUN4 strain (Lineage 8, GenBank: EF635006.1), JX strain (Lineage 8, GenBank: JX317649.1), HN07-1 strain (Lineage 8, GenBank: KX766378.1), CH-1R strain (Lineage 8, GenBank: EU807840.1), BJ-4 strain (Lineage 5, GenBank: AF331831.1) and HNhx strain (Lineage 1, GenBank: KX766379.1), which are deposited in the Wang Yu'e research group of the College of Veterinary Medicine, Southwest University. Among them, the HUN4 strain is used in Examples 2-5.

[0038] The PEDV virus used in the following examples is the CV777 (GenBank: AF353511.1) strain, which is deposited in the research group of Wang Yu'e, College of Veterinary Medicine, Southwest University; The PDCoV virus used in the following examples is the HKU15 (GenBank: KM012168.1) strain, which is deposited in the research group of Wang Yu'e, College of Veterinary Medicine, Southwest University; The TGEV virus used in the following examples is the H165 strain, which is preserved in the research group of Wang Yu'e at the College of Veterinary Medicine of Southwest University and has been published in the document "Autophagy Negatively Regulates Transmissible Gastroenteritis Virus Replication".

[0039] Example 1: Effect of Ebselen on cell viability of Marc145 cells Marc145 cells (African green monkey embryonic kidney cells) were inoculated into 96-well plates. When the cell density reached 70%, the culture medium was replaced with 10% FBS DMEM medium containing Ebselen (containing 100 U / mL penicillin and 50 μg / mL streptomycin). The concentrations of Ebselen were 200 μmol / L, 100 μmol / L, 50 μmol / L, 25 μmol / L, 12.5 μmol / L, 6.25 μmol / L, 3.125 μmol / L and 1.5625 μmol / L, respectively. A control group and a blank group were set up. After 24 hours of culture, the culture medium was discarded, and 10 μL CCK8 was added to each well. After further culture for 1.5 hours, the absorbance at 450 nm was measured using an enzyme reader and substituted into the cell viability calculation formula. The cell viability calculation formula is: Cell viability = [OD(drug added) - OD(blank)] / [OD(0 drug added) - OD(blank)] × 100%, and finally the effect of Ebselen on cell activity was calculated using GraphPad Prism software.

[0040] The results are as follows Figure 1 As shown in the figure, Ebselen concentration below 25 μmol / L had no significant effect on cell viability.

[0041] Example 2: Ebselen inhibits PRRSV RNA levels Marc145 cells were inoculated into 12-well plates. When the cell density reached 70%, PRRSV was infected at a dose of MOI = 0.1 for 1 hour, and then the culture medium was changed to 2% FBS DMEM medium supplemented with 20 μmol / L, 10 μmol / L, 5 μmol / L, 2.5 μmol / L and 1.25 μmol / L Ebselen for 24 hours, and the samples were collected. The effects of drugs on viral RNA levels were detected by RNA extraction, reverse transcription and qPCR.

[0042] RNA extraction: Total RNA from cell culture was extracted using the Simply P total RNA extraction kit from Hangzhou Bioer Technology Co., Ltd.

[0043] Reverse transcription: TAKARA's PrimeScript RT Master Mix reagent was used for reverse transcription. The reaction conditions were: 37°C for 15 min, 85°C for 5 s, and stored at 4°C.

[0044] Fluorescence quantitative PCR: Specific detection primers were designed for the N gene of PRRSV, and the ChamQUniversal SYBR qPCR Master Mix reagent of Novozymes was used for fluorescence quantitative PCR test. The reaction conditions were: 95°C for 30s; 95°C for 10s, 60°C for 30s annealing, and 40 cycles; the fluorescence signal was collected in this step.

[0045] The results are as follows Figure 2 As shown in the figure, after treatment with 1.25 μmol / L, 2.5 μmol / L, 5 μmol / L, 10 μmol / L and 20 μmol / L of Ebselen, the RNA level of PRRSV was 0.86, 0.50, 0.25, 0.17 and 0.11 times that of the control group. The results show that Ebselen inhibits the RNA level of PRRSV in a dose-dependent manner.

[0046] Example 3: Ebselen inhibits protein expression of PRRSV Marc145 cells were seeded into 12-well plates. When the cell density reached 70%, PRRSV was infected at a dose of MOI = 0.1 for 1 h, and then the medium was replaced with 2% FBS DMEM medium supplemented with 20 μmol / L, 10 μmol / L, 5 μmol / L, 2.5 μmol / L and 1.25 μmol / L Ebselen and continued to be cultured for 24 h, and samples were collected. The effects of drugs on viral N protein expression were detected by collecting cells, lysing proteins and Western blot.

[0047] The results are as follows Figure 3 As shown in the figure, after treatment with 1.25 μmol / L, 2.5 μmol / L, 5 μmol / L, 10 μmol / L and 20 μmol / L of Ebselen, the expression levels of PRRSV N protein were 1.14, 1.05, 0.76, 0.41 and 0.34 times that of the control group. The results show that Ebselen inhibits the protein expression of PRRSV in a dose-dependent manner.

[0048] Example 4: Ebselen inhibits the production of PRRSV progeny viruses Marc-145 cells were cultured at 3 × 10 5Each well was inoculated in a 24-well plate. After the cells adhered to the wall, the culture medium in the plate was discarded and infected with PRRSV HuN-4 strain with MOI=0.1. One hour after infection, the virus solution was discarded and replaced with 2% FBS DMEM containing 20 μmol / L, 10 μmol / L, 5 μmol / L, 2.5 μmol / L and 1.25 μmol / L Ebselen. After culturing in a 37℃, 5% CO2 incubator for 5 days, the positive rate of cytopathic effect in each well was observed and recorded. The virus titer (TCID50) value in the cell culture medium of each test group was calculated by the Reed-Muench method.

[0049] The results are as follows Figure 4 As shown, the TCID of PRRSV was significantly increased after treatment with 1.25 μmol / L, 2.5 μmol / L, 5 μmol / L, 10 μmol / L and 20 μmol / L of Ebselen. 50 The concentrations of 10^7.18 / ml decreased to 10^6.26, 10^6.03, 10^5.56, 10^4 and 10^3.65 / ml respectively. The results showed that Ebselen inhibited the production of PRRSV progeny viruses in a dose-dependent manner.

[0050] Example 5: Ebselen has no direct effect in killing PRRSV DMEM culture medium containing 10 μmol / L, 20 μmol / L, and 40 μmol / L Ebselen was incubated with MOI=0.1 PRRSV at room temperature for 2 h, and then the cells were co-inoculated for 1 h. After replacing the complete culture medium and continuing to culture for 12 h, RNA was extracted to detect the difference in N gene expression of PRRSV.

[0051] The results are as follows Figure 5 As shown, pre-incubation of Ebselen with PRRSV had no significant effect on the RNA level of PRRSV, proving that Ebselen has no direct effect of killing PRRSV.

[0052] Example 6: Ebselen mainly plays a role in the PRRSV replication stage Marc145 cells were seeded into 12-well plates. When the cell density reached 70%, PRRSV was infected at a dose of MOI=0.1 and replaced with 2% FBS DMEM medium (containing penicillin 100 U / mL, streptomycin 50 μg / mL) containing 10 μmol / LEbselen after 1 h. The cells were collected at 1h, 2h, 3h, 6h, 12h and 24h, and RNA was extracted. The effects of different treatment times on the expression of PRRSV N gene were detected by fluorescence quantitative PCR.

[0053] The results are as follows Figure 6 As shown, the treatment groups showed significant inhibition of PRRSV 12h and 24h after infection, confirming that Ebselen mainly exerts its inhibitory effect in the replication stage of PRRSV.

[0054] Example 7: Ebselen has a significant inhibitory effect on different lineages of PRRSV Marc145 cells were inoculated into 12-well plates. When the cell density reached 70%, different PRRSV strains were infected at a dose of MOI=0.1, namely HUN4 (Lineage 8), JX (Lineage 8), HN07-1 (Lineage 8), CH-1R (Lineage 8), BJ4 (Lineage 5) and HNhx (Lineage 1). After 1 hour, the culture medium was replaced with complete medium supplemented with 10 μmol / L Ebselen. After 24 hours, samples were collected to determine the effect of Ebselen on different PRRSV strains.

[0055] The results are as follows Figure 7 As shown, Ebselen has a significant inhibitory effect on different PRRSV strains.

[0056] Example 8: Ebselen has a significant inhibitory effect on common porcine coronaviruses Vero E6 cells (African green monkey kidney cells) were inoculated into 96-well plates. When the cell density reached 70%, the culture medium was replaced with 10% FBS DMEM medium containing Ebselen (containing 100 U / mL penicillin and 50 μg / mL streptomycin). The concentrations were 20 μmol / L, 10 μmol / L, 5 μmol / L, 2.5 μmol / L, 1.25 μmol / L, 0.625 μmol / L, and 0.3125 μmol / L, respectively. A control group and a blank group were set up. After culturing for 24 hours, the culture medium was discarded, and 10 μL CCK8 was added to each well. After continuing to culture for 1.5 hours, the absorbance at 450 nm was measured using an enzyme reader and substituted into the cell viability calculation formula. The cell viability calculation formula is: Cell viability = [OD(drug added) - OD(blank)] / [OD(0 drug added) - OD(blank)] × 100%. Finally, GraphPad The effect of Ebselen on cell viability was calculated by Prism software.

[0057] The results are as follows Figure 8 As shown in the figure, Ebselen concentration below 20 μmol / L had no significant effect on cell viability.

[0058] PK15 cells (porcine kidney cells) were inoculated into 96-well plates. When the cell density reached 70%, the culture medium was replaced with 10% FBS DMEM medium containing Ebselen (containing 100 U / mL penicillin and 50 μg / mL streptomycin) at concentrations of 20 μmol / L, 10 μmol / L, 5 μmol / L, 2.5 μmol / L, 1.25 μmol / L, and 0.625 μmol / L, respectively. A control group and a blank group were set up. After culturing for 24 hours, the culture medium was discarded, and 10 μL CCK8 was added to each well. After continuing to culture for 1.5 hours, the absorbance at 450 nm was measured using a microplate reader and substituted into the cell viability calculation formula. The cell viability calculation formula is: cell viability = [OD(drug added) - OD(blank)] / [OD(0 drug added) - OD(blank)] × 100%. Finally, GraphPad Prism software was used to calculate the effect of Ebselen on cell activity.

[0059] The results are as follows Fig. 9 As shown in the figure, Ebselen concentration below 20 μmol / L had no significant effect on cell viability.

[0060] Vero E6 cells were inoculated into 12-well plates. When the cell density reached 70%, the PEDVCV777 strain was infected with MOI=0.1 for 1 h, and then the medium was replaced with 2% FBS DMEM medium supplemented with 10 μmol / L Ebselen for 24 h, and the samples were collected. The effects of drugs on PEDV RNA levels were detected by RNA extraction, reverse transcription, and qPCR; PK15 cells were inoculated into 12-well plates. When the cell density reached 70%, the PDCoV HKU15 strain was infected with MOI=0.1 for 1 h, and then the medium was replaced with 2% FBS DMEM medium supplemented with 10 μmol / L Ebselen for 24 h, and the samples were collected. RNA extraction, reverse transcription and qPCR were used to detect the effects of drugs on PDCoV RNA levels; PK15 cells were inoculated into 12-well plates, and when the cell density reached 70%, they were infected with TGEVH165 strain at a dose of MOI=0.1 for 1 hour, and then the culture medium was replaced with 10 μmol / L Ebselen and cultured for 24 hours, and samples were collected. RNA extraction, reverse transcription and qPCR were used to detect the effects of drugs on TGEV RNA levels.

[0061] Test results such as Fig.10As shown in the figure, after treatment with 10 μmol / L Ebselen, the RNA levels of PEDV, PDCoV and TGEV were 0.217, 0.123 and 0.475 times that of the control group. This result shows that Ebselen significantly inhibits the RNA levels of PEDV, PDCoV and TGEV.

[0062] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Use of ebselen in the preparation of a medicine or feed additive for preventing and / or treating porcine viral diseases, characterized in that: The disease includes at least one of porcine blue ear disease and porcine epidemic diarrhea.

2. Use of ebselen according to claim 1 in the preparation of a medicine or feed additive for preventing and / or treating porcine viral diseases, characterized in that: The porcine reproductive and respiratory syndrome virus (PRRSV) is a porcine reproductive and respiratory syndrome virus (PRRSV). The porcine epidemic diarrhea includes porcine epidemic diarrhea caused by porcine epidemic diarrhea virus PEDV, porcine deltacoronavirus PDCoV and / or porcine transmissible gastroenteritis virus TGEV.

3. The use of ebselen in the preparation of a drug for resisting porcine virus infection, characterized in that: The virus includes at least one of porcine reproductive and respiratory syndrome virus PRRSV, porcine epidemic diarrhea virus PEDV, porcine deltacoronavirus PDCoV and porcine transmissible gastroenteritis virus TGEV.

4. The use according to claim 3, characterized in that: The anti-swine virus infection comprises at least one of inhibiting viral gene expression, inhibiting viral protein expression and inhibiting the production of progeny virus particles.

5. The use of ebselen in the preparation of anti-swine virus drugs, characterized in that: The virus includes at least one of porcine reproductive and respiratory syndrome virus PRRSV, porcine epidemic diarrhea virus PEDV, porcine deltacoronavirus PDCoV and porcine transmissible gastroenteritis virus TGEV.

6. The use according to claim 5, characterized in that: The anti-swine virus drug is a pig virus replication inhibitor.

7. The use according to any one of claims 3 to 6, characterized in that: The porcine reproductive and respiratory syndrome virus PRRSV comprises at least one of HUN4 strain, JX strain, HN07-1 strain, CH-1R strain, BJ4 strain and HNhx strain; The porcine deltacoronavirus PDCoV includes the HKU15 strain; The porcine epidemic diarrhea virus PEDV includes the CV777 strain; The porcine transmissible gastroenteritis virus TGEV includes the H165 strain.

8. The use according to any one of claims 3 to 6, characterized in that: The effective concentration of ebselen is 2.5-20 μmol / L.

9. The use according to any one of claims 3 to 6, characterized in that: The ebselen includes ebselen and a pharmaceutically acceptable salt thereof; The medicine or feed additive comprises ebselen and at least one pharmaceutically acceptable excipient; The dosage form of the medicine is a gastrointestinal administration dosage form or an injection dosage form.

10. The use according to claim 9, characterized in that: The gastrointestinal tract administration dosage form is tablets, capsules, granules, pills or dripping pills.

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