Application of sclareol in preparation of medicine for resisting porcine reproductive and respiratory syndrome virus

Abstract

The invention discloses application of sclareol in preparation of drugs for resisting porcine reproductive and respiratory syndrome virus, and belongs to the field of veterinary drugs. According to the invention, Marc-145 cells are taken as host cells, a plurality of porcine reproductive and respiratory syndrome virus strains are selected for infection, and the antiviral performance of sclareol is tested; experimental results show that 30-50 [mu] M sclareol has no obvious toxicity to host cells, and can significantly inhibit infection of different porcine reproductive and respiratory syndrome virus strains in the host cells. The sclareol has good activity of resisting porcine reproductive and respiratory syndrome virus infection, and the sclareol is a medicine with a natural source, has the advantages of safety, few side effects, low metabolic residue and no pollution, and provides a reliable basis for treatment, prevention and control of porcine reproductive and respiratory syndrome.

Description

Technical Field

[0001] The invention relates to the field of veterinary medicine, in particular to the application of sclareol in the preparation of anti-porcine reproductive and respiratory syndrome virus medicine. Background Art

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is an acute infectious disease caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). PRRSV replicates mainly in pig alveolar macrophages and lymphoid organs, and can induce long-term viremia and persistent infection. PRRSV can infect pigs of different ages and breeds. Infected piglets mainly show respiratory symptoms, while pregnant sows may experience symptoms such as premature birth, abortion, stillbirth and mummification.

[0003] PRRSV belongs to the family Arteriviridae of the order Nidovirales and is a single-stranded positive-strand RNA virus. The virus particles are spherical or elliptical, with a diameter of about 50-60nm and a smooth surface. Its genomic RNA is wrapped by nucleocapsid protein, and the surface glycoprotein and membrane protein are embedded in the lipid bilayer membrane to form a complete virus particle. The in vitro survival ability of PRRSV is affected by factors such as temperature, pH value and lipid solvent. The total length of the PRRSV genome is about 15.4kb, containing multiple open reading frames (ORFs), namely ORF1 to ORF7. Among them, ORF1a and ORF1b are the largest open reading frames in the genome, accounting for about 75% of the entire genome, and mainly encode proteins related to RNA replication and transcription. ORF2a, ORF2b, ORF3 to ORF7 and ORF5a encode GP2a, GP2b, GP3, GP4, GP5, M membrane protein and N nucleocapsid protein, respectively.

[0004] PRRSV can be divided into two genotypes: European PRRSV (PRRSV-1, based on Lelystad) and North American PRRSV (PRRSV-2, based on VR-2332), with a nucleotide homology of about 60%. Due to the high variability and strong recombination ability of PRRSV-1, the risk of producing new strains after infection is high, so more attention is needed. Although vaccination is the main strategy for preventing and controlling PRRSV infection, there is currently no authorized vaccine for PRRSV-1 in my country, and the existing PRRSV-2 vaccine cannot provide effective cross-immunity protection for pigs infected with PRRSV-1 strains. Therefore, antiviral drugs play a key role in the prevention and control of PRRS. Further research is still urgently needed to develop effective antiviral strategies to reduce the economic losses caused by virus transmission.

[0005] Sclareol, also known as sclareol, is a diterpenoid compound mainly derived from the capitula of Sclareol and tobacco leaves. As a natural product, sclareol has a variety of biological activities, including anti-inflammatory, bactericidal and anti-cancer. In addition, sclareol can also be used as an industrial fragrance and a raw material for the synthesis of ambergris. At present, there are few studies on the antiviral effects of sclareol, and there are no reports on the inhibition of porcine reproductive and respiratory syndrome virus. Summary of the invention

[0006] The purpose of the present invention is to provide an application of sclareol in the preparation of anti-porcine reproductive and respiratory syndrome virus drugs to solve the problems existing in the above-mentioned prior art. In Marc-145 cells, 30-50 μM sclareol has a significant inhibitory effect on the infection of different porcine reproductive and respiratory syndrome virus strains.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The invention provides application of sclareol in preparing products for resisting porcine reproductive and respiratory syndrome virus.

[0009] Preferably, the invention comprises use in preparing products for resisting porcine reproductive and respiratory syndrome virus infection.

[0010] Preferably, the concentration of sclareol in the product is 30-50 μM.

[0011] Preferably, the concentration of sclareol in the product is 50 μM.

[0012] The invention also provides the use of sclareol in preparing a medicine for treating porcine reproductive and respiratory syndrome.

[0013] Preferably, the porcine reproductive and respiratory syndrome is a porcine respiratory infectious disease caused by porcine reproductive and respiratory syndrome virus infection.

[0014] Preferably, the use comprises inhibiting the replication of porcine reproductive and respiratory syndrome virus and / or improving the survival rate of pigs infected with porcine reproductive and respiratory syndrome virus.

[0015] Preferably, the concentration of sclareol in the drug is 30-50 μM.

[0016] The present invention discloses the following technical effects:

[0017] The present invention develops the antiviral effect of sclareol on porcine reproductive and respiratory syndrome virus infection. As a potential antiviral drug, sclareol has the following advantages compared with traditional chemical antiviral drugs:

[0018] 1. Safe, with few side effects: Sclarea alcohol is a natural extract derived from Sclarea, and its main component is diterpene diterpenoid alcohol. Unlike hormones, antibiotics and chemical synthetic drugs, it has no obvious toxic side effects on the body's physiological functions.

[0019] 2. Low drug residue and no pollution; the drug is easily metabolized and absorbed by the animal body, and the animal excrement does not pollute the environment.

[0020] 3. Significant antiviral activity: At a concentration of 50 μM, clarysol can significantly inhibit the infection of porcine reproductive and respiratory syndrome virus in Marc-145 cells.

[0021] 4. Potential clinical application value: This antiviral drug has good anti-porcine reproductive and respiratory syndrome virus infection activity, providing a reliable basis for the treatment and prevention of diseases caused by the virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The results of the safe concentration determination of Marc-145 cells with different concentrations of sclareol; CellViability is cell viability, ns means P>0.05, * means P<0.05, *** means P<0.001;

[0024] Figure 2The results of the activity of sclareol in inhibiting PRRSV1-DV-EGFP virus infection in Marc-145 cells are shown in Figure 1; A is a fluorescence image; B is a TCID 50 Test results; C is the absolute fluorescence quantitative test result; Titer is titer, RNACopies is the viral RNA content, ns means P>0.05, * means P<0.05, *** means P<0.001;

[0025] Figure 3 The results of the activity of sclareol in inhibiting HP-PRRSV virus infection on Marc-145 cells are shown in Figure 1; A is a fluorescence image; B is a TCID 50 Test results; C is the absolute fluorescence quantitative test result; Titer is titer, RNACopies is the viral RNA content, ns means P>0.05, ** means P<0.01, *** means P<0.001;

[0026] Figure 4 The results of the activity of sclareol in inhibiting NADC30 virus infection in Marc-145 cells are shown in Figure 1. A is a fluorescence image; B is a TCID 50 Detection results; C is the absolute fluorescence quantitative detection result; Titer is the titer, RNACopies is the viral RNA content, ns means P>0.05, * means P<0.05, *** means P<0.001. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The experimental materials used in the present invention are as follows:

[0033] Viruses: HP-PRRSV (TJ strain) and NADC30 strain were isolated and preserved by the inventor's research group, and have been published in "Ruan Shengnan. Pathogenic characteristics of porcine reproductive and respiratory syndrome and antiviral activity of antimicrobial peptides [D]. Huazhong Agricultural University, 2024."; PRRSV1-DV-EGFP is a recombinant strain carrying an enhanced green fluorescent tag constructed by the inventor's research group based on the PRRSV1-DV strain (GenBank: MT311646.1) through a reverse genetics system;

[0034] The applicant promises to release the above-mentioned virus strain to the public within 20 years from the filing date of this invention.

[0035] Cells: African green monkey embryonic kidney cells (Marc-145), purchased from ATCC;

[0036] Antibody: PRRSV mouse monoclonal antibody was prepared and preserved by the inventor's research group according to the method in "Huang Xiaojing, Li Rui, Qiao Songlin, et al. Preparation and identification of monoclonal antibody against GP5 protein of porcine reproductive and respiratory syndrome virus [J]. Chinese Journal of Veterinary Science, 2019, 49(11): 1374-1382."

[0037] Sclareol: molecular formula is C 20 H 36 O2, CAS: 515-03-7, the structural formula is as follows:

[0038]

[0039] Sclareol solution: 95% pure sclareol (purchased from Shaanxi Ciyuan Biotechnology Co., Ltd.) was dissolved in serum-free DMEM to prepare a sclareol solution with a storage concentration of 100 μM, which was sterilized by filtration through a 0.22 μm filter membrane and stored at -30°C after aliquoting;

[0040] The CCK8 detection kit was purchased from Biosharp; the reverse transcription kit was purchased from Nanjing Novozyme Biotechnology Co., Ltd.; the Real-time fluorescence quantitative PCR reagent (2×Universal SYBR Green FastqPCR Mix) was purchased from ABclonal Technology Co., Ltd.;

[0041] The primers were ordered from Qingke Biotechnology Co., Ltd. and the sequences are as follows:

[0042] Upstream primer: 5'-ATGGCCAGCCAGTCAATCA-3' (SEQ ID NO.1)

[0043] Downstream primer: 5'-TCGCCCTAATTGAATAGGTG-3' (SEQ ID NO. 2).

[0044] The detection method involved in the embodiment of the present invention is specifically as follows:

[0045] 1.TCID 50 Determination

[0046] Marc-145 cells were cultured at 2×10 5 / mL was inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 until the cell confluence reached about 80%. The culture medium was discarded, and the collected PRRSV virus supernatant was diluted 10-fold in a maintenance solution containing 2% FBS (10 -1 Up to 10 -8 100 μL of diluted virus solution was added to each well, and 8 wells were set up for each dilution. Virus titer was calculated according to the Reed-Muench method.

[0047] 2. Indirect Immunofluorescence Experiment

[0048] Marc-145 cells were inoculated in a 24-well plate. When the cell density reached about 90%, they were washed twice with PBS. Then, diluted virus solution containing 0.1 MOI PRRSV was added to the wells, cultured at 37°C for 2 hours, then washed twice with PBS again, and cultured for another 24 hours with 2% DMEM maintenance solution. When cytopathic effect (CPE) was observed, the culture medium was discarded and gently rinsed twice with PBS.

[0049] Fixation: Fix with 4% paraformaldehyde for 15 min at room temperature, then wash three times with PBS, with an interval of 5 min each time.

[0050] Permeabilization: The cells were permeabilized with 0.1% Triton X-100 for 10 min at room temperature, followed by three washes with PBS, each with an interval of 5 min.

[0051] Blocking: Block with 5% BSA at room temperature for 2 h, then wash three times with PBS, with an interval of 5 min each time.

[0052] Primary antibody: Add appropriate amount of primary antibody, incubate at 37°C for 1 h, and then wash three times with PBST, with an interval of 5 min each time.

[0053] Secondary antibody: Add fluorescent secondary antibody (1:500) under light-protected conditions, incubate at room temperature for 45 min, and then wash three times with PBST, with an interval of 5 min each time.

[0054] Nuclear staining: Add appropriate amount of DAPI in dark conditions, stain for 10 min at room temperature, and finally wash three times with PBS. Observe the fluorescence under an inverted fluorescence microscope and take photos.

[0055] 3. Absolute fluorescence quantitative detection

[0056] Marc-145 cells were inoculated in a 24-well plate and treated according to the corresponding method in the embodiment. After 48 hours of infection, the cell plate was placed at -80°C and repeatedly frozen and thawed 3 times to fully lyse the cells and release the virus into the supernatant. The lysed supernatant was collected into a sterile EP tube and centrifuged at 4000rpm and 4°C for 10 minutes. The supernatant was recollected and stored at -30°C for standby use. 200 μL of viral supernatant was taken and total RNA was extracted by trizol method. After RNA extraction, reverse transcription was performed immediately to generate cDNA. Then, RT-qPCR was performed using the synthesized cDNA. The reaction conditions were 95°C pre-denaturation for 30s, 95°C denaturation for 5s, 60°C annealing for 30s, and a total of 45 cycles.

[0057] Example 1 Toxicity determination of sclareol on Marc-145 cells

[0058] Marc-145 cells were cultured at 2×10 5 Cells were inoculated into 96-well plates at 100 μL / mL, and 100 μL of cell suspension was added to each well. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 until the cell confluence reached about 80%, and the culture medium was discarded. Drug treatment group, cell control group and blank control group were set up. Drug treatment group: different concentrations of sclareol (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM) diluted with 2% DMEM maintenance solution were added; cell control group: cell maintenance solution containing 0.1% DMSO was added; blank control group: no cells and drugs were added. 100 μL of liquid was added to each well, and 4 replicates were set for each concentration. The 96-well culture plate was placed in a 37°C, 5% CO2 incubator for 48 hours. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated in a 37°C, 5% CO2 incubator for 1 hour. The OD value of each well was measured at a wavelength of 450 nm using an enzyme reader. Cell viability calculation formula:

[0059] Cell viability = [OD(drug added) - OD(blank)] / [OD(normal) - OD(blank)] × 100%.

[0060] Among them, OD(drug added) is the absorbance of the drug treatment group wells after drug addition, OD(blank) is the absorbance of the blank group wells without cells, and OD(normal) is the absorbance of the normal cell control group wells.

[0061] The experimental results are as follows Figure 1 As shown in the figure, compared with the normal control group cells, the concentration of sclareol in the range of 6.25-50μM had no significant effect on the viability of Marc-145 cells. When the concentration reached 100μM, the cell viability decreased significantly. Therefore, in the subsequent antiviral experiments, the drug concentration was controlled below 50μM.

[0062] Example 2 The inhibitory effect of sclareol on Marc-145 cells infected with PRRSV1-DV-EGFP virus

[0063] Marc-145 cells were seeded in a 96-well plate, 100 μL of cell suspension was added to each well, and the plate was cultured in a 37°C, 5% CO2 incubator until the cell density reached about 80%. After discarding the culture medium, 100 μL of different concentrations (10 μM, 20 μM, 30 μM, 40 μM, 50 μM) of sclareol solution diluted in a maintenance solution containing 2% FBS was added to each well, and the plate was placed in a 37°C incubator to pretreat the cells for 2 h.

[0064] Subsequently, 0.1MOI of PRRSV1-DV-EGFP virus suspension was added to each well and incubated at 37°C for 2h. After the incubation, the mixture of drugs and viruses was discarded, the cells were washed twice with PBS, and the culture medium containing different concentrations of clarysol or the maintenance medium containing 2% FBS was replaced. Continue to incubate in a 37°C incubator for 48h. At the same time, the experiment set up a PRRSV1-DV-EGFP virus control group and a normal cell control group (Control) without virus treatment, with 3 replicates in each group. After the culture was completed, fluorescent images were taken separately, and the cell plates were repeatedly frozen and thawed 3 times to collect the supernatant for TCID 50 Detection and absolute fluorescence quantification.

[0065] The experimental results are as follows Figure 2 As shown in the figure, according to the infection of fluorescent virus, sclareol has a significant inhibitory effect on PRRSV virus. The inhibitory effect of sclareol at a concentration of 50 μM on PRRSV1-DV-EGFP virus is most obvious, and no obvious fluorescence is seen. As the concentration decreases, its inhibitory effect gradually weakens ( Figure 2 (A). TCID 50 The test results showed that compared with the control group, sclareol treatment significantly reduced the viral titer in the cell supernatant ( Figure 2 Similarly, RT-qPCR analysis also confirmed the inhibitory effect of sclareol ( Figure 2 These results indicate that sclareol has a significant inhibitory effect on the infection of PRRSV-1 in Marc-145 cells.

[0066] Example 3 The inhibitory effect of sclareol on HP-PRRSV infection in Marc-145 cells

[0067] Marc-145 cells were inoculated in a 96-well plate, 100 μL of cell suspension was added to each well, and the plate was placed in a 37°C, 5% CO2 incubator for culture until the cell density reached about 80%. After discarding the culture medium, 100 μL of different concentrations (10 μM, 20 μM, 30 μM, 40 μM, 50 μM) of sclareol solution diluted in a maintenance solution containing 2% FBS was added to each well, and the cells were pretreated in a 37°C incubator for 2 hours. The experiment set up an HP-PRRSV virus control group and a normal cell control group (Control) that was not treated with the virus, with 3 replicates in each group.

[0068] Subsequently, 0.1MOI of HP-PRRSV virus suspension was added to each well and incubated at 37°C for 2h. After the incubation, the mixture of drugs and viruses was discarded, the cells were washed twice with PBS, and the culture medium containing different concentrations of clarysol or the maintenance medium containing 2% FBS was replaced. Continue to incubate in a 37°C incubator for 24h. After the culture is completed, the supernatant is discarded for indirect immunofluorescence experiments. In addition, the same operation steps were performed in a 24-well plate. After incubation in a 37°C incubator for 48h, the cell plate was repeatedly frozen and thawed 3 times to collect the supernatant for TCID 50 Detection and absolute fluorescence quantification.

[0069] The experimental results are as follows Figure 3 As shown in the indirect immunofluorescence results, sclareol has a significant inhibitory effect on HP-PRRSV virus. When the concentration of sclareol is 50 μM, the inhibitory effect on HP-PRRSV virus is most obvious, and no obvious fluorescence is seen. As the concentration decreases, the inhibitory effect gradually weakens ( Figure 3 (A). TCID 50 The test results showed that compared with the control group, sclareol treatment significantly reduced the viral titer in the cell supernatant ( Figure 3 Similarly, RT-qPCR analysis also confirmed the inhibitory effect of sclareol ( Figure 3 These results all indicate that sclareol has a significant inhibitory effect on the infection of HP-PRRSV strains in Marc-145 cells.

[0070] Example 4 Inhibitory effect of sclareol on Marc-145 cells infected with NADC30 virus

[0071] Marc-145 cells were seeded in a 96-well plate, 100 μL of cell suspension was added to each well, and the plate was cultured in a 37°C, 5% CO2 incubator until the cell density reached about 80%. After discarding the culture medium, 100 μL of different concentrations (10 μM, 20 μM, 30 μM, 40 μM, 50 μM) of sclareol solution diluted in a maintenance solution containing 2% FBS was added to each well, and the cells were pretreated in a 37°C incubator for 2 hours. The experiment set up a NADC30 virus control group and a normal cell control group (Control) that was not treated with the virus, with 3 replicates in each group.

[0072] Subsequently, 0.1 MOI of NADC30 virus suspension was added to each well and incubated at 37°C for 2 hours. After the incubation, the mixture of drugs and viruses was discarded, the cells were washed twice with PBS, and the culture medium containing different concentrations of clary alcohol or the maintenance medium containing 2% FBS was replaced. Continue to incubate in a 37°C incubator for 24 hours. After the culture is completed, the supernatant is discarded for indirect immunofluorescence experiments. In addition, the same operation steps were performed in a 24-well plate. After incubation in a 37°C incubator for 48 hours, the cell plate was repeatedly frozen and thawed 3 times to collect the supernatant for TCID 50 Detection and absolute fluorescence quantification.

[0073] The experimental results are as follows Figure 4 As shown in the indirect immunofluorescence results, sclareol has a significant inhibitory effect on NADC30 virus. The inhibitory effect of sclareol at a concentration of 50 μM on NADC30 virus is significant. As the concentration decreases, the inhibitory effect gradually weakens ( Figure 4 (A). TCID 50 The test results showed that compared with the control group, sclareol treatment significantly reduced the viral titer in the cell supernatant ( Figure 4 Similarly, RT-qPCR analysis also confirmed the inhibitory effect of sclareol ( Figure 4 These results indicate that sclareol has a significant inhibitory effect on the infection of NADC30 strain in Marc-145 cells.

[0074] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of sclareol in the preparation of products against porcine reproductive and respiratory syndrome virus.

2. The use according to claim 1, characterized in that Including the use in the preparation of products for resisting porcine reproductive and respiratory syndrome virus infection.

3. The use according to any one of claims 1 to 2, characterized in that: The concentration of sclareol in the product is 30-50 μM.

4. The use according to claim 3, characterized in that The concentration of sclareol in the product is 50 μM.

5. Application of clarysol in the preparation of drugs for the treatment of porcine reproductive and respiratory syndrome.

6. The use according to claim 5, characterized in that The porcine reproductive and respiratory syndrome is a respiratory infectious disease of pigs caused by porcine reproductive and respiratory syndrome virus infection.

7. The use according to claim 5, characterized in that This includes inhibiting the replication of porcine reproductive and respiratory syndrome virus and / or increasing the survival rate of pigs infected with porcine reproductive and respiratory syndrome virus.

8. The use according to any one of claims 5 to 7, characterized in that: The concentration of sclareol in the drug is 30-50 μM.

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