Application of Bromocriptine in resisting porcine reproductive and respiratory syndrome virus

Abstract

The invention provides an application of Bromocriptine in inhibition of porcine reproductive and respiratory syndrome virus (PRRSV). According to the invention, the antagonism of the Bromocriptine on the PRRSV (porcine reproductive and respiratory syndrome virus) and the action mechanism of the Bromocriptine are discovered. Researches find that Bromocriptine can reduce the hydrolysis effect of PLA1A protein on membrane phospholipid by down-regulating the expression of the PLA1A protein, so that the cell membrane fluidity is reduced, virus particles are prevented from being released to the outside of a host cell through a membrane fusion or budding process, and the specific inhibition on PRRSV release is realized. Therefore, the anti-PRRSV activity is exerted. The Bromocriptine provided by the invention is applied to inhibition of the PRRSV and has important significance in the field of resisting the PRRSV, and a new alternative medicine is provided for prevention and treatment of the PRRSV.

Description

Technical Field

[0001] This invention belongs to the field of biological product preparation technology, specifically relating to the application of bromocriptine in inhibiting porcine reproductive and respiratory syndrome virus. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ear disease, is a highly contagious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV). Its main characteristics include reproductive disorders in sows and respiratory symptoms in pigs of all ages. PRRS first broke out in North America in the late 1980s and was first discovered to be prevalent in China in 1995. In 2006, a highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) outbreak occurred in China, causing significant economic losses to the country's pig farming industry.

[0003] PRRSV, as an RNA virus, exhibits diverse and highly mutable strains. This leads to insufficient heterologous protection in existing commercial vaccines, and vaccine development often lags behind strain mutations and the emergence of new strains. Furthermore, vaccine strains may experience virulence reversion or recombination with field strains, posing safety risks. Therefore, screening effective components from natural drug libraries, peptides, and small molecule compounds to inhibit viral infection and proliferation is an effective solution to the current challenges in PRRSV control. However, the vast number of viruses, with significant differences in structural characteristics, infection mechanisms, and pathogenic pathways, results in highly specific drug inhibitory effects. A drug effective against one virus may be ineffective against another, or even completely ineffective due to target mismatch. Currently, the screening of specific inhibitory drugs for PRRSV still has significant shortcomings, and a mature and effective drug control system has not yet been established. Therefore, targeted drug screening research is urgently needed.

[0004] Furthermore, cellular state has a significant impact on the viral infection process. Cell membrane fluidity is one of the core characteristics of cells maintaining normal physiological functions, and its dynamic balance directly regulates the viral infection and release process: viral adsorption to host cells depends on the effective recognition and binding of receptors on the cell membrane surface, and appropriate membrane fluidity can provide the necessary spatial environment for receptor aggregation; during the viral entry phase, membrane fluidity supports the smooth completion of endocytosis or membrane fusion; after viral replication, it also needs to utilize cell membrane fluidity to release the virus through budding and other mechanisms. However, current research on PRRSV prevention and control has not fully utilized the cell membrane fluidity-related regulatory mechanisms to develop specific intervention methods. Summary of the Invention

[0005] This invention provides the application of bromocriptine in inhibiting porcine reproductive and respiratory syndrome virus, thereby overcoming the shortcomings of the prior art.

[0006] The present invention first provides one use of bromocriptine in the preparation of products for inhibiting porcine reproductive and respiratory syndrome virus.

[0007] The present invention also provides an article for inhibiting porcine reproductive and respiratory syndrome virus, comprising a pharmacologically effective concentration of bromocriptine; Preferably, the amount of Bromocriptine used is 1.6-51.5 μM.

[0008] Furthermore, the formulation also contains components that reduce the content of phospholipase PLA1A protein.

[0009] This invention discovers the antagonistic effect and mechanism of action of bromocriptine against porcine reproductive and respiratory syndrome virus (PRRSV). Bromocriptine concentrations above 1.6 μM can inhibit viral proliferation by half, and even at concentrations as high as 51.5 μM, it does not significantly affect cell viability. Furthermore, this invention found that bromocriptine may exert its anti-PRRSV activity by downregulating PLA1A protein expression, reducing its hydrolytic effect on membrane phospholipids, leading to decreased cell membrane fluidity, and hindering the release of viral particles from the host cell through membrane fusion or budding. This results in specific inhibition of PRRSV release. The bromocriptine provided by this invention is of great significance for inhibiting PRRSV and in the field of anti-PRRSV treatment, providing a new drug option for PRRSV prevention and control. Attached Figure Description

[0010] Figure 1A schematic diagram illustrating the mechanism by which phospholipase PLA1A regulates PRRSV replication and its specific mechanism, provided in Example 1 of this invention: where, Figure 1 -a represents the effect of PLA1A knockdown on PRRSV N protein levels as detected by Western Blot. Figure 1 -b indicates TCID-based 50 To investigate the effect of PLA1A knockdown on PRRSV viral titers; Figure 1 -c represents the effect of PLA1A knockdown on PRRSV infection (N protein positive signal) based on IFA detection; Figure 1 -d represents the specific role stage of PLA1A in the PRRSV life cycle based on RT-qPCR analysis; Figure 1 -e indicates the knockdown efficiency of PLA1A protein in Western Blot detection of Figure D. Figure 1 -f indicates TCID-based 50 To investigate the effect of PLA1A knockdown on cell supernatant and intracellular PRRSV titers; Figure 1 -g represents the effect of gradient expression of PLA1A on cell membrane fluidity detected using the TMA-DPH probe; Figure 1 -h represents the effect of gradient expression of PLA1A on cell membrane fluidity based on Laurdan dye detection; Figure 1 -i represents the PLA1A protein gradient overexpression levels in plots G and H detected by Western Blot. Figure 1 -j represents the effect of TMA-DPH probe-based detection of PRRSV infection and PLA1A knockdown on cell membrane fluidity; Figure 1 -k represents the PLA1A knockdown efficiency of PRRSV N protein levels detected by Western Blot in Figure J.

[0011] Figure 2 The natural small molecule drug targeting phospholipase PLA1A, as provided in Example 1 of this invention, is screened via molecular docking: wherein, Figure 2 -a represents the predicted three-dimensional structure of phospholipase PLA1A based on AlphaFold3; Figure 2 -b represents the potential binding region for small molecule drugs in the target protein PLA1A, predicted using PYMOL software. Figure 2 -c represents the predicted potential binding pocket for PLA1A based on the Ramachandran plot; Figure 2 -d is a schematic diagram of the top 103 anti-PRRSV active small molecule compounds screened based on IFA; Figure 2 -e represents Bromocriptine, the drug with the most significant antiviral effect selected by IFA. Figure 2 -f is for TCID 50To investigate the effect of bromocriptine on PRRSV viral titers; Figure 2 -g represents a three-dimensional model of the molecular docking between Bromocriptine and PLA1A, constructed and displayed using PYMOL software.

[0012] Figure 3 A schematic diagram illustrating the inhibition of PRRSV replication by Bromocriptine according to Embodiment 1 of the present invention: Figure 3 -a is the chemical structural formula of Bromocriptine; Figure 3 -b represents the cytotoxicity assay of Bromocriptine in MARC-145 cells; Figure 3 -c represents the assay of bromocriptine's anti-PRRSV activity in MARC-145 cells based on luciferase (NanoBiT); Figure 3 -d indicates TCID-based 50 Viral titers in Bromocriptine-treated MARC-145 cells were detected at different time points; Figure 3 -e represents the levels of target proteins PLA1A and PRRSV N in MARC-145 cells treated with Bromocriptine at different time points, as detected by Western Blot. Figure 3 -f indicates the cytotoxicity assay of bromocriptine in PAMs cells; Figure 3 -g represents the assay of bromocriptine's anti-PRRSV activity in PAMs cells based on luciferase (NanoBiT). Figure 3 -h indicates TCID-based 50 Detect the viral titer in PAM cells treated with Bromocriptine at different time points; Figure 3 -i represents the level of target proteins PLA1A and PRRSV N in PAMs cells treated with Bromocriptine at different time points, detected by Western Blot.

[0013] Figure 4 This is a schematic diagram illustrating the stages of Bromocriptine's inhibition of PRRSV replication and its mechanism of PRRSV regulation, as provided in Embodiment 1 of the present invention: [Diagram showing the process by which Bromocriptine inhibits PRRSV replication and regulates PRRSV] Figure 4 -a is a schematic diagram of Bromocriptine treatment at different stages of the viral infection cycle. Figure 4 -b represents the phase of Bromocriptine's anti-PRRSV action based on luciferase analysis. Figure 4 -c represents the effect of bromocriptine on cell membrane fluidity detected using Laurdan fluorescent dye; Figure 4-d indicates TCID-based 50 To determine the effect of bromocriptine on the release phase of PRRSV. Detailed Implementation

[0014] This invention, through molecular docking screening, discovered that bromocriptine can interact with phospholipases and inhibit PRRSV proliferation by affecting cell membrane fluidity. Bromocriptine may reduce the hydrolytic effect of PLA1A protein on membrane phospholipids by downregulating its expression, leading to decreased cell membrane fluidity and hindering the release of viral particles from the host cell through membrane fusion or budding, thereby specifically inhibiting PRRSV release and exerting anti-PRRSV activity. Therefore, this invention addresses the issue from the perspective of preventing or reducing cell membrane fluidity, and through drug screening and verification, discovered the antiviral effect of bromocriptine, providing a new approach to intervening in PRRSV infection.

[0015] Information on the materials and methods used in the embodiments of this invention is as follows: 1.1 Viruses and Cells The clinical isolate JXwn06 of PRRSV was previously isolated and preserved by the Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs of China Agricultural University (hereinafter referred to as "this laboratory"). It was isolated in 2006 from a pig farm in Jiangxi Province where a highly pathogenic type of PRRS occurred. It is a highly pathogenic strain, and its GenBank accession number is EF641008.

[0016] PRRSV Saves the Poison RvJX-Nsp2 325 HiBiT is a recombinant virus constructed in the laboratory in the early stage. It is based on JXwn06 and has the HiBiT sequence of the small subunit of complementary luciferase inserted into it. It is disclosed in Chinese patent CN115161287A.

[0017] Both MARC-145 cells and porcine primary alveolar macrophages (PAMs) were used for PRRSV infection. MARC-145 cells were a monkey-derived kidney cell line that was previously preserved in our laboratory. PAMs were obtained by alveolar lavage of specific pathogen-free (SPF) piglets.

[0018] 1.2 Molecular Biology Reagents DMEM medium, RPMI-1640 medium, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific; the Nano-Glo HiBiT Lytic Detection System reagent was purchased from Promega Corporation (Wisconsin, USA); PRRSV N protein monoclonal antibody N35 was prepared in our laboratory, and GAPDH monoclonal antibody was purchased from Huaan Biotechnology Co., Ltd. (HUABIO, HZ, CN); accession number CGMCCNO.3238, disclosed in Chinese patent CN101661042B. The natural small molecule drug Bromocriptine was purchased from Selleck Biotechnology Co., Ltd. (Selleck, BJ, USA).

[0019] 1.3 Cell Culture Reagents DMEM cell maintenance medium (2%): Add 2% fetal bovine serum, penicillin (100 U / ml) and streptomycin (100 μg / ml) to DMEM cell culture medium and store at 4°C.

[0020] PAMs cell culture medium (10%): 10% fetal bovine serum, penicillin (100 U / ml) and streptomycin (100 μg / ml) were added to 1640 cell culture medium and stored at 4℃.

[0021] 1640 cell maintenance medium (2%): 2% fetal bovine serum, penicillin (100 U / ml) and streptomycin (100 μg / ml) were added to the 1640 cell culture medium and stored at 4°C.

[0022] PBS: Weigh 4.00 g NaCl, 0.72 g Na2HPO4, 0.12 g KH2PO4 and 0.10 g KCl respectively. After they are completely dissolved, add ultrapure water to make up to 500 mL. After autoclaving, store at 4℃ for later use.

[0023] However, in addition to the reagents mentioned above, other alternative reagents in the field of molecular biology can also be used.

[0024] The present invention will now be described in detail with reference to the embodiments and accompanying drawings. Example 1

[0025] 1. Regulation of PRRSV replication by phospholipase PLA1A and its specific mechanism 1.1 Effect of knockdown of phospholipase PLA1A on PRRSV replication (1) PRRSV JXwn06 infection experiment after si-PLA1A transfection: Well-cultured MARC-145 cells were evenly seeded into 12-well plates. When the cell confluence reached 50%, the siRNA:RNAiMaX transfection reagent was diluted at a ratio of 1:2 and mixed. After standing at room temperature for 15 min, the mixture was evenly added to the cell surface. After incubation in a cell culture incubator for 36 h, JXwn06 was seeded into a confluent monolayer of MARC-145 cells at MOI=0.1 and incubated at 37℃ for 1 h. The culture medium was discarded, and DMEM medium containing 2% fetal bovine serum was added. Samples were collected at different time phases (0 h, 12 h, 24 h, 36 h, 48 h) and stored at -80℃ for subsequent Western Blot, IFA and TCID. 50 Test results. Figure 1 As shown in AC, knockdown of phospholipase PLA1A significantly reduced PRRSV N protein levels and viral titers at 24h, 36h, and 48h post-infection.

[0026] 1.2 The specific mechanism by which phospholipase PLA1A regulates PRRSV replication (1) Effect of siRNA interference on the PLA1A gene in cells on the PRRSV infection stage: ① Adsorption phase: The PLA1 gene of MARC-145 cells was interfered with according to the above method. After 36 h, the cells were pre-cooled at 4℃ for 30 min, and PRRSV was seeded at MOI=0.1. The cells were then incubated at 4℃ for another 1 h. The virus solution was discarded, and the cells were washed three times with sterile PBS. RNA was extracted from the cells and reverse transcribed. The relative quantification of the adsorbed PRRSV N gene was performed by RT-qPCR.

[0027] ② Internalization stage: After interfering with the PLA1A gene of MARC-145 cells for 36 h as described above, the cells were pre-cooled at 4℃ for 30 min, inoculated with PRRSV at MOI=0.1, and incubated at 4℃ for 1 h. The virus solution was discarded, and the cells were washed three times with sterile PBS, then replaced with DMEM medium containing 2% fetal bovine serum and incubated at 37℃ for 2 h. The medium was discarded, and the cells were washed three times with citric acid solution (5 min each time) to remove virus particles adsorbed on the cell surface but not internalized. After collecting the cells, RNA was extracted and reverse transcribed. The PRRSV N gene that had been internalized into the cells was relatively quantified by RT-qPCR.

[0028] ③ Replication Phase: After interfering with the PLA1A gene of MARC-145 cells for 36 h as described above, PRRSV was inoculated at an MOI of 0.1 and incubated at 4°C for 1 h. The virus solution was discarded, and the cells were washed three times with sterile PBS. The medium was then replaced with DMEM containing 2% fetal bovine serum and cultured at 37°C for another 12 h. After collecting the cells, RNA was extracted and reverse transcribed. The relative quantification of the PRRSV N gene in the cells and supernatant was performed by RT-qPCR.

[0029] ④ Release Phase: After interfering with the PLA1A gene of MARC-145 cells for 36 h as described above, PRRSV was inoculated at MOI=0.1 and incubated at 4℃ for 1 h. The virus solution was discarded, and the cells were washed three times with sterile PBS. The medium was then replaced with DMEM containing 2% fetal bovine serum and cultured at 37℃ for another 24 h. Cells and supernatant were collected separately for two assays: one part of the cells and supernatant was used to extract RNA and reverse transcribe it, and the PRRSV N gene was quantitatively analyzed by RT-qPCR; the other part of the cells and supernatant was subjected to three freeze-thaw cycles at -80℃ and analyzed by TCID45. 50 Viral titers in cells and supernatant were measured separately, and the viral budding rate was calculated using the formula (viral budding rate = number of viral particles in supernatant / number of viral particles in cells). Results are as follows: Figure 1 As shown in DF: Knockdown of phospholipase PLA1A significantly reduced PRRSV N protein levels at 24 h, 36 h, and 48 h post-infection, and also significantly reduced viral titers at 24 h and 36 h post-infection.

[0030] (2) Phospholipase PLA1A regulates cell membrane fluidity: ① Overexpression group: PLA1A-EGFP recombinant plasmid was transfected at gradient concentrations (0 μg, 0.25 μg, 0.5 μg, 0.75 μg, 1 μg, 2 μg) (0 μg group was the empty vector control). After 24 h of transfection, PRRSV was inoculated into the cells at MOI=1 and cultured in a 37℃ incubator for another 24 h. ② Knockdown group: si-PLA1A was transfected (si-NC negative control was set up at the same time). After 24 h of transfection, PRRSV was inoculated into the cells at MOI=0.1 and cultured in a 37℃ incubator for another 24 h. The cell supernatant of each group was discarded and the cells were gently washed 3 times with sterile PBS. Then, cell membrane fluidity-specific dyes Laurdan and TMA-DPH with a final concentration of 0.5-5 μM were added to each group of cells and incubated at 37℃ for 30 min in the dark. After incubation, wash the cells again 2-3 times with sterile PBS to remove unbound free dye. Detect the fluorescence signal using a fluorescence polarimeter, measure its excitation / emission spectrum, and calculate the generalized polarization value (GP value, formula: GP = (I0 + I0)). 440 -I490 ) / (I 440 +I 490 ), where I 440 The fluorescence intensity at 440 nm, I 490 The fluorescence intensity at 490 nm is used; the GP value is negatively correlated with mobility. For TMA-DPH, the fluorescence polarization degree (P value, formula: P=(I)) is measured. ∥ -I ⊥ ) / (I ∥ +I ⊥ ), where I ∥ I represents the fluorescence intensity in the parallel direction. ⊥ (The fluorescence intensity is in the vertical direction; the P-value is negatively correlated with mobility). Results are as follows: Figure 1 As shown in GI: after gradient overexpression of PLA1A and infection with the virus for 24 h, the fluorescence polarization value gradually increased with the increase of PLA1A expression level, indicating that the cell membrane fluidity gradually increased; conversely, when PLA1A was knocked down, the fluorescence polarization value increased significantly, indicating that the cell membrane fluidity decreased.

[0031] 3. Bromocriptine, a natural drug targeting PLA1A, was obtained through molecular docking screening, and its inhibitory effect on PRRSV replication was tested. 3.1 Virtual screening of small molecule inhibitors targeting phospholipase PLA1A Molecular docking simulations were performed using PYMOL software to target the protein PLA1A and its ligand molecules. Small molecule drugs from the Selleck Biotech compound library (https: / / www.selleck.cn / screening-libraries.) were selected for molecular docking, comprising 3196 FDA drug libraries, 10480 natural products, and drug-food homology libraries. The docking algorithm was Lamarck's genetic algorithm, the docking mode was semi-flexible, the exhaustiveness was set to 16, and the maximum number of output conformations was set to 9. The conformation with the lowest binding free energy was selected as the most reasonable binding mode. Finally, top-ranked small molecule drugs were screened, such as... Figure 2 As shown in AC, subsequent biological experiments will be conducted to verify this.

[0032] 3.2 IFA screening and validation of small molecule drugs against PRRSV in cells After initial screening using molecular docking to identify small molecule inhibitors targeting PLA1A, we purchased the top 103 compounds (Selleck). MARC-145 cells were pretreated with the inhibitor at a final concentration of 10 μM for 2 hours, followed by inoculation with PRRSV JXwn06 strain at an MOI of 0.1. One hour post-infection, the viral solution was discarded and replaced with maintenance medium containing 40 μM of the inhibitor, with DMSO used as a control. After 24 hours, cells were fixed with pre-cooled anhydrous ethanol at room temperature for 15 min, then diluted PRRSV N protein monoclonal antibody was added, and incubation was performed at 37 °C for 1 h. Cells were washed three times with PBS, and then incubated with diluted FITC-labeled goat anti-mouse secondary antibody at 37 °C for 30 min in the dark. Cells were washed three times with PBS. Finally, the green fluorescence signal of each group of cells was observed using fluorescence microscopy, and the inhibitory effect of the drug on PRRSV replication was determined by comparing the fluorescence intensity. Results are as follows: Figure 2 As shown in DF: Among 103 drugs, Bromocriptine had the most significant antiviral effect, not only inhibiting the expression of PRRSV's N protein, but also significantly reducing viral titer.

[0033] Example 2: Cytotoxicity assay of Bromocriptine in MARC-145 cells and PAMs cells

[0034] Different concentrations of bromocriptine (1.56 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM) were added to 96-well plates containing a monolayer of MARC-145 cells or PAM cells, and incubated for 48 h. Then, 10 μL of CCK8 solution was added to each well, and the plates were incubated at 37°C for 1 h. The absorbance at 490 nm was measured. Zeroing wells (2% maintenance solution) and control wells (DMSO with the same drug concentration) were also included. Results are as follows: Figure 3 As shown in B and 3F: the drug concentration of 51.5 μM had little effect on the activity of MARC-145 cells; while the drug concentration of 25.15 μM had little effect on the activity of PAMs cells.

[0035] 1. Assay of anti-PRRSV activity of bromocriptine in MARC-145 cells and PAMs Fluorescence intensity: MARC-145 cells or PAMs cells confined to a monolayer in a 96-well plate were seeded with RvJX-Nsp2 at an MOI of 0.1. 325The HiBiT strain was incubated at 37°C for 1 h. After incubation, the unadsorbed virus solution in the wells was discarded, and the maintenance medium was replaced: for MARC-145 cells, the medium was replaced with cell maintenance medium containing different concentrations of bromocriptine (0.24 μM, 0.48 μM, 0.97 μM, 1.95 μM, 3.9 μM, 7.81 μM, 15.6 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM); for PAMs cells, the medium was replaced with cell maintenance medium containing different concentrations of bromocriptine (2.5 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, 20 μM). A DMSO control group was set up for both groups. After incubation at 37°C for 36 h, the samples were subjected to three freeze-thaw cycles at -80°C. After the samples were completely thawed, 50 μL of viral lysis buffer was added to a black 96-well plate, followed by an equal volume (50 μL) of Nano-Glo® HiBiT Lytic Reagent. The plates were then vortexed at room temperature for 10 min. Finally, the fluorescence intensity of each well was detected and recorded using a Spark® multi-mode microplate reader. By comparing the fluorescence values ​​of different concentrations of Bromocriptine with the control group, the inhibitory effect of Bromocriptine on PRRSV replication and its dose-dependent relationship were analyzed. The results are as follows: Figure 3 As shown in C and 3G: Bromocriptine in MARC-145 cells and PAMs at IC50 50 The values ​​were 1.6 μM and 4.101 μM, respectively.

[0036] Virus titer: JXwn06 cells were inoculated with confluent MARC-145 and PAMs cells at MOI=0.1, incubated for 1 h, the culture medium was discarded, and cell maintenance medium containing 10 μM bromocriptine was added. Cell plates were collected at different time points after culture and cooled to -80℃. After three freeze-thaw cycles, the virus solution was collected and serially diluted 10-fold with 2% maintenance medium. 10 -1 ~10 -7 Dilutes were inoculated into 96-well plates containing a confluent monolayer of MARC-145, 100 μL per well, with each dilution inoculated in quadruplicate. After incubation at 37 ℃ for 48 h, indirect immunofluorescence was performed, and the results were observed and recorded under a fluorescence microscope. The viral titer was calculated using the Reed-Muench method. Results are as follows: Figure 3 As shown in D and 3H, Bromocriptine significantly reduced viral titers at different time points after PRRSV infection in PAMs and MARC-145 cells.

[0037] 2. The stages and mechanisms of action of bromocriptine in inhibiting PRRSV replication 2.1 Bromocriptine inhibits PLA1A expression, thereby inhibiting PRRSV replication. Western Blot: JXwn06 cells were seeded at an MOI of 0.1 to form a confluent monolayer of MARC-145 cells. After 1 h of incubation, the culture medium was discarded, and 10 μM of bromocriptine was added as cell maintenance medium. Cell pellets were collected at 0 h, 12 h, 24 h, 36 h, and 48 h after culture. RIPA protein lysis buffer containing 1 mM PMSF was added, and the mixture was incubated on ice for 30 min. The mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. 5× loading buffer was added, and the mixture was boiled for 10 min. SDS-PAGE was performed, and the protein was transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 h. Anti-PRRSV N antibody was used as the primary antibody, and GAPDH was used as the internal control gene. The membrane was incubated at room temperature for 2 h. Horseradish peroxidase-labeled goat anti-mouse IgG was used as the secondary antibody, and the membrane was incubated at room temperature for 1 h. High-sensitivity ECL chemiluminescence solution was added, and the membrane was developed in a dark room and chemically exposed using a gel imaging system. Results are as follows: Figure 3 As shown in E and 3I, bromocriptine significantly inhibited the expression levels of PRRSV N protein and its target protein PLA1A in both MARC-145 and PAMs cells, suggesting that bromocriptine may antagonize viral replication by downregulating PLA1A expression. However, whether bromocriptine inhibits PRRSV proliferation through other pathways requires further investigation.

[0038] 2.2 Based on RvJX-Nsp2 325 -HiBiT assay shows that bromocriptine inhibits the replication phase of PRRSV. Virus elimination (Virucidal): Combine Bromocriptine (final concentration 10 μM) with RvJX-Nsp2 325 The HiBiT strain (MOI=0.1) was co-incubated at 37°C for 2 h, and then the mixture was seeded into 96-well plates containing a monolayer of MARC-145 cells and incubated at 37°C for 1 h. After incubation, the supernatant in the wells was discarded, and fresh cell maintenance medium containing 2% FBS was added. After culturing for another 36 h, samples were collected and fluorescence intensity was measured. A control group (DMSO) was included in the experiment.

[0039] Pre-treated cells: MARC-145 cells that had grown into a monolayer were pretreated with bromocriptine (final concentration 10 μM) for 2 h, and then inoculated with RvJX-Nsp2. 325-HiBiT strain (MOI=0.1), infected at 37℃ for 1 h. After infection, the virus solution in the wells was discarded, and fresh cell maintenance medium containing 2% FBS was added. After culturing for 36 h, samples were collected and fluorescence intensity was measured; cells with an equal volume of DMSO were added as a control group.

[0040] Adsorption and entry phase (During-treated): Virus RvJX-Nsp2 was introduced into a 2% maintenance solution. 325 HiBiT was diluted to MOI=0.1, and the final concentration of Bromocriptine was prepared to 10 μM using the diluted virus solution. The mixture was then inoculated into a 96-well plate with a monolayer of MARC-145 and cultured for 1 h. The supernatant was discarded, and fresh maintenance medium containing 2% FBS was added. After culturing for 36 h, the samples were collected and the fluorescence intensity was measured. The control was prepared by adding the same volume of DMSO.

[0041] Replication phase (Post-treated): The virus RvJX-Nsp2 325 HiBiT was seeded into a confluent monolayer of MARC-145 cells at MOI=0.1 and allowed to incubate for 1 h. The supernatant was discarded, and fresh maintenance medium containing 10 μM Bromocriptine was added. After culturing for 36 h, samples were collected and fluorescence intensity was measured. The control was prepared by adding the same volume of DMSO.

[0042] The results are as follows Figure 4 As shown in AB, Bromocriptine mainly plays a role in the viral replication phase.

[0043] 2.3 Based on TCID 50 Detecting the effects of bromocriptine on the replication and release phases of PRRSV Add 10 μM bromocriptine and JXwn06 virus strain (MOI = 0.1) to 48-well plates confluent with a monolayer of MARC-145 cells, and incubate at 37°C for 1 h. After incubation, discard the culture medium and immediately add cell maintenance medium containing 10 μM bromocriptine, and continue culturing under the same conditions for 36 h. After culturing, collect the cell supernatant and adherent cells from each well (cell samples were added to an equal volume of maintenance medium), and perform three freeze-thaw cycles at -80°C. Collect the virus solution, perform 10-fold serial dilutions with cell maintenance medium containing 2% FBS, and select 10... -1 ~10 -7 For dilution, 100 μL of diluted virus solution was added to each well, with four replicates for each dilution. The viral titer was then calculated using the Reed-Muench method. Results are as follows: Figure 4As shown in C: Bromocriptine plays a role in both viral replication and release phases.

[0044] 2.4 Detection of the effect of bromocriptine on cell membrane fluidity based on Laurdan fluorescent dye MARC-145 cells that had grown into a monolayer in 24-well plates were incubated with 10 μM bromocriptine and JXwn06 virus at an MOI of 0.1 for 1 h at 37°C. After incubation, the medium was discarded, and cell maintenance medium containing 10 μM bromocriptine was immediately added, and the cells were cultured for another 24 h. The cell supernatant was discarded, and the cells were gently washed three times with sterile PBS. Subsequently, the cell membrane fluidity-specific dye Laurdan was added to each group of cells at a final concentration of 0.5–5 μM, and the cells were incubated at 37°C for 30 min in the dark. After incubation, the cells were washed 2–3 times again with sterile PBS to remove unbound free dye. Fluorescence signals were detected using a fluorescence polarimeter, and their excitation / emission spectra were measured to calculate the generalized polarization value (GP value). Simultaneously, three control groups were set up to ensure the validity of the results: a blank control with only an equal volume of DMSO (no virus, no drug) containing Bromocriptine; a drug control with a final concentration of 10 μM Bromocriptine (without inoculation with the JXwn06 strain); and a virus control with an equal volume of DMSO (only inoculated with the JXwn06 strain at MOI=0.1, no drug). All other procedures for the control groups were consistent with those for the experimental groups. Results are as follows: Figure 4 As shown in Figure D: Bromocriptine can significantly reduce cell membrane fluidity.

[0045] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Application of Bromocriptine in the preparation of products for inhibiting porcine reproductive and respiratory syndrome virus.

2. A product for inhibiting porcine reproductive and respiratory syndrome virus, characterized in that, The product contains a pharmacologically effective concentration of bromocriptine.

3. The article of claim 2, characterized in that, The amount of Bromocriptine used is 1.6-51.5 μM.

4. The article of claim 2, characterized in that, The product also contains components that reduce the content of phospholipase PLA1A protein.

5. The use of the product according to claim 2 in reducing the content of phospholipase PLA1A protein.

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