Application of lysophosphatidic acid inhibitors in the preparation of drugs for inhibiting PRRSV replication

Abstract

The present invention belongs to the field of biotechnology and discloses the use of lysophosphatidic acid inhibitors in the preparation of drugs for inhibiting PRRSV replication. Through metabolomics analysis and screening, the present invention discovered that the natural metabolite lysophosphatidic acid (LPA) significantly promotes PRRSV replication. Targeted inhibition of LPA can effectively inhibit viral replication in infected pigs, alleviate clinical symptoms such as high fever and cough caused by porcine respiratory and reproductive syndrome (PRRS), significantly reduce viral loads in virus-infected tissues such as the lungs, spleen, and inguinal lymph nodes, and alleviate pathological damage to infected tissues. Therefore, lysophosphatidic acid inhibitors have great application prospects in the prevention and treatment of porcine reproductive and respiratory syndrome (PRRS).

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of a lysophosphatidic acid inhibitor in preparing a drug for inhibiting PRRSV replication. Background Art

[0002] The causative agent of porcine reproductive and respiratory syndrome (PRRS) is porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped, positive-sense, single-stranded RNA virus belonging to the order Nidovirales and the family Arteriviridae. Pigs are the sole reservoir for PRRSV infection, with infection most severe in pregnant sows and piglets. PRRSV can cause respiratory problems and immune system suppression in pigs of all ages; pregnant sows experience miscarriage, stillbirth, mummification, and weak fetuses; while piglets experience symptoms such as diarrhea, swollen eyes, and paralysis of the limbs. Secondary infection leading to exhaustion is the primary cause of death in piglets, resulting in significant economic losses for the domestic and international pig farming industries.

[0003] Due to its biological characteristics, such as high transmissibility, high variability, and persistent infection, PRRSV is difficult to control and eliminate. Currently, there is no clinically effective drug for the treatment of PRRS. Control of PRRSV primarily relies on purification and vaccination. However, due to the limitations of the domestic pig industry and the breeding environment, biosafety control, monitoring, and purification conditions remain insufficient to address the threat of PRRSV transmission. Furthermore, commercial vaccines are not effective, with poor cross-protection between different strains. Furthermore, the replication of live viruses in pigs carries the risk of reversion to stronger virulence and recombination with wild-type viruses into new strains. Therefore, the industry urgently needs a drug that can effectively inhibit and eliminate PRRSV. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies and defects of the prior art and provide the use of a lysophosphatidic acid inhibitor in the preparation of a drug for inhibiting PRRSV replication.

[0005] The purpose of the present invention is achieved through the following technical solutions: the use of lysophosphatidic acid inhibitors in the preparation of drugs for inhibiting PRRSV replication is based on the fact that the inventors of the present invention found through metabolomics analysis and screening that the natural metabolite lysophosphatidic acid (LPA) has a significant promoting effect on the replication of PRRSV. Targeted inhibition of LPA can effectively inhibit viral replication in infected pigs, alleviate clinical symptoms such as high fever and cough caused by porcine respiratory and reproductive syndrome, significantly reduce the viral load in virus-infected tissues such as the lungs, spleen, and inguinal lymph nodes, and reduce pathological damage to infected tissues.

[0006] The lysophosphatidic acid inhibitor is preferably HA130 having the structural formula shown in (1);

[0007]

[0008] The drug for inhibiting PRRSV replication is a drug that antagonizes porcine reproductive and respiratory syndrome virus replication, can target and downregulate LPA levels, antagonize PRRSV replication, and alleviate the symptoms of porcine reproductive and respiratory syndrome.

[0009] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0010] (1) The present invention discovered for the first time that the natural metabolite lysophosphatidic acid (LPA) has a significant promoting effect on the replication of PRRSV. Targeted inhibition of LPA can effectively inhibit viral replication in infected pigs, alleviate clinical symptoms such as high fever and cough caused by porcine respiratory and reproductive syndrome, significantly reduce the viral load in virus-infected tissues such as the lungs, spleen, and inguinal lymph nodes, and alleviate pathological damage to infected tissues.

[0011] (2) The present invention discloses for the first time that HA130 has anti-PRRSV activity. HA130 was used to successfully inhibit PRRSV replication in a passageable porcine alveolar macrophage cell line (iPAMs), and the inhibition was dose-dependent, demonstrating the potential of HA130 in treating PRRSV. Furthermore, in the piglet treatment experiment, HA130, as a natural metabolite molecule, showed the advantages of being non-toxic and having no side effects in the control group; in the piglets challenged with the virus, HA130 treatment significantly alleviated the piglets' symptoms of fever, cough, and anorexia; and HA130 treatment greatly reduced the viral load in the lungs, spleen, and inguinal lymph nodes of the infected piglets, and reduced tissue damage in the lungs and spleen. HA130 shows great potential as a PRRSV therapeutic drug, and its targeted natural metabolite molecule has a clear structure, is easy to prepare, is non-toxic, and has no side effects, and has good application prospects in the prevention and treatment of porcine reproductive and respiratory syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results of non-targeted metabolomics and non-targeted lipidomics analysis of pig serum infected and uninfected with PRRSV 3, 7, 10, and 21 days ago are shown in Figure 1. A is the serum metabolomics model establishment and analysis process; B is the volcano plot of differential metabolites in the serum metabolome at 3, 7, 10, and 21 dpi (days post infection), with the horizontal axis being log2 (foldchange) and the vertical axis being log 10(q value), differential metabolites were screened based on the conditions of metabolite fold change ≥1.2 or ≤0.8 and q value <0.05. The blue and red dots are significantly up- and down-regulated metabolite molecules, respectively; C is the volcano plot of differential metabolites in serum lipid groups at 3, 7, 10, and 21 dpi, with the horizontal axis being log2 (fold change) and the vertical axis being log 10 (qvalue), differentially expressed lipid molecules were screened based on the conditions of metabolite fold change ≥1.2 or ≤0.83 and q value <0.05. The blue and red dots are the lipid molecules that are significantly up-regulated and down-regulated, respectively; D is the heat map of the expression changes of all differentially expressed metabolites and lipid molecules in pig serum at 3, 7, 10, and 21 dpi. Each row represents a significantly changed differential molecule, with blue indicating down-regulation and red indicating up-regulation.

[0013] Figure 2 The results of the effect of HA130 on PRRSV in iPAMs are shown in Figure 2. A shows the cytotoxicity test results of LPA and HA130 on iPAMs. B shows the effect of different concentrations of LPA on PRRSV replication by WB and the grayscale scanning results. C shows the effect of different concentrations of HA130 on PRRSV replication by WB and the grayscale scanning results. D shows the TCID 50 Detect the effect of different concentrations of LPA on virus titer; E is TCID 50 The effects of different concentrations of HA130 on viral titer were detected. The results showed that HA130 had an inhibitory effect on PRRSV in iPAMs in a dose-dependent manner.

[0014] Figure 3 The figure shows the statistical data of the piglet treatment experiment and the viral load measurement results; among them, A is the piglet temperature monitoring result; B is the piglet serum LPA concentration; C is the viral load detection of the lung tissue of the piglets in the challenge group and the challenge treatment group; D is the viral load detection of the spleen of the piglets in the challenge group and the challenge treatment group; E is the viral load detection of the inguinal lymph nodes of the piglets in the challenge group and the challenge treatment group.

[0015] Figure 4 This is a photo of the pathological section results of the piglet treatment experiment. DETAILED DESCRIPTION

[0016] The present invention will be described in further detail below with reference to the embodiments and accompanying drawings.

[0017] The experimental process of the present invention is summarized as follows: First, metabolomics mass spectrometry was performed on the obtained PRRSV-positive and -negative sera. The metabolomics data were analyzed using bioinformatics methods, and a significantly upregulated metabolite, lysophosphatidic acid (LPA), was screened. Its level was highly positively correlated with the viral load of PRRSV, and may play an important role in the infection and replication process of PRRSV. Therefore, the present invention verified the promoting effect of LPA on PRRSV replication in a passageable porcine alveolar macrophage cell line (iPAMs), and selected the drug HA130, which targets LPA, to determine its inhibitory effect on PRRSV replication. Finally, animal experiments were conducted, and the drug was administered to treat piglets infected with PRRSV to verify the clinical therapeutic effect of HA130.

[0018] The materials used in this experiment are as follows:

[0019] (1) Cells and viruses

[0020] The highly pathogenic PRRSV strain Li11, African green monkey embryonic kidney cells Marc-145, and immortalized porcine alveolar macrophages (iPAMs) are maintained in this laboratory. The strains and cells are conventional strains and cells that have been disclosed in the existing technology (see "Wei Y, et al. Porcine TRIM21RING-finger E3 ubiquitin ligase is essential for anti-PRRSV activity. Veterinary Microbiology 256 (2021)").

[0021] (2) Main reagents

[0022] LPA used for in vitro experiments was purchased from Avanti Polar Lipids, and HA130 used for both in vivo and in vitro experiments was purchased from MedChemExpress; the enhanced CCK8 kit was purchased from Beyotime Biotechnology Co., Ltd.; the anti-PRRSVN protein antibody (JN-0401) was purchased from Jinnuo Diagnostics, South Korea; the internal control GAPDH mouse monoclonal antibody and goat anti-mouse secondary antibody were purchased from Abcam; the reverse transcription reagent PrimeScriptTM RT Master Mix was purchased from Taraka, PerfectStart Green qPCR SuperMix was purchased from Quanshijin Company, and the LPA ELISA detection kit was purchased from Signalway Antibody (SAB); all other routine reagents were of analytical grade.

[0023] Example 1

[0024] Ten pigs were divided into a control group (5 pigs) and a challenge group (10 pigs). The highly pathogenic PRRSV strain Li11 was used for challenge. Pig serum was collected 3, 7, 10, and 21 days after challenge for non-targeted metabolomics and non-targeted lipidomics testing. The results are as follows: Figure 1 As shown, a significantly upregulated metabolic molecule, lysophosphatidic acid (LPA), was screened out. Its level was highly positively correlated with the viral load of PRRSV, and may play an important role in the infection and replication process of PRRSV.

[0025] Example 2

[0026] Effects of LPA and its inhibitor HA130 on PRRSV replication in iPAMs

[0027] (1) Cytotoxicity test of LPA and HA130 on iPAMs

[0028] The CCK8 cytotoxicity assay was used to determine the cytotoxicity of LPA and HA130 on iPAMs as follows:

[0029] 6 × 10 5 A cell suspension (100 μL / well) of 100 cells / mL was added, leaving one column unseeded. The cells were cultured at 37°C and 5% CO2 until fully confluent. The medium was aspirated, and the cells were washed three times with PBS. LPA was diluted to different concentrations (5 μM, 10 μM, 20 μM, 50 μM, 100 μM, and 500 μM) using RPMI-1640 medium, with three replicates per concentration, using 100 μL / well. After 48 hours of incubation at 37°C and 5% CO2, the solution was aspirated, the cells were washed three times with PBS, and a 10% (v / v) CCK8 solution prepared in RPMI-1640 medium was added to a 96-well plate (100 μL / well). The cells were incubated at 37°C and 5% CO2 for 1 hour. The absorbance (λ = 450 nm) was measured using a microplate reader, and the cell viability after 48 hours of treatment with different concentrations of the solution was calculated.

[0030] Similarly, HA130 was dissolved in DMSO and diluted to different concentrations (10nM, 50nM, 100nM, 200nM, 500nM, and 1000nM), with three replicates per concentration, 100μL / well. After incubation at 37°C, 5% CO₂ for 48 hours, the solution was aspirated, the cells were washed three times with PBS, and a 10% CCK-8 solution prepared in RPMI-1640 medium was added to a 96-well plate (100μL / well). Incubation was continued at 37°C, 5% CO₂ for 1 hour. The absorbance (λ = 450nm) was measured using a microplate reader, and cell viability after 48 hours of treatment with different concentrations of the solution was calculated.

[0031] The results are as follows Figure 2As shown in Figure A, the maximum safe concentration of LPA for iPAMs is 100 μM, and the maximum safe concentration of HA130 is 200 nM.

[0032] (2) Effects of LPA and HA130 on PRRSV replication in iPAMs—Western blot

[0033] 6 × 10 5 1 mL of cell suspension was inoculated at a cell concentration of 10 cells / mL and cultured at 37°C, 5% CO2 until the cells were fully confluent. The culture medium in the 6-well plate was discarded and washed 3 times with PBS. 1640 culture medium with LPA concentrations of 0 μM (negative control), 0 μM (positive control), 25 μM, 50 μM, and 100 μM was added to the 5 wells seeded with cells and treated for 1 hour. The treatment solution was discarded, and PRRSV virus solution (MOI = 0.1) was added to the wells except the negative control. After infection for 1 hour, the supernatant was discarded, and the plate was washed 3 times with PBS and replaced with 1640 culture medium containing 2% (v / v) FBS. The plate was cultured at 37°C, 5% CO2 for 24 hours.

[0034] The treatment solution was discarded, and the cells were washed three times with PBS. 200 μL of RIPA lysis buffer was added to each well and lysed on ice for 10 min. The cell pellets were mixed by pipetting and then transferred to 1.5 mL sterile EP tubes. The cells were centrifuged at 12000 r / min for 5 min, 180 μL of supernatant was aspirated and mixed with 36 μL of 5× loading buffer (5 times the concentration of loading buffer), and the protein samples for western blot were obtained by water bath at 100°C for 10 min.

[0035] The same method was used to obtain protein samples pretreated with 0 nM (negative control), 0 nM (positive control), 50 nM, 100 nM, and 200 nM HA130 and infected with PRRSV (MOI=0.1) for 24 h.

[0036] Electrophoresis was performed using a 10-well, 10% precast gel. The loading order was as follows: marker, 0 μM (negative control), 0 μM (positive control), 25 μM, 50 μM, and 100 μM LPA-treated samples. The electrophoresis instrument was programmed to run at 80 V for 30 minutes and 120 V for 1 hour. After electrophoresis, a semi-dry transfer system was used to assemble the transfer system in the following order: black side (negative electrode) - sponge - filter paper - protein gel - PVDF membrane - filter paper - sponge - white side (positive electrode). After assembly, use a clean test tube or glass rod to remove bubbles. The transfer instrument was programmed to run at 15 V for 40 minutes. After transfer, the PVDF membrane was removed, washed with TBST for 5 minutes, and blocked in 5% skim milk powder at room temperature for 1 hour.

[0037] Pour off the blocking solution and wash three times with TBST (5 minutes each). Then remove the membrane and wrap it in clean plastic wrap. Cut the membrane containing the 15kDa PRRSV-N protein and the 36kDa GAPDH and place it in the corresponding primary antibody. Incubate on a shaker at 40 rpm and 4°C overnight. After incubation, recover the primary antibody and wash three times with TBST (5 minutes each). Place both membranes in a solution of HRP-labeled goat anti-mouse IgG secondary antibody diluted in TBST (1:10,000 dilution) and incubate on a shaker at 40 rpm at room temperature for 1 hour. Recover the secondary antibody dilution and wash the membrane three times with TBST (5 minutes each). Soak the membrane in TBST. Prepare the ECL developer in the dark and develop the gel using a gel scanning system for imaging.

[0038] The same method was used to detect PRRSV-N protein and GAPDH bands in samples pretreated with 0 nM (negative control), 0 nM (positive control), 50 nM, 100 nM, and 200 nM HA130.

[0039] The results are as follows Figure 2 As shown in Figures B and C, western blot (WB) assays were used to examine PRRSV proliferation after LPA and HA130 treatment. Following LPA treatment, the color of the corresponding PRRSV-N protein-sized band gradually increased with increasing drug concentration; whereas, following HA130 treatment, PRRSV-N protein content gradually decreased with increasing drug concentration. This indicates that LPA promotes PRRSV proliferation, while its inhibitor, HA130, inhibits PRRSV replication, and both actions are dose-dependent.

[0040] (3) Effects of LPA and HA130 on PRRSV replication in iPAMs—TCID 50

[0041] 6 × 10 51 mL of cells was inoculated into each well at a cell concentration of 10 cells / mL. 1640 medium containing LPA at concentrations of 0 μM (negative control), 0 μM (positive control), 25 μM, 50 μM, and 100 μM was added to five wells seeded with cells. DMSO-dissolved HA130 was added to the other five wells seeded with cells at concentrations of 0 nM (negative control), 0 nM (positive control), 50 nM, 100 nM, and 200 nM, respectively. After 1 hour of drug treatment, the treatment solution was discarded. PRRSV virus solution (MOI = 0.1) was then added to the wells except the negative control. After 1 hour of infection, the supernatant was discarded, the cells were washed three times with PBS, and then replaced with 1640 medium containing 2% (v / v) FBS. The cells were cultured at 37°C and 5% CO2 for 24 hours. The cell culture plate was removed and sealed with sealing film. The culture was frozen and thawed three times at -80°C. The culture was collected into a sterile 1.5 mL EP tube and centrifuged in a high-speed centrifuge at 10,000 r / min at 4°C for 5 min. The supernatant was collected into a new 1.5 mL EP tube and stored at -80°C.

[0042] Press 6×10 5 Cells were seeded in 5 96-well plates at a concentration of 100 μL per well and cultured at 37°C and 5% CO2 until the cells were fully confluent. The 96-well plates with cells were washed 3 times with PBS and the collected samples were diluted 10-fold to 0-10 -7 8 gradients, add the diluted samples to the cell wells of the corresponding dilution multiples, repeat 8 wells for each gradient, 100 μL per well, and culture at 37°C, 5% CO2 for 5 to 7 days.

[0043] The number of lesion wells in each dilution gradient was observed and recorded under a microscope, and the viral titer of PRRSV infection 24 h after iPAMs were treated with different concentrations of LPA and its inhibitor HA130 was calculated using the Reed-Muench method.

[0044] The results are as follows Figure 2 As shown in Figures D and E, after LPA treatment of iPAMs, the viral titer of PRRSV was higher 24 hours after infection, while HA130 treatment could reduce the viral titer of PRRSV, and both were dose-dependent.

[0045] Example 3

[0046] The therapeutic effect of HA130 on piglets infected with PRRSV

[0047] (1) Piglet treatment experimental design

[0048] Forty-eight four-week-old commercial piglets (Large White pigs, purchased from Wen's Food Group Co., Ltd.) were tested negative for PRRSV, PCV2, PRV, and CSFV antibodies and antigens by ELISA and PCR. They were randomly divided into four groups (12 pigs per group):

[0049] The first group was the blank control group, in which each pig was injected with an equal volume of DMEM to the challenge dose. The drug-treated group was injected with an equal volume of empty solvent (10% DMSO + 40% PEG3000 + 5% Tween-80 + 45% saline) at the beginning of treatment.

[0050] The second group was the drug-treated control group, in which each pig was injected with an equal volume of DMEM to the challenge dose and an equal volume of HA130 solution during the treatment phase;

[0051] The third group was the challenge group, and each pig was intramuscularly injected with 2 mL of the virus with a titer of 1×10 -6 The highly pathogenic PRRSV strain Li11 virus solution with TCID50 / ml was injected simultaneously with an equal volume of empty solvent (10% DMSO + 40% PEG3000 + 5% Tween-80 + 45% saline) in the drug-treated group.

[0052] The fourth group was the challenge treatment group, and each pig was injected intramuscularly with 2 mL of the titer of 1x10 -6 The highly pathogenic PRRSV strain Li11 virus liquid with TCID50 / ml was injected intramuscularly with a dose of 1 mg / kg body weight of HA130 solution every day for three consecutive days starting when the pig's body temperature rose and obvious fever symptoms were detected.

[0053] HA130 solution formula: 10% DMSO + 40% PEG3000 + 5% Tween-80 + 45% saline + HA130.

[0054] Experiment and sampling arrangements:

[0055] After the piglets were transported to the disinfected experimental site, they were randomly divided into 4 groups and placed in different sites. They were allowed to acclimate to the environment with free access to water and food for 2 days. On the 3rd day, the third and fourth groups were intramuscularly injected with the virus, and the first and second groups were injected with an equal volume of DMEM. After the infection, the rectal temperature of the piglets was monitored daily. After the third and fourth groups of piglets showed high fever symptoms (≥40°C), HA130 treatment was started. The first and second groups of piglets were intramuscularly injected with 1 mg / kg body weight of HA130 solution for three consecutive days. The rectal temperature and clinical symptoms of the piglets in each group were continuously monitored, including body weight, skin, eating habits, mental state, etc.

[0056] Piglet serum was collected 1, 3, 5, and 7 days after the end of administration to detect changes in lysophosphatidic acid levels. Three piglets were randomly selected from each group and killed to collect lung, spleen, and inguinal lymph node samples, three tissues susceptible to PRRSV infection and pathological changes, to measure viral load and examine tissue pathology.

[0057] (2) Clinical symptoms

[0058] No piglets died during the experiment, and the body temperature monitoring results were as follows: Figure 3 As shown in Figure A, on the second day after the challenge, the third and fourth groups of piglets injected with PRRSV virus solution all developed high fever symptoms (≥40°C). Therefore, on days 2-4 after the challenge, the control group and the challenge treatment group were treated with HA130, while the other two groups were injected with an equal volume of solvent. The results showed that HA130 treatment reduced the high fever symptoms of the treated piglets. On the third day after the start of treatment, the body temperature of the treated piglets had dropped below 40.5°C and was approaching normal body temperature.

[0059] (3) Serum LPA content detection

[0060] Piglet serum was collected 1, 3, 5, and 7 days after the end of administration, and serum LPA levels were detected using the SAB LPA ELISA Kit as follows:

[0061] Use sample diluent to dissolve the LPA standard provided by the kit and dilute it step by step to seven concentration gradients of 4000pg / mL, 2000pg / mL, 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, and 62.5pg / mL. Set up blank wells in a 96-well plate, add gradient concentration standards and serum samples in sequence, 50μL per well, then add 50μL HRP-Conjugate to each well except the blank well, gently tap to mix, cover with film, and incubate at 37°C for 1h; discard the solution, add 250μL wash buffer to each well, let it stand for 1min, and then aspirate it. Repeat this washing 5 times. After the last wash, turn the well plate upside down on absorbent paper and let it stand for 3min; add 100μL Substrate to each well except the blank well. Solution (substrate solution), incubate in the dark at 37°C for 20 minutes. When the high-concentration standard in the first three wells turns a distinct blue, add 50 μL Stop Solution (stop buffer) to each well. Use a microplate reader to measure the absorbance at 450 nm, draw a standard curve, and calculate the LPA concentration in the sample wells.

[0062] The results are as follows Figure 3As shown in Figure B, the LPA level in the PRRSV-infected group was consistent with the metabolomics results. PRRSV infection upregulated the host serum LPA content, while HA130 treatment effectively reduced the serum LPA content, making the serum LPA content of PRRSV-infected piglets tend to normal levels.

[0063] (4) Viral load detection of lung, spleen, and inguinal lymph node tissue samples

[0064] Three piglets were randomly selected from each group 1, 3, 5, and 7 days after the end of administration. The lung, spleen, and inguinal lymph node samples, three tissues susceptible to PRRSV infection and pathological changes, were collected and the expression level of PRRSV-N mRNA was determined by qRT-PCR. The details are as follows:

[0065] After thawing, approximately 1 g of tissue sample was transferred to a tissue disruptor tube, 1 mL of Trizol was added, and sterilized ceramic beads were added. The sample was placed in a tissue disruptor and vigorously shaken for 3 times (20 s / time). The sample was then placed on ice for 20 min. The sample was centrifuged at 12,000 rpm for 5 min, and the supernatant was transferred to a clean RNase-free EP tube. 200 μL of chloroform was added, the sample was vortexed vigorously, and the sample was allowed to stand for stratification. The sample was then centrifuged at 12,000 rpm for 10 min. The supernatant was transferred to a new RNase-free EP tube, an equal volume of isopropanol was added, the sample was mixed, and the sample was precipitated at -20°C for 1 h. The sample was centrifuged at 12,000 rpm for 15 min, and the supernatant was discarded to obtain the RNA precipitate. The sample was rinsed twice with 75% alcohol, air-dried for 5 min, and dissolved in 50 μL of RNase-free water. The concentration and OD value were measured. Reverse transcription was performed according to the Taraka PrimeScript™ RT Master Mix (Perfect Real Time) instructions. The reverse transcription system consisted of 2 μL of 5× PrimeScript RT Master Mix, 500 ng of total RNA, and RNase-free ddH₂O added to 10 μL. After thorough mixing, the reverse transcription reaction was performed in a PCR instrument using the following protocol: 37°C for 15 min; 85°C for 5 s. The product was frozen at −80°C for future use.

[0066] Using the above cDNA as a template, real-time fluorescence quantitative PCR was performed to detect the expression levels of PRRSV-N and GAPDH mRNA in tissue samples. The primer sequences are as follows:

[0067] PRRSV-NF: 5'-TAAGATCATCGCCCAACAAA-3'

[0068] PRRSV-NR: 5'-TCGGCAAACTAAACTCCACA-3';

[0069] GAPDH-F: 5'-CCTTCCGTGTCCCTACTGCCAAC-3'

[0070] GAPDH-R: 5'-GACGCCTGCTTCACCACCTTCT-3'.

[0071] PCR reactions were performed according to the instructions for PerfectStart Green qPCR SuperMix from Quanshijin. The reaction system consisted of 1 μL cDNA, 5 μL 2× PerfectStart Green qPCR SuperMix, 0.2 μL Passive Reference Dye (50×), 0.2 μL each of 10 μM upstream and downstream primers, and 3.4 μL RNase-free water. The system was prepared for three replicates per sample, and 10 μL was added to each well of a 384-well plate. The reaction was performed on an ABI 7300 Real-time PCR instrument with the following protocol: initial denaturation at 95°C for 2 min; 95°C for 15 s, and 61°C for 31 min, for a total of 40 cycles. A 2% PCR reaction was used based on the CT value. -ΔΔCT The expression levels of PRRSV-N and GAPDH mRNA in each tissue sample were calculated by the method.

[0072] The results are as follows Figure 3 As shown in Figures C, D, and E, the PRRSV-N mRNA levels in the lungs, spleens, and inguinal lymph nodes of piglets after HA130 treatment were significantly reduced compared with those in the challenge group, indicating that HA130 has the effect of inhibiting PRRSV replication in vivo.

[0073] (5) Histopathological changes

[0074] Remove a small piece of tissue no larger than 4 mm × 2 cm from the collected lung, spleen, and inguinal lymph nodes. 2 The tissue blocks were fixed with 4% paraformaldehyde, dehydrated with graded alcohol, transparentized with xylene, embedded in paraffin, trimmed, sectioned, and stained with hematoxylin and eosin. Lung tissue sections were observed under a microscope.

[0075] The results are as follows Figure 4As shown: The lung, spleen, and inguinal lymph node tissue sections of the drug-treated control group showed no lesions, indicating that HA130 has no significant effect on host organs. Compared with the mock control group, the lung tissue sections of the challenge group showed a large number of red blood cells between the cells around the blood vessels, which is a diffuse hemorrhage phenomenon after infection. In addition, pathological features characteristic of porcine reproductive and respiratory syndrome (PRRS) such as alveolar hypertrophy, alveolar wall epithelial macrophage proliferation, alveolar septal thickening, inflammatory cell infiltration, and spleen hemorrhage were observed. In contrast, the lung tissue of the challenge treatment group showed no obvious hemorrhage spots around the blood vessels, and the alveolar wall and alveolar size were normal. There was no obvious hemorrhage in the spleen of the treatment group. There was no significant difference in inguinal lymph node between the groups. These results indicate that HA130 treatment effectively reduces PRRSV-induced tissue damage.

[0076] The above-described embodiments represent only a few of the many implementation methods of the present invention. Although the description is relatively specific and detailed, it should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and such modifications and improvements are within the scope of protection of the present invention.

Claims

1. Use of a lysophosphatidic acid inhibitor in the preparation of a drug for inhibiting PRRSV replication, characterized in that: The lysophosphatidic acid inhibitor is HA130.

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