Streptomyces sp. lm-35, metabolites thereof and use thereof in the preparation of a medicament for the treatment or prevention of porcine reproductive and respiratory syndrome virus infection

Abstract

This invention belongs to the field of antiviral drugs for livestock and poultry, and discloses Streptomyces LM-35, its metabolites, and their application in the preparation of drugs for treating or preventing porcine reproductive and respiratory syndrome virus (PRRSV) infection. Anti-PRRSV activity studies were conducted using the fermentation supernatant of Streptomyces LM-35 at the cellular and in vivo levels, revealing significant anti-PRRSV activity both in vivo and in vitro. Pigs fed with Streptomyces LM-35 fermentation broth achieved a survival rate of 80%; this effectively controls the adverse effects of PRRSV infection on the body and can be used for PRRSV prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of antiviral microecological preparations for livestock and poultry, specifically involving Streptomyces LM-35, its metabolites, and their application in the preparation of drugs for the treatment or prevention of porcine reproductive and respiratory syndrome virus infection. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Its typical clinical features include reproductive disorders in pregnant sows (such as abortion, stillbirth, and weak piglets) and respiratory symptoms in pigs of all ages (such as coughing, dyspnea, and pneumonia). PRRSV induces persistent immunosuppression by targeting key immune cells such as alveolar macrophages, significantly increasing the risk of secondary bacterial or viral co-infections. Furthermore, the virus has an extremely high genetic mutation rate (especially in the ORF5 and Nsp2 gene regions), resulting in low antigenic matching between existing commercial vaccine strains and circulating wild-type strains, leading to limited cross-protective efficacy. Therefore, there is an urgent need to develop novel prevention and control strategies with broad-spectrum antiviral activity, novel mechanisms of action, and low susceptibility to inducing drug resistance. Streptomyces spp., as the most representative genus of Actinobacteria with the richest secondary metabolites, can synthesize natural compounds with diverse structures and broad biological activities (such as macrolides, anesamcinolones, and nucleoside derivatives). Some of these metabolites have been shown to have the potential to regulate the host's innate immune response or directly inhibit viral replication, providing important resources and new pathways for the development of original antiviral drugs targeting PRRSV.

[0003] Related studies have shown that secondary metabolites produced by Streptomyces can exert antiviral effects through multiple mechanisms, including directly disrupting the structural integrity of viral particles, targeting and inhibiting key steps in viral replication (such as genome replication, protein translation, and viral assembly), and bidirectionally regulating the host's innate and adaptive immune responses. For example, Streptomyces lavendulae can efficiently synthesize 1-deoxynojirimycin (DNJ) under specific solid-state fermentation conditions. This compound specifically inhibits the activity of host α-glucosidase I / II, blocking the N-linked glycosylation modification of the porcine reproductive and respiratory syndrome virus (PRRSV) envelope glycoprotein GP5, leading to abnormal spatial conformation and inhibited maturation of GP5, thereby significantly inhibiting the fusion of the viral envelope with the host cell membrane and reducing viral invasion efficiency. Compared with traditional chemically synthesized antiviral drugs, active metabolites derived from Streptomyces have comprehensive advantages such as high natural structural diversity, novel targets, excellent biocompatibility, and low risk of drug resistance. Furthermore, by systematically optimizing key fermentation parameters such as carbon-nitrogen source ratio, inorganic salt concentration, pH gradient, dissolved oxygen level, and induction timing, the potency and stability of the target active product can be significantly improved. Combining this with genome mining platforms (such as antiSMASH) for precise prediction and functional annotation of secondary metabolic gene clusters, supplemented by heterologous expression and gene knockout verification, can efficiently elucidate potential antiviral biosynthetic pathways, accelerating the discovery, structural confirmation, and mechanistic elucidation of novel lead compounds. Therefore, screening and identifying Streptomyces strains with potent and specific anti-PRRSV activity not only helps expand the antiviral natural product resource library but also has significant scientific value and application prospects for developing safe and sustainable new prevention and control strategies and alternative treatments.

[0004] This study used a high-throughput cytopathic effect (CPE) inhibition screening system to obtain a Streptomyces strain (tentatively designated LM-35) that showed significant inhibitory activity against PRRSV replication. Quantitative analysis by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) confirmed that no DNJ or other known inhibitors targeting GP5 glycosylation (such as castanospermine and swainsonine) were detected in its active metabolite profile. This result breaks through the existing cognitive framework that "Streptomyces' anti-PRRSV activity mainly depends on DNJ mediation" and provides new clues for further revealing the non-classical mechanism of antiviral activity of Streptomyces metabolites. Summary of the Invention

[0005] The purpose of this invention is to address the problem of the difficulty in preventing and treating porcine reproductive and respiratory syndrome virus (PRRSV) infection by providing a Streptomyces strain LM-35 that is resistant to PRRSV infection. The preservation number of this Streptomyces strain is: CCTCC NO: M 2026442.

[0006] Another object of the present invention is to provide the use of Streptomyces LM-35 or its fermentation supernatant in the preparation of medicaments for the treatment or prevention of porcine reproductive and respiratory syndrome virus infection.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] The applicant has screened a Streptomyces strain from its own Streptomyces library that has a significant inhibitory effect on porcine reproductive and respiratory syndrome virus infection. This strain was deposited on March 13, 2026, at the China Center for Type Culture Collection (CCTCC), with the classification name: Streptomyces LM-35, accession number CCTCC NO: M 2026442, address: Wuhan University, Wuhan, China.

[0009] Streptomyces LM-35 is a Gram-positive bacterium that forms spores. Its hyphae consist of substrate hyphae and aerial hyphae, which can develop into spiral or linear sporophytes. The spores often have wart-like or spiny patterns on their surface. Colonies on ISP medium are 5-10 mm in diameter. The hyphae of this genus are septate, and the colonies are dense and dry. Upon maturity, they reproduce by conidia. Studies have shown that the optimal growth temperature is 28℃, and the optimal pH is neutral to slightly alkaline. 16S analysis revealed that this bacterium shares 99.9% similarity with Streptomyces.

[0010] Further experiments by the applicant revealed that 4-methylcatechol, one of the active ingredients in the fermentation supernatant of LM-35, exhibits significant anti-PRRSV activity. 4-methylcatechol has the molecular formula C7H8O2, a molecular weight of 124.14, CAS number 452-86-8, and its structural formula is as follows:

[0011] .

[0012] The scope of protection of this invention includes:

[0013] Fermentation supernatant of Streptomyces LM-35.

[0014] One of the active ingredients in the fermentation supernatant described above is 4-methylcatechol.

[0015] A compound, wherein the active ingredient comprises Streptomyces LM-35 with accession number CCTCC NO: M 2026442 and / or the fermentation supernatant of Streptomyces LM-35.

[0016] The use of Streptomyces LM-35, the fermentation supernatant of Streptomyces LM-35, and / or the above-mentioned compound in the preparation of drugs for the treatment or prevention of porcine reproductive and respiratory syndrome virus infection.

[0017] Application of Streptomyces LM-35, fermentation supernatant of Streptomyces LM-35 and / or the above-mentioned compound in the preparation of porcine reproductive and respiratory syndrome vaccine.

[0018] Application of Streptomyces LM-35, fermentation supernatant of Streptomyces LM-35 and / or the above-mentioned compound in the preparation of pig feed additives.

[0019] Compared with the prior art, the advantages of the present invention are as follows:

[0020] This invention is the first to isolate a Streptomyces strain LM-35 that can produce 4-methylcatechol. The fermentation supernatant of Streptomyces LM-35 was used to conduct anti-infection studies on PRRSV at the cellular and animal levels, and it was found that it has significant anti-PRRSV activity in vivo and in vitro.

[0021] The Streptomyces LM-35 provided by this invention, as a potential antiviral microecological agent, has the following advantages compared with vaccines and traditional antiviral chemical drugs:

[0022] 1. Streptomyces has low production costs, strong resistance to adverse conditions, and is convenient to store, transport, and use.

[0023] 2. No toxic side effects, no residue, antiviral, promotes growth, green and safe.

[0024] 3. It has the advantages of being easy to use, safe, without immune stress, and highly economical.

[0025] 4. The Streptomyces LM-35 fermentation broth of this invention can reduce the infectivity of PRRSV cells at the cellular level, inhibit PRRSV infection of cells, and reduce viral proliferation on cells, and can be used to prevent and control PRRSV infection.

[0026] 5. The Streptomyces LM-35 fermentation broth of this invention, when administered orally to experimental pigs, can effectively protect against morbidity and mortality caused by PRRSV infection. 80% of the pigs in the control group died, while the survival rate of pigs fed with Streptomyces LM-35 fermentation broth reached 80%. It can effectively control the adverse effects of PRRSV infection on the body and can be used for the prevention and control of PRRSV. Attached Figure Description

[0027] Figure 1 Screening of Streptomyces fermentation supernatant with PRRSV resistance;

[0028] A represents the effect of RT-PCR detection of different strains of Streptomyces supernatant on the proliferation of PRRSV-WH3 virus; B represents the effect of RT-PCR detection of different strains of Streptomyces supernatant on the proliferation of PRRSV-GFP virus.

[0029] Figure 2 The effect of Streptomyces LM-35 fermentation supernatant on the proliferation of different PRRSV strains in primary PAM cells;

[0030] In this study, A represents the effect of Streptomyces LM-35 fermentation supernatant on PRRSV-NADC30 proliferation in primary PAM cells; B represents the effect of Streptomyces LM-35 fermentation supernatant on PRRSV-WH3 proliferation in primary PAM cells; C represents the effect of Streptomyces LM-35 fermentation supernatant on HP-PRRSV proliferation in primary PAM cells; and D represents the effect of Streptomyces LM-35 fermentation supernatant on PRRSV-GFP proliferation in primary PAM cells.

[0031] Figure 3 The effect of different concentrations of Streptomyces LM-35 fermentation supernatant on Marc145 cytotoxicity.

[0032] Figure 4 The effect of Streptomyces LM-35 fermentation supernatant on the proliferation of recombinant PRRSV-GFP in Marc145 cells.

[0033] Figure 5 The effect of Streptomyces LM-35 fermentation supernatant on PRRSV-WH3 virus proliferation in Marc145 cells;

[0034] In this study, A represents the effect of LM-35 fermentation supernatant on the proliferation of PRRSV-WH3 in Marc145 cells detected by RT-PCR; B represents the effect of LM-35 fermentation supernatant on the proliferation of PRRSV-WH3 in Marc145 cells detected by TCID50; and C represents the effect of LM-35 fermentation supernatant on the expression of N protein in PRRSV-WH3 in Marc145 cells detected by Western blot.

[0035] Figure 6 The effect of the Streptomyces metabolite 4-methylcatechol on the proliferation of different PRRSV strains in Marc145 cells;

[0036] In this study, A represents the effect of the metabolite 4-methylcatechol on the proliferation of PRRSV-WH3 in Marc145 cells; B represents the effect of the metabolite 4-methylcatechol on the proliferation of PRRSV-NADC30 in Marc145 cells; C represents the effect of the metabolite 4-methylcatechol on the proliferation of PRRSV-GFP in Marc145 cells; and D represents the effect of the metabolite 4-methylcatechol on the proliferation of HP-PRRSV in Marc145 cells.

[0037] Figure 7 The effect of feeding Streptomyces LM-35 fermentation supernatant on the survival rate of PRRSV-infected pigs. Detailed Implementation

[0038] To better understand the content of this invention, the following detailed description, in conjunction with specific embodiments, further illustrates the invention. However, the scope of protection of this invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and conditions in the embodiments of this invention are conventional methods. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the field; unless otherwise specified, the reagents or materials are all derived from commercial sources.

[0039] The GenBank accessions for PRRSV-WH3, PRRSV-NADC30, and HP-PRRSV used in this invention are: HM853673.2, JN654459.1, and HQ401282.1, respectively.

[0040] The PRRSV-GFP strain is a recombinant virus constructed by inserting a green fluorescent protein (GFP) coding sequence into the PRRSV-NADC30 backbone. Its GFP expression is stable and does not affect the basic replication characteristics of the virus.

[0041] Example 1:

[0042] Screening for Streptomyces strains resistant to PRRSV infection

[0043] 1. Culture of Marc-145 cells

[0044] Marc145 cells frozen in liquid nitrogen were rapidly thawed in a 37°C water bath. Once the remaining ice crystals in the cryovial had completely melted, they were immediately transferred to a centrifuge tube containing 10 mL of pre-warmed RPMI-1640 complete medium containing 10% fetal bovine serum (FBS) and gently mixed. The cells were then centrifuged at 300 × g at 4°C for 5 min, the supernatant was discarded, and the cell pellet was resuspended in 10% FBS-RPMI-1640 medium. The cells were then seeded into T25 cell culture flasks and placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for static culture. Once the cells have adhered and merged to form a uniform monolayer (80–90%), discard the old culture medium and gently wash three times with calcium- and magnesium-free PBS (pH 7.2–7.4). Add 0.25% trypsin-EDTA solution (pre-warmed to 37°C) and digest for 60–90 minutes. Observe under a microscope until the intercellular spaces increase and the edges retract but are not completely detached. Immediately add an equal volume of pre-warmed medium containing 10% FBS-RPMI-1640 to terminate the digestion. Gently pipette to prepare a single-cell suspension, centrifuge at 300×g and 4°C for 5 minutes, discard the supernatant, resuspend in fresh complete culture medium, and seed into new T25 culture flasks or cell culture plates at the required density for later use.

[0045] 2. Preparation of Streptomyces fermentation supernatant

[0046] The applicant's laboratory retained multiple Streptomyces glycerol cultures and streaked them. First, the glycerol cultures from the -80°C freezer were removed, and a small amount of the culture was streaked evenly onto a solid culture dish containing ISP-2 (yeast malt extract agar). The dish was then incubated upside down at 28°C for 48 hours. After 48 hours, the dish was removed, and a single colony was inoculated into a conical flask containing 50 ml of sterile ISP-2 (yeast malt extract without agar) liquid medium. The flask was then incubated on a shaker at 28°C for 48 hours. A plate test showed the viable bacterial count was approximately 10⁻⁶. 5 CPU / mL, then inoculated into 50ml of ISP-2 liquid medium at a bacterial culture-to-medium ratio of 1:1000. The conical flask was incubated on a shaker at 28℃ for 48h, followed by plate testing to determine the bacterial count. The viable count was approximately 10. 5 Collect the supernatant at CPU / mL. First, transfer the bacterial culture into a 50ml centrifuge tube in an aseptic operating table, centrifuge at 3500r / min for 10min at 4℃. After centrifugation, slowly pour the supernatant into a new aseptic tube in an aseptic operating table and discard the precipitated bacterial cells. Then, collect the supernatant and filter it in batches through a 0.22μM filter in an aseptic operating table. Finally, store the filtered fermentation supernatant at -80℃ for later use in the following examples.

[0047] 3. Screening of PRRSV-resistant Streptomyces supernatant

[0048] To identify Streptomyces supernatant with PRRSV-resistant activity, a dual-strain screening was performed using PRRSV-WH3 and PRRSV-GFP strains. The prepared Streptomyces supernatant was added to RPMI-1640 medium at a final concentration of 2% (v / v) and thoroughly mixed before being inoculated into Marc145 cells. After 6 hours of treatment, the cells were inoculated with PRRSV-WH3 and PRRSV-GFP strains, respectively. Two hours after infection, the medium was changed, and fresh 2% concentration of fermentation supernatant was inoculated again into a mixture of fermentation supernatant and RPMI-1640 medium. Virus fluid was collected after 32 hours for RT-PCR detection.

[0049] The results are as follows Figure 1 As shown, tests revealed that samples 6 (LM-35) and 8 (LM-T4) exhibited antiviral activity against both strains of PRRSV, with sample 6 showing the best antiviral activity. While sample 14 showed antiviral activity in the test, cell observation revealed significant cell damage, resulting in false positives. Therefore, LM-35 was selected as the final test strain. The final Streptomyces strain LM-35 was deposited on March 13, 2026, at the China Center for Type Culture Collection (CCTCC), with the classification name: Streptomyces LM-35, accession number: CCTCC NO: M 2026442, address: Wuhan University, Wuhan, China.

[0050] Tests revealed that *Streptomyces* LM-35 is a Gram-positive bacterium. This bacterium forms spores, and its hyphae consist of both substrate and aerial hyphae. The aerial hyphae can develop into spiral or linear sporophytes, and the spores often have wart-like or spiny patterns on their surface. Colonies on ISP medium are 5-10 mm in diameter. The hyphae of this genus are septate, and the colonies are dense and dry. Upon maturity, they reproduce by conidia. Studies have shown that the optimal growth temperature for this bacterium is 28℃, and the optimal pH is neutral to slightly alkaline. 16S analysis revealed that this bacterium has a 99.9% similarity to *Streptomyces*.

[0051] 4. Anti-PRRSV treatment of Streptomyces LM-35 fermentation supernatant in Marc 145 cells and alveolar macrophages (PAM)

[0052] Marc 145 cells in logarithmic growth phase were injected with 1×10⁻⁶ cells. 5Cells were evenly seeded at a density of 500 μL / mL in 24-well cell culture plates and incubated in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity. After the cells adhered and merged to form a uniform monolayer (80–90%), the old culture medium was discarded, and the cells were gently washed three times with calcium- and magnesium-free PBS (pH 7.2–7.4) pre-warmed to 37°C. Then, 500 μL of RPMI-1640 complete medium (containing 10% FBS) with 10 μL / mL Streptomyces LM-35 fermentation supernatant was added to each well for pretreatment. A blank control group was set up, which was treated with an equal volume of complete medium without Streptomyces LM-35 fermentation supernatant. After 6 h of pretreatment, the supernatant was discarded, and the cells were washed three times with PBS. 500 μL of a co-incubation mixture containing Streptomyces fermentation supernatant (final concentration 10 μL / mL) and PRRSV (MOI = 0.1) was added to each well, and the cells were incubated at 37°C and 5% CO2 for 2 h for virus adsorption and infection. After adsorption, the mixture was discarded, and the cells were washed three times with PBS to remove unadsorbed virus. Then, 500 μL of fresh complete culture medium (containing the same concentration of Streptomyces LM-35 fermentation supernatant) was added to each well, and the cells were incubated at 37°C and 5% CO2 for another 24 h. The treatment procedure for primary porcine alveolar macrophages (PAM) was completely consistent with that for Marc-145 cells, including seeding density, pretreatment time, virus adsorption conditions, and subsequent culture parameters.

[0053] The results are as follows Figure 2 As shown, primary PAM cells were treated with Streptomyces LM-35 fermentation supernatant and then inoculated with PRRSV-NADC30, PRRSV-WH3, PRRSV-GFP, and HP-PRRSV, respectively. RT-PCR detection revealed that the PRRSV load in the supernatant was significantly reduced, indicating that Streptomyces LM-35 fermentation supernatant significantly inhibited the proliferation of PRRSV in primary PAM cells.

[0054] 5. Cytotoxicity assay of Streptomyces LM-35 fermentation supernatant against Marc145 (CCK-8)

[0055] The cytotoxicity of Streptomyces LM-35 fermentation supernatant to Marc145 cells was assessed using the CCK-8 Cell Proliferation and Virulence Assay Kit (Beyotime, Cat. No. C0037). The specific procedures are as follows: Marc145 cells in logarithmic growth phase were seeded at a suitable density of 8 × 10³ cells per well in a 96-well plate (100 μL per well). The cells were cultured at 37°C and 5% CO2 for 24 h until adherence and a uniform monolayer formed. The old culture medium was discarded, and the cells were gently washed twice with pre-warmed (37°C) calcium- and magnesium-free PBS. Fresh complete culture medium containing a series of volume concentrations of Streptomyces LM-35 fermentation supernatant was added to each well. A solvent control group (containing no Streptomyces LM-35 fermentation supernatant) and a blank control group (containing only culture medium) were set up. After culturing for another 24 h, 10 μL of CCK-8 working solution (culture medium: CCK-8 = 10:1) was added to each well, and the cells were incubated at 37°C in the dark for 2 h. The absorbance (OD) of each well was measured at 450 nm using a BMG LabTech PolarStar Omega multi-mode microplate reader. 450 Cell viability (%) is calculated using the following formula: Cell viability = [(OD200%) / (OD200%) 450 (sample) − (OD) 450 Blank)] / [(OD) 450 Solvent control) − (OD 450 [Blank)]×100%.

[0056] The results are as follows Figure 3 As shown, the survival rate of Marc145 cells was above 90% in the fermentation supernatant of Streptomyces LM-35 at a concentration of 10% (i.e., 100 μL / mL), and the fermentation supernatant at a concentration of 10% or less was non-toxic to the cells.

[0057] 6. Cellular fluorescence lesion assay

[0058] Marc145 cells were fed at a rate of 1×10 5 The virus was evenly seeded in 24-well plates. After 24 h, the plates were washed three times with sterile PBS and then inoculated with either anti-PRRSV positive drug (1-Palmitoyl-sn-glycero-3-phosphocholine) or DMEM medium containing 2% (v / v) Streptomyces LM-35 fermentation supernatant. After 4 h of incubation, the PRRSV-GFP recombinant virus strain was inoculated at 0.1 MOI. After 3 h, the viral inoculum was removed, washed three times with PBS, and then inoculated with either anti-PRRSV positive drug or DMEM medium containing 2% (v / v) Streptomyces LM-35 fermentation supernatant. After 32 h, the cytopathic effect was observed and photographed under a fluorescence microscope.

[0059] The results are as follows Figure 4As shown, the expression level of PRRSV-GFP fluorescence was significantly reduced after treatment of Marc145 cells with the fermentation supernatant of Streptomyces LM-35, indicating that the fermentation supernatant of Streptomyces LM-35 significantly inhibited the proliferation of PRRSV in Marc 145 cells.

[0060] 7. Determination of viral load in cells and viral fluids

[0061] Marc145 cells were fed at a rate of 1×10 5 The virus inoculum was evenly spread in 24-well plates. After 24 h, the plates were washed three times with sterile PBS. The plates were then inoculated with either anti-PRRSV positive drug (1-Palmitoyl-sn-glycero-3-phosphocholine) or DMEM medium containing 2% (v / v) Streptomyces LM-35 fermentation supernatant. After 4 h of incubation, the PRRSV-GFP recombinant strain was inoculated at 0.1 MOI. After 3 h, the viral inoculum was removed, washed three times with PBS, and then inoculated with either anti-PRRSV positive drug or a mixture of supernatant containing 2% LM-35 fermentation supernatant and DMEM medium. After 32 hours of complete cytopathic effect, cells in 24-well plates were subjected to three freeze-thaw cycles at -80°C (each freeze-thaw cycle at -80°C for at least 30 min, followed by rapid thawing at 37°C in a water bath until completely liquefied) to fully lyse the cells and release intracellular viral particles. The cells were then centrifuged at 1200 × g for 10 min at 4°C, and the supernatant was carefully collected and stored at -80°C for subsequent TCID administration. 50 Viral load was detected using RT-PCR. Viral load was determined according to the one-step real-time quantitative RT-PCR (qRT-PCR) method recommended in the National Standard of the People's Republic of China for Diagnostic Techniques of Porcine Reproductive and Respiratory Syndrome (GB / T 18090—2023).

[0062] Weston blot sample cell processing is the same as RT-PCR cell processing. After complete cytopathic effect, the supernatant is discarded, and the cells are washed three times with PBS. Then, 100 μL of cell lysis buffer (containing protease inhibitors) is added. After lysis on ice for 10 minutes, the mixture of cells and lysis buffer is collected and centrifuged at 3000 rpm for 5 minutes at 4°C. The supernatant is then added to 1× protein loading buffer and boiled in a water bath at 100°C for 10 minutes. Subsequently, the samples are centrifuged at 5000 rpm for 5 minutes at 4°C. After centrifugation, the samples are stored at -20°C for subsequent Weston blot experiments.

[0063] The results are as follows Figure 5 As shown, RT-PCR and TCID 50The results showed that treatment of Marc145 cells with LM-35 fermentation supernatant significantly reduced PRRSV-WH3 viral proliferation, and the effect of LM-35 fermentation supernatant on PRRSV-WH3 proliferation in Marc145 cells was gradient-dependent. Figure 5 (A and B) indicates that the fermentation supernatant of mold LM-35 significantly inhibited the proliferation of PRRS virus particles in Marc 145 cells. Weston blot results showed that treatment of Marc145 cells with LM-35 fermentation supernatant significantly reduced the expression of PRRSV-WH3-N protein (). Figure 5 (C), further demonstrating the inhibitory effect of LM-35 fermentation supernatant on PRRSV.

[0064] Example 2:

[0065] LS-MS reveals anti-PRRSV components in Streptomyces fermentation supernatant

[0066] The target Streptomyces LM-35 and six Streptomyces strains without PRRSV resistance were streaked simultaneously. Single colonies were picked and placed in 50 ml of ISP liquid medium. After incubation at 28 °C for 24 h on a shaker, the culture was centrifuged at 3500 r / min for 10 min. The supernatant was collected and filtered through a 0.22 μM filter. The supernatant was then aliquoted into 15 ml centrifuge tubes, 5 ml per tube, and stored at -80 °C for subsequent LS-MS experiments.

[0067] Raw LS-MS data were processed using Compound Discoverer 3.1 (retention time tolerance 0.2 min, mass tolerance 5 ppm, S / N ≥ 3) to screen for stable peaks (TIC intensity ≥ 0.01% and present in 50% of samples). Characteristic peaks were matched with adducts [M+H]⁺ / [M−H]⁻ and validated using mzCloud and mzVault databases (score ≥ 70). Statistical analysis was performed using R / Python. After normalization by natural logarithm or CLR, differential metabolites were screened using PCA / OPLS-DA (log2FC ≥ 2, q < 0.05).

[0068] Finally, 208 differentially expressed molecules were screened and validated in cells. Marc145 cells were then treated with 10 μM of the small molecule for 12 h, followed by inoculation with PRRSV-NADC30 virus. The viral load in the cells was detected by RT-PCR. The results showed that the differentially expressed small molecule 4-methylcatechol had significant anti-PRRSV activity in Marc145 cells, and that 4-methylcatechol had broad-spectrum antiviral activity against PRRSV.

[0069] The results are as follows Figure 6As shown, Marc145 cells were treated with different concentrations of 4-methylcatechol (5, 10, 15, 20 μM) and inoculated with different PRRSV strains. Viral fluid was collected 32 h after infection, and viral load was detected by RT-PCR. It was found that 4-methylcatechol showed broad-spectrum anti-PRRSV activity in Marc145 cells in a dose-dependent manner.

[0070] Example 3:

[0071] Application of Streptomyces LM-35 fermentation broth supernatant in the preparation of drugs for the prevention of porcine reproductive and respiratory syndrome virus infection.

[0072] 1. Laboratory animal facilities and preparation of laboratory animals

[0073] One week before the start of the animal experiments, the experimental animal room was thoroughly cleaned and fumigated for disinfection. Two days before the experiments, the experimental piglets were transported to the animal room, and 10 healthy PRRSV-negative piglets aged 40 days were randomly divided into groups and fed in the animal room for 2 days. The piglets' feeding status was observed to ensure that each piglet could eat and drink normally.

[0074] 2. Animal Experiment Design

[0075] Ten PRRSV-negative piglets (40-day-old weaned piglets) were randomly divided into two groups: a control group and an experimental group, with five piglets in each group. The five piglets in the control group were numbered 1, 2, 3, 4, and 5, while the five piglets in the experimental group were numbered 6, 7, 8, 9, and 10. All animals were housed in an animal facility conforming to GMP animal laboratory standards. The control group was not fed Streptomyces LM-35 fermentation broth supernatant; the experimental group was fed Streptomyces LM-35 fermentation broth supernatant. After 10 days of feeding, the piglets were intranasally infected with the highly pathogenic PRRSV strain PRRSV-WH3 at an infectious dose of 10... 4 TCID 50 The experimental groups are shown in Table 1.

[0076] Table 1 Grouping of Animal Experiments with Probiotic Preparations Against PRRSV Infection

[0077] .

[0078] 3. Effect of LM-35 fermentation broth supernatant on mortality rate in PRRSV-infected pigs

[0079] Following PRRSV infection, the health status of pigs was observed daily, and clinical symptoms were recorded. Results are shown in Table 2: In the control group, one pig died on day 11, one on day 18, and two on day 20, with a survival rate of 20%; in the experimental group, no deaths occurred, with a survival rate of 80%. Results are shown in Table 2 and... Figure 7 As shown.

[0080] Table 2 Record of PRRSV-infected piglet mortality

[0081] .

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An isolated Streptomyces strain ( Streptomyces The Streptomyces species described is LM-35 (spp.), and its preservation number is CCTCCNO: M 2026442.

2. The fermentation supernatant of Streptomyces LM-35 as described in claim 1.

3. The fermentation supernatant according to claim 2, one of its effective components is 4-methylcatechol.

4. A compound, wherein the active ingredient of the compound comprises Streptomyces LM-35 as described in claim 1 and / or the fermentation supernatant as described in claim 2.

5. The use of the fermentation supernatant of claim 2 and / or a compound containing the fermentation supernatant of claim 2 in the preparation of a medicament for treating or preventing porcine reproductive and respiratory syndrome virus infection.

6. The use of the fermentation supernatant of claim 2 and / or a compound containing the fermentation supernatant of claim 2 in the preparation of pig feed additives.

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