7-GTDF from Archidendron clypearia extract inhibits PRRSV entry and interacts with the CD163 receptor

Abstract

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a major threat to global swine production, with no clinically approved specific therapeutics. Archidendron clypearia (AC), a medicinal plant traditionally used for human upper respiratory infections, exhibits broad-spectrum antiviral activity. This study systematically evaluated the antiviral efficacy of AC against PRRSV, elucidated its pharmacodynamic material basis through comprehensive chemical profiling, and explored the targeted molecular mechanisms of its core active component, 7-GTDF.

Results

Screening of 30 herbal ethanol extracts based on a PRRSV-EGFP fluorescence phenotype identified AC as possessing significant anti-PRRSV activity. In Marc-145 cells and porcine alveolar macrophages (PAMs), AC inhibited the replication of PRRSV-Li11 and PRRSV-CHR6 at both the mRNA and protein levels. Time-of-addition assays indicated that AC acts by inactivating the virus and interfering with early infection stages. Phytochemical analysis identified 40 constituents in AC, with 7-O-galloyltricetiflavan (7-GTDF) quantified via high-performance liquid chromatography (HPLC) as the predominant compound, accounting for 37.59% of the total peak area. Network pharmacology combined with experimental validation demonstrated that AC suppresses the PRRSV-induced overexpression of TNF-α, IL-1β, and IL-6 in PAMs by modulating the TLR4/NF-κB signaling pathway. Subsequent screening of the primary compounds in AC revealed that 7-GTDF exhibits significant anti-PRRSV activity. Molecular docking and molecular dynamics simulations indicated a stable interaction between 7-GTDF and the host CD163 receptor. Finally, cellular thermal shift assay (CETSA) confirmed this target engagement, demonstrating that 7-GTDF binds to the CD163 receptor and reduces its thermal stability.

Conclusions

These data indicate that AC and its constituent 7-GTDF inhibit PRRSV infection through mechanisms involving CD163 targeting and inflammatory pathway modulation. These findings provide a pharmacological basis for evaluating AC as a potential antiviral candidate in swine health management.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-026-05489-6.

Introduction

Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), imposes a substantial global economic burden on the swine industry, resulting in annual losses exceeding $1.2 billion in the U.S. alone [1]. This pathogen induces severe respiratory distress driven by upper airway inflammation and pulmonary consolidation in neonatal pigs, and reproductive failure associated with arteritis-mediated vascular damage in sows [2–4]. The high genetic diversity of PRRSV [5], combined with its capacity for persistent infection and sophisticated immune evasion strategies [6], collectively undermines the efficacy of existing commercial vaccines [7, 8]. These challenges underscore the urgent need to develop novel antiviral therapies that inhibit viral replication and target the complex immunopathological mechanisms underlying PRRSV infection.

Current antiviral strategies targeting PRRSV focus on disrupting its replication cycle through multi-stage interventions [9]. These strategies target viral entry, replication, and assembly to block infection at critical junctures. For instance, CD163-targeted molecules (e.g., truncated CD163 proteins, neutralizing antibodies) interfere with viral attachment and uncoating by blocking GP5-CD163 interactions, while toll-like receptor (TLR) agonists (e.g., poly(I:C), SZU101) activate innate immunity to enhance interferon (IFN) production. Synthetic oligonucleotides—including phosphorothioate-modified oligonucleotides (PMOs) and small interfering RNAs (siRNAs)—destabilize viral RNA or inhibit translation, and nanobodies against NSP4/NSP9 disrupt transcription-replication complex formation. Notably, miRNA-based inhibitors (e.g., miR-181, let-7f-5p) downregulate host receptors like CD163 by binding to their 3’ untranslated regions (3’UTRs), reducing viral entry efficiency.

CD163 is a cornerstone of PRRSV infection. Notably, studies have demonstrated that CD163-knockout (KO) pigs exhibit complete resistance to PRRSV, characterized by undetectable viremia and an absence of tissue viral replication [10]. Heterozygous dams carrying CD163-KO fetuses also protect offspring from vertical transmission [11], highlighting its translational potential for breeding PRRS-resistant livestock. These advancements not only deepen our understanding of PRRSV pathogenesis but also establish CD163 as a prime target for next-generation antiviral therapies and vaccines.

In traditional Chinese veterinary medicine, PRRS is classified as a “warm disease” or “epidemic disease,” characterized by exogenous invasion of damp-heat epidemic toxins and internal disruption of Qi-blood balance in Zang-fu organs [12]. Therapeutic principles emphasize “strengthening host defenses while expelling pathogens,” with formulations aimed at heat-clearing, detoxification, pulmonary enhancement, dampness drainage, or Qi-tonification and Yin-nourishment. Emerging evidence supports traditional Chinese medicine’s (TCM) potential in developing PRRS antiviral strategies [13]. For example, Huashi Baidu Granules, a TCM formula, suppress pulmonary edema in PRRS-affected pigs by resolving dampness [14]. Four classical heat-clearing-detoxifying formulas, including Banxia Xiexin Decoction, Sanhuang Xiexin Decoction, Huanglian Jiedu Decoction, and Qingwen Baidu Powder, also exhibit significant in vitro antiviral activity against PRRSV [15]. These findings establish TCM theory as a robust framework for identifying botanicals with anti-PRRSV efficacy.

Based on this framework, 30 TCMs classified as “heat-clearing-detoxifying (Qing-re-jie-du)” and "spleen-fortifying and dampness-resolving (Jian-pi-chu-shi)" were screened for their ability to inhibit PRRSV replication in Marc-145 cells, aiming to identify promising herbal candidates. Archidendron clypearia (Jack) I.C. Nielsen (AC) was identified as exhibiting significant antiviral activity and potent anti-inflammatory effects against PRRSV-induced inflammation. Its active monomer, 7-GTDF, provided biophysical and computational evidence of engagement with the CD163 receptor, suggesting a highly probable mechanism for inhibiting viral entry.

Materials and methods

Cell culture and virus preparation

Marc-145 cells (African green monkey embryonic kidney cells, CL-0566) were sourced from Wuhan Pusenuo Life Science and Technology Co., Ltd. (Wuhan, China) and maintained in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Hyclone, USA) at 37 °C in a humidified incubator with 5% CO₂. Primary porcine alveolar macrophages (PAMs) were provided by the National Pig Technology Innovation Center South China Branch (Guangzhou, China). PAMs were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, USA) containing 10% FBS, 1% penicillin-streptomycin, and 2 mM L-glutamine (Hyclone, USA) at 37 °C with 5% CO₂. The highly pathogenic PRRSV-2 strain GDBY1 (GenBank accession no. GQ374442.1) and the classical PRRSV-2 strain CH-1a (GenBank accession no. AY032626.1) were provided by the National Pig Technology Innovation Center South China Branch (Guangzhou, China). The specific laboratory-adapted passages of these strains utilized in the current experiments are designated as PRRSV-Li11 and PRRSV-CHR6, respectively. Virus stocks were quantified via the 50% tissue culture infective dose (TCID₅₀) assay and standardized using the Reed-Muench method [16].

Chemical reagents

Dexamethasone (LOT: JS252843) served as the positive control for anti-inflammatory activity. Catechin (LOT: S01hb191501), epigallocatechin (LOT: m07hb177342), myricitrin (LOT: D11IB234751) and hyperoside (LOT: JB301671) were all purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). 5,7,3’,4’,5’-Pentahydroxyflavan (BBP07052, BioBioPha Co., Ltd., China), 7-O-galloyltricetiflavan (7-GTDF) and di-O-galloyltricetiflavan (a mixture of 7,4’-di-O-galloyltricetiflavan and 7,3’-di-O-galloyltricetiflavan) were isolated from AC according to the method [17]. Identification results are shown in Fig. S4.

Antibodies

Primary antibodies were sourced as follows: anti-CD163 (Abcam, UK), anti-PRRSV N (clone 4A5; MEDIAN Diagnostics, South Korea), NF-κB p65 (D14E12 XP^® Rabbit mAb; CST, USA), Phospho-NF-κB p65 (Ser536; 93H1 Rabbit mAb; CST, USA), Phospho-IκBα (Ser32; 14D4 Rabbit mAb; CST, USA), GAPDH (14C10 Rabbit mAb; CST, USA), β-Tubulin (CST, USA), Vinculin (CST, USA) and IκBα (44D4; CST, USA). All primary antibodies were diluted 1:3000 for Western blot (WB) analysis. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (SA00001-2; Proteintech, USA) was used as the secondary antibody at 1:10000 dilution.

Preparation of 30 traditional Chinese medicine ethanol extracts

Dried Chinese medicines (Table S1, 100 g each) were macerated in 95% ethanol (Guangzhou Dongzheng Chemical) at a 1:4 (w/v) ratio overnight at room temperature. The mixture was filtered through filter paper, and the supernatant was collected after centrifugation at 12,000 × g for 15 min. The crude extract was concentrated under reduced pressure using a rotary evaporator (Yamato, Japan) to yield a concentrated solution. The concentrated solution was then freeze-dried using an Alpha 1–4 LDplus freeze dryer (CHRIST, Germany) to obtain a yellowish-brown powder. Dried extracts were stored in airtight containers at 4 °C until further use.

Cytotoxicity assay

Cells were seeded in 96-well plates at a density of 5 × 10³ cells/cm² allow to adhere overnight. Subsequently, the cells were divided into an untreated mock group and various drug-treated groups. The drug-treated groups were incubated with the AC extract at specified concentrations ranging from 12.5 to 100 μg/mL, diluted in a maintenance medium supplemented with 2% FBS (ExCell Bio, China). The mock group received an equivalent volume of the maintenance medium alone. After 48 h of incubation, cell viability was assessed using the CellTiter 96^® Aqueous One Solution MTS assay (Promega, USA). according to the manufacturer's protocol. Finally, absorbance was measured at 490 nm using a BioTek Epoch2 microplate reader (BioTek Instruments, USA).

Anti-PRRSV activity

Marc-145 cells were seeded in 12-well plates at a density of 1 × 10⁵ cells/well and incubated overnight at 37 °C in a humidified incubator with 5% CO₂ to allow adherence. To identify potential antiviral candidates, the cells were inoculated with PRRSV expressing enhanced green fluorescent protein (PRRSV-EGFP) at a multiplicity of infection (MOI) of 0.1 and concurrently treated with 30 different herbal ethanol extracts. Concurrently with viral inoculation, cells were treated with AC extract at gradient concentrations (10–100 µg/mL) in maintenance medium DMEM supplemented with 2% FBS and 1% penicillin-streptomycin. After 48 h of incubation, cytopathic effects (CPE) and fluorescence intensity were observed and recorded under an inverted fluorescence microscope (Nikon Eclipse Ti2-U, Nikon Corporation, Tokyo, Japan). EGFP fluorescence intensity was imaged using an IVIS Lumina III imaging system (PerkinElmer, USA) with excitation/emission wavelengths of 488/520 nm.

Viral titer determination

Cell supernatants were collected from each well and serially diluted in maintenance medium containing 2% FBS using a 10-fold dilution series (10⁻¹ to 10⁻⁶). 100 µL aliquots of the diluted virus were transferred to 96-well plates pre-seeded with Marc-145 cells. Each dilution was tested in six replicates to ensure statistical reliability. Plates were incubated at 37 °C under 5% CO₂ for 72 h, after which CPE were quantified under an inverted microscope. CPE severity was recorded as follows: ≥50% cell lysis (≥ 50% CPE) was designated as positive (+), and < 50% CPE as negative (−). The TCID₅₀ was calculated using the Reed-Muench method, with results expressed as TCID₅₀ per milliliter.

Qualitative analysis of AC

The total chemical profile of AC extract was analyzed by liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-Q-TOF-MS). Structural elucidation was performed on a ZenoTOF 7600 Q-TOF mass spectrometer (SCIEX, Framingham, MA, USA) coupled with an Acquity HPLC system. Chromatographic conditions mirrored the quantitative HPLC method. The mobile phase comprised (A) acetonitrile (Fisher Chemical; same lot as above) and (B) water with 0.1% (v/v) formic acid (Aladdin Biotech; Lot D1502086), with a flow rate of 0.3 mL/min. The mass spectrometer was operated in positive and negative electrospray ionization (ESI±) modes. Spray voltage: 5000 V (ESI+) and − 4500 V (ESI-); spray gas (GS1): 55 psi; auxiliary heating gas (GS2): 50 psi; ion source temperature: 550 °C; curtain gas (CUR): 35 psi; collision energy (CE): 30 eV; declustering potential (DP): 80 V; collision cell pressure: 10 psi. The mass-to-charge ratio (m/z) scan range was 50–1500. Nitrogen (N₂) was used as the nebulizer and auxiliary gas. Data acquisition was performed using Analyst® TF software (SCIEX) in information-dependent acquisition (IDA) mode.

Quantitative analysis of AC

The quantitative analysis of AC extract was performed using a high-performance liquid chromatography-diode array detector (HPLC-DAD) system. An Agilent 1260 HPLC system (Agilent Technologies, USA) was used. Separation was achieved on an Agilent ZORBAX Eclipse Plus C18 column (4.6 × 150 mm, 3.5 μm particle size; Agilent Technologies). The mobile phase consisted of (A) acetonitrile (Fisher Chemical, USA; Lot F24OBF203) and (B) water with 0.2% (v/v) phosphoric acid (Aladdin Biotech, China; Lot 2127420), with a flow rate of 0.3 mL/min. A gradient elution program was applied: 0–10 min (10% A), 10–65 min (10–25% A), 65–70 min (25–10% A), 70–75 min (10% A) (re-equilibration). Dual-wavelength detection was performed at 273 nm and 208 nm. Column temperature was maintained at 25 °C ± 0.5 °C, and the injection volume was 5 µL.

Six reference standards (Table 1) were dissolved in HPLC-grade methanol (Fisher Chemical; Lot F25P5D207) to prepare a mixed stock solution (1.0 mg/mL), which was serially diluted with methanol to generate calibration standards (0.01–100 µg/mL). AC extract (10 mg) was dissolved in HPLC-grade methanol to a final concentration of 1.0 mg/mL. All solutions were filtered through 0.22 μm nylon membrane filters (Merck Millipore, Germany) prior to injection. Quantification was performed using external standard calibration curves (R² ≥ 0.999), with peak areas correlated to concentrations. Relative quantitative analysis of non-standard components was conducted using the proportional relationship: (Peak area of component A)/(Concentration of A) = (Peak area of component B)/(Concentration of B).

Table 1.

Information on the reference standards

Name	Source	LOT NO	Purity (HPLC)
Gallic acid	Chengdu Pufei De Biotech Co., Ltd.	151009	≥ 98%
Epigallocatechin	Chengdu Musi Bio-Technology Co., Ltd.	MUST-21042810	98.71%
Gallocatechin	Chengdu Musi Bio-Technology Co., Ltd.	MUST-21041804	98.57%
Epigallocatechin gallate	Shanghai Yuanye Bio-Technology Co., Ltd.	C07J8Y39406	≥ 98%
Gallocatechin gallate	Shanghai Yuanye Bio-Technology Co., Ltd.	M13GB146375	≥ 99%
Quercitrin	National Institutes for Food and Drug Control	XS2J-0JMS	92.7%

Effects of AC on the PRRSV life cycle

To evaluate the direct virucidal effect of AC on PRRSV, PRRSV-Li11 (MOI = 0.1) was pre-incubated with the AC extract (60 μg/mL) in cell-free DMEM at 37 °C for 2 h. Marc-145 cells, cultured to 80% confluence in 24-well plates and washed twice with pre-chilled PBS, were then inoculated with this virus-extract mixture. The mock-infected group received DMEM supplemented with 2% FBS alone, while the PRRSV-infected control group was inoculated with virus that had been pre-incubated with an equivalent volume of extract-free DMEM. Following a 2-hour incubation at 37 °C for viral adsorption, the inoculum was removed, and the cells were washed three times with pre-chilled PBS. Fresh maintenance medium was added, and the cultures were incubated for 48 hours. At 48 hours post-infection (hpi), supernatants and cell lysates were collected and stored at –80 °C for TCID₅₀ determination and Western blot analysis, respectively.

To assess the impact of AC on PRRSV adsorption, Marc-145 cells were cultured to 80% confluence in 24-well plates. After being washed with pre-chilled PBS, the cells were simultaneously incubated with PRRSV-Li11 at an MOI of 0.1 and AC at a concentration of 60 μg/mL. This co-incubation was performed at 4 °C for 2 hours to specifically permit viral attachment while blocking internalization. Subsequently, the cells were washed three times with pre-chilled PBS to remove unadsorbed viruses, followed by the addition of a maintenance medium consisting of DMEM supplemented with 2% FBS. At 48 hours post-infection, supernatants were collected, and viral titers were quantified as TCID₅₀ using an endpoint dilution assay.

To evaluate the effect of AC on PRRSV replication, Marc-145 cells were cultured to 80% confluence in 24-well plates. After being washed twice with pre-chilled PBS, the cells were inoculated with PRRSV-Li11 at an MOI of 0.1. This inoculation was carried out at 37 °C for 2 hours to facilitate viral adsorption. Following three washes with PBS to remove unattached virions, the cells were treated with AC at a concentration of 60 μg/mL at specific time points of 2, 4, 8, or 12 hours post-infection. At 48 hours post-infection, supernatants were collected, and viral titers were quantified as TCID₅₀ using an endpoint dilution assay.

To determine the effect of AC when co-administered with PRRSV, Marc-145 cells were cultured to 80% confluence in 24-well plates. After being washed with pre-chilled PBS, the cells were simultaneously inoculated with PRRSV-Li11 at an MOI of 0.1 and treated with a maintenance medium either containing AC at 60 μg/mL or lacking the extract for the control group. Following a 2-hour incubation, the cells were washed three times with PBS to remove unbound virions. Fresh maintenance medium, correspondingly with or without the AC, was then added to the respective wells. At 48 hours post-infection, supernatants were collected, and viral titers were quantified as TCID₅₀ using an endpoint dilution assay.

Quantitative real-time PCR (qPCR)

To compare the expression levels of proinflammatory factors after drug and viral treatments, total RNA was extracted from treated Marc-145 cells or PAMs using the EZ-press RNA Purification Kit (EZBioscience, China). RNA concentration and purity were measured with a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). First-strand cDNA was synthesized by reverse transcription using the PrimeScript™ RT Reagent Kit (TAKARA Co., Ltd., Japan), which contains gDNA Eraser to eliminate genomic DNA contamination, following the manufacturer’s instructions. Gene-specific primers (sequences listed in Table S2) were designed for target genes and the reference gene (GAPDH). Quantitative real-time PCR was performed using SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus; TAKARA Co., Ltd., Japan) on an Applied Biosystems 7500 real-time PCR system (Thermo Fisher Scientific, USA). Reactions were carried out in triplicate, with each 20 µL mixture containing 10 µL SYBR Premix, 0.8 µL each primer (10 µM), 2 µL cDNA template, and 6.4 µL nuclease-free water. Relative gene expression levels were calculated using the 2 ^−ΔΔCt method, where ΔΔCt = (Ct_target - Ct_GAPDH) _experimental - (Ct_target - Ct_GAPDH) _control, and normalized to the GAPDH.

Western blot (WB) analysis

Cultured cells were washed twice with ice-cold PBS (pH 7.4) and lysed on ice for 30 min in cell lysis buffer (Beyotime Institute of Biotechnology, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF; Beyotime Institute of Biotechnology, China) and a protease/phosphatase inhibitor cocktail (Beyotime Institute of Biotechnology, China). Lysates were centrifuged at 14,000 × g for 15 min at 4 °C to pellet cellular debris. The supernatant was transferred to a new tube, mixed with 5 × SDS-PAGE loading buffer (YaMei Biotech, China) containing 10% (v/v) β-mercaptoethanol, and boiled at 95 °C for 10 min to denature proteins. Samples were either immediately loaded onto 10–12.5% SDS-polyacrylamide gels (Urandi Biotech, China) or stored at −80 °C.

Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Roche Diagnostics, USA) using a wet-transfer system (Bio-Rad Laboratories, USA) at 200 mA for 1 h at 4 °C. Membranes were blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich, USA; A9418) in Tris-buffered saline with Tween-20 (TBST; 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20; Yamei Biotech, China) for 1 h at room temperature. Primary antibodies were applied overnight at 4 °C. After three washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (e.g., goat anti-rabbit IgG-HRP; Beyotime Institute of Biotechnology, China) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology, China) and quantified via densitometry using ImageJ software (v1.53t, National Institutes of Health, USA).

Enzyme-linked immunosorbent assay (ELISA)

To evaluate the effect of AC on cytokine secretion, cell culture supernatants were collected exclusively from the treated PAMs. The supernatants were centrifuged at 1,000 × g for 20 min at 4 °C to pellet cellular debris and subsequently stored at -80 °C until analysis. The concentrations of TNF-α, IL-1β, and IL-6 were quantified using porcine-specific commercial ELISA kits (J&L Biological, China) according to the manufacturer's instructions.

Structural characterization of 7-GTDF

To identify the isolated compound 7-GTDF (Fig. S4), nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analyses were performed. A 5 mg sample of 7-GTDF was dissolved in 0.5 mL of methanol-d₄ (99.8% deuteration; Shanghai Aladdin Biochemical Technology Co., Ltd., China) with sonication until completely dissolved. The solution was transferred to a 5 mm NMR tube (SYNTHWARE, Taiwan, China ) and analyzed on a Bruker AVANCE III HD 500 MHz spectrometer (Bruker Corporation, Germany) equipped with a 5 mm TCI cryoprobe, operated at 25 °C. For further structural confirmation, 1 mg of 7-GTDF was dissolved in 2 mL of HPLC-grade acetonitrile (Thermo Fisher Scientific, USA) and filtered through a 0.22 μm nylon membrane filter (Jinteng, China). The filtrate was transferred to an HPLC vial and analyzed using an Acquity HPLC system Agilent 1260 Infinity RRLC system coupled with a ZenoTOF 7600 mass spectrometer (SCIEX, Framingham, MA, USA).

Network pharmacology targets for PRRS treatment

To identify potential pharmacological targets of the active ingredients in AC for treating PRRS, a network pharmacology approach was employed. Potential targets of the active compounds were predicted and filtered using a probability score > 0 in the SwissTargetPrediction database. Concurrently, PRRS-associated genes were retrieved from the GeneCards database via a keyword search for "Porcine Reproductive and Respiratory Syndrome" and subsequently screened based on their relevance scores. These PRRS-related targets were then intersected with the predicted drug targets using Venny 2.1.0 to generate a refined overlapping gene set. The overlapping targets were imported into the STRING database (version 12.0) to construct a protein-protein interaction (PPI) network. This network was further visualized and analyzed using Cytoscape 3.10.0, where the CytoHubba plugin was utilized to identify hub targets, and topological properties (such as degree centrality and betweenness) were quantified using the NetworkAnalyzer tool. This integrated analysis resulted in the construction of a visualized "compound-target-disease" regulatory network. For functional annotation, the shared drug-disease targets were subjected to Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis via the DAVID platform.

Molecular docking of 7-GTDF with the porcine CD163 receptor

To investigate the specific interaction between 7-GTDF and the porcine CD163 receptor, which serves as a key entry mediator for PRRSV, molecular docking simulations were performed. The initial structural information for 7-GTDF was retrieved from the PubChem database. Subsequently, geometric optimization of the compound was conducted using the Chem3D module of ChemBioOffice Ultra 13.0.2 via energy minimization and molecular mechanics refinement. The target protein structure of CD163, designated by the identifier 5HRJ, was downloaded from the RCSB Protein Data Bank. This structure was processed in PyMOL 2.5.2 to remove crystalline water molecules, bound ligands, and non-protein heteroatoms. Semi-flexible molecular docking was then executed using AutoDock Vina 1.1.2. The receptor structure was prepared by defining flexible side chains and establishing a specific grid box encompassing the active site. Finally, docking poses were ranked based on their binding affinity scores expressed in kcal/mol.

Molecular dynamics (MD) simulation of 7-GTDF and the porcine CD163 receptor

To investigate the interaction between 7-GTDF and the porcine CD163 receptor (Sus scrofa), MD simulations were performed using GROMACS 2022.3. The protein and ligand were parameterized using the AMBER14SB and GAFF2 force fields, respectively. The system was solvated in a 1.0 nm TIP3P water box, and charge neutrality was achieved by adding 0.15 M NaCl using GROMACS gmx genion [18]. Energy minimization, NVT, and NPT equilibration were conducted sequentially. Production MD simulations were run for 100 ns with a 2 fs time step. Post-simulation analyses were performed to calculate RMSD, RMSF, hydrogen bonds, radius of gyration, and SASA. Binding free energy was computed using the MM-PBSA method. All protocols adhered to standard benchmarks to ensure accuracy.

Cellular thermal shift assay (CETSA)

To detect the interaction between 7-GTDF and its target proteins in cells, CETSA was performed. Frozen PAMs were rapidly thawed and seeded at 1 × 10⁶ cells/mL. After overnight incubation, cells were washed with ice-cold PBS and harvested. Pellets were resuspended in ice-cold IP lysis buffer supplemented with 1 mM PMSF and incubated for 1 h to inhibit protease activity. Subsequently, cells were treated with 7-GTDF (50 μM) or DMSO (0.1% v/v). Samples were aliquoted into PCR tubes and heated at different temperatures (37 to 70 °C) for 10 min using a thermal cycler, followed by cooling. After heating, samples were centrifuged at 20,000 × gfor 20 min to pellet denatured proteins. The supernatants were mixed with 5× SDS-PAGE loading buffer, boiled, and analyzed by SDS-PAGE and Western blotting.

Statistical analysis

All experiments were performed with at least three independent replicates, and data are presented as the mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Pairwise comparisons were analyzed using an unpaired Student’s t-test, while multiple-group comparisons were evaluated using a one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. A p-value < 0.05 was considered to indicate statistical significance.

Results

Anti-PRRSV activity of thirty extracts and the anti-PRRSV activity of AC​ was evaluated in vitro

The anti-PRRSV activity of 30 Chinese medicine extracts (tested at 200 µg/mL), measured by expression of enhanced green fluorescent protein (EGFP) as a viral reporter, is shown in Fig. S1. Based on assessment of fluorescence intensity (green signal indicating viral infection) and cytotoxicity (cell morphology under white light relative to mock-infected controls), we selected seven extracts for multi-concentration evaluation (Fig. S2).

In this initial screening, all 30 extracts were evaluated for anti-PRRSV activity at a concentration of 200 μg/mL, and the results are shown in Fig. S1. Based on the assessment of fluorescence intensity and cytotoxicity, seven extracts were selected for subsequent multi-concentration evaluation, with the results illustrated in Fig. S2. To further evaluate the anti-PRRSV activity of the seven selected extracts, their effects on cell viability and inhibition rates against PRRSV-Li11 were assessed at a concentration of 25 μg/mL. As illustrated in Fig. 1A and B, AC exhibited no significant cytotoxicity toward Marc-145 cells versus the 0 μg/mL group (p = 0.3365) and demonstrated the highest viral inhibition rate. Near-complete suppression of viral fluorescence was observed at 12.5–100 µg/mL, with cell viability remaining above 90% (Fig. 1C). Figure 1D, G show that the 50% cytotoxic concentration (CC₅₀) of AC was > 400 µg/mL for Marc-145 cells and 117.1 µg/mL for PAMs. In Marc-145 cells, the half-maximal effective concentration (EC₅₀) of AC against PRRSV-Li11 and PRRSV-CHR6 strains was 12.61µg/mL and 21.30 µg/mL, respectively (Fig. 1E, F). In PAMs, the EC₅₀ values were 63.52 µg/mL for PRRSV-Li11 and 36.85 µg/mL for PRRSV-CHR6 (Fig. 1H, I). The selectivity index (SI ) for each virus-cell combination is shown in Table 2. Given its favorable safety profile and potent antiviral activity, AC was selected for further studies. Based on the cytotoxicity and antiviral efficacy profiles (Table 2), a unified concentration of 60 µg/mL was selected for subsequent AC extract-related assays.

Fig. 1.

Antiviral efficacy and cytotoxicity profiling of AC against PRRSV in Marc-145 and PAMs. A. Cytotoxicity of seven extracts at 25 µg/mL in Marc-145 cells. The evaluated extracts included Archidendron clypearia (AC), Dryopteris crassirhizoma (DC), Mori Folium (MF), Cyperi Rhizoma (CR), Granati Pericarpium (GP), Polygoni Chinensis (PC), Perillae Caulis (PCs). B. Anti-PRRSV activity of seven extracts at 25 µg/mL in Marc-145 cells. C. Relative fluorescence intensity of AC at gradient concentrations of 0 to 200 µg/mL with PRRSV-EGFP at a dose of 100 TCID₅₀ in Marc-145 cells at 48 hpi; green fluorescence indicates viral load. D. Cytotoxicity of AC in Marc-145 cells. E-F. EC₅₀ values of AC against PRRSV-Li11 (E) and PRRSV-CHR6 (F) in Marc-145 cells at 48 hpi. G. Cytotoxicity of AC in PAMs. H-I. EC₅₀ values of AC against PRRSV-Li11 (H) and PRRSV-CHR6 (I) in PAMs at 48 hpi. Values represent mean ± SD (n = 3). Statistical significance was analyzed using Student’s t-test or one-way ANOVA. **, p < 0.01; ****, p < 0.0001 versus the 0 µg/mL group, scale bar = 200 μm.

Table 2.

SI of AC against PRRSV infection in Marc-145 cells and PAMs

Cells	CC50 (µg/mL)	PRRSV-Li11		PRRSV-CHR6
		EC50 (µg/mL)	SI	EC50 (µg/mL)	SI
Marc-145	> 400	12.61	> 31.72	21.30	> 18.78
PAM	117.1	63.52	1.84	36.85	3.18

AC reduced N gene and protein expression of PRRSV in Marc-145 cells and PAMs

In both Marc-145 cells and PAMs, AC progressively reduced N gene expression as the treatment concentration increased, compared with the PRRSV-infected control group (Fig. 2A-D). At the protein level, AC significantly downregulated the N protein expression in PRRSV-Li11/CHR6-infected Marc-145 cells and PAMs, with the reduction becoming more pronounced at higher concentrations and the maximum inhibitory effect observed at 60 μg/mL (p < 0.0001 vs. the 0 μg/mL control) (Fig. 2E, G, I, K). To further evaluate viral replication, TCID₅₀ titers in the cell culture supernatants were measured. The results demonstrated a significant decrease in viral titers across both cell types following AC treatment (Fig. 2F, H, J, L).

Fig. 2.

Inhibitory effects of AC on PRRSV replication. A-D. Relative N gene expression in Marc-145/PAMs infected with PRRSV-Li11 or PRRSV-CHR6 at 24 hpi and treated with graded concentrations of AC. E, G, I, K. N protein expression in Marc-145 cells and PAMs under similar treatment conditions. F, H, J, L. TCID₅₀ titers in cell culture supernatants from the corresponding experimental groups. Values represent mean ± SD (n = 3). Statistical significance was analyzed using Student’s t-test or one-way ANOVA. ^####, p < 0.0001 versus the mock-infected group; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 versus the PRRSV-infected control group; ns, not significant.

AC inhibited PRRSV entry by inactivating viral particles and suppressing early infection stages

To determine the stage of PRRSV infection targeted by AC, we performed a time-of-addition assay (Fig. 3A). The results demonstrated that AC directly inactivated PRRSV particles (Fig. 3B, F). AC significantly inhibited the PRRSV adsorption phase (p= 0.0062 versus the PRRSV-infected control group; Fig. 3G), and viral titers in supernatants differed significantly from the PRRSV group (p= 0.0031versus the PRRSV-infected control group; Fig. 3C). Notably, when AC was added 4 hpi, a stage when PRRSV had completed adsorption and entered cells, it did not inhibit viral replication or N protein expression, yielding a p-value greater than 0.05 (p> 0.05 versus the PRRSV-infected control group;Fig. 3D, H). However, simultaneous exposure of Marc-145 cells to PRRSV and AC significantly suppressed the N protein expression and viral replication (Fig. 3E, I). These results indicate that AC primarily exerts its antiviral effects by directly inactivating PRRSV and blocking the early stages of infection.

Fig. 3.

Inhibitory effects of AC on the PRRSV life cycle. A. Experimental design detailing Marc-145 cells infected with PRRSV-Li11 at an MOI of 0.1 and simultaneously treated with 60 μg/mL of AC. Cell lysates were collected for WB, and supernatants were harvested at 24 hpi for viral titer quantification via TCID₅₀ assay. B-E. Viral titer dynamics in direct virucidal activity (B), viral adsorption inhibition (C), viral replication inhibition (D), and co-treatment (E) assays. F-I. Validation of N protein expression in direct inactivation (F), adsorption (G), replication (H), and co-treatment (I) experiments. Values represent mean ± SD (n = 3). Statistical significance was analyzed using Student’s t-test or one-way ANOVA. ^####, p < 0.001 versus the mock-infected group; **, p < 0.01; ****, p < 0.0001 versus the PRRSV-infected control group; ns, not significant.

Qualitative and quantitative analysis of phytochemicals in AC

Qualitative phytochemical profiling of AC extracts identified 40 bioactive compounds (Fig. S3 and Table S4). Furthermore, quantitative analysis was performed for 11 major constituents (Fig. 4 and Table 3). Together, these 11 compounds represented 88.06% of the total chromatographic peak area, with 7-GTDF alone accounting for 37.59%. 4 3

Fig. 4.

HPLC chromatograms of 11 compounds of AC. The red and black traces represent the mixed reference standards and the AC extract, respectively. The identified compounds are as follows: 1. Gallic acid; 2. Epigallocatechin; 3. Gallocatechin; 4. Catechin; 5. 5,7,3’,4’,5’-Pentahydroxyflavan; 6. Myricitrin; 7. Hyperoside; 8. Quercitrin; 9. 7-O-galloyltricetifavan; 10. 7,4’-di-O-galloyltricetifavan; 11. 7,3’-di-O-galloyltricetifavan.

Table 3.

Contents of characteristic compounds in AC

No.	Compound	Retention time (min)	Relative peak area (%)	Peak area (mAU·min)	Asymmetry (EP)	Resolution (EP)	Content of crude drug (mg/g)
1	Gallic acida	3.953	1.39	4.1832	0.8	13.69	7.484078
2	Epigallocatechina	8.223	0.17	0.5212	0.82	22.67	0.171216
3	Gallocatechina	18.867	0.28	0.8311	0.71	1.62	0.645804
4	Catechin	31.06	1.55	4.6621	0.9	9.74	3.622667
5	5,7,3’,4’,5’-Pentahydroxyflavan	34.827	2.31	6.9443	0.97	9.83	5.396
6	Myricitrin	43.12	1.41	4.2415	1.25	2.76	14.52596
7	Hyperoside	45.263	0.2	0.593	0.85	1.58	2.030902
8	Quercitrina	47.387	0.2	0.6169	1	3.67	2.112706
9	7-O-galloyltricetiflavan	51.827	37.59	113.1542	1	10.44	87.92525
10	7,4’-di-O-galloyltricetiflavan	61.66	26.09	78.5387	1.05	1.99	61.02761
11	7,3’-di-O-galloyltricetiflavan	63.517	16.87	50.7898	1.04	n.a.	39.46565

^aConfirmation in comparison with authentic standards.

Network pharmacology analysis reveals multi-target mechanisms of AC against PRRS

Database screening yielded 533 PRRS-associated targets and 582 potential targets corresponding to the active components in AC. Intersection analysis revealed 72 overlapping targets (Fig. 5A, B), forming a “Component-Target-Disease” network comprising 113 nodes (58 compounds, 42 targets, and 13 PRRS-related diseases) and 299 edges. This network highlighted complex interactions among AC components, PRRS-related targets, and disease pathways. Topological analysis of the PPI network (Fig. 5C) indicated that 7-GTDF, the most abundant component in AC, exhibited a high degree of connectivity, suggesting a potential central regulatory role within the network (Table 4). Furthermore, putative core targets (Table 5) and pathways predominantly enriched in immune modulation and inflammatory responses, such as TNF signaling and innate immune response, were identified (Fig. 5D and E). These systemic network predictions served as a framework to guide subsequent experimental evaluations. Consequently, in-depth experimental validation was directed toward two critical physiological axes: first, the systemic immune-modulatory response, assessed via the PRRSV-induced TLR4-NF-κB inflammatory cascade; and second, the direct antiviral mechanism, exploring the potential specific interaction between 7-GTDF and the viral entry mediator, CD163 receptor.

Fig. 5.

Network pharmacology analysis of AC in treating PRRS. A. Venn diagram of cross-targets between AC and PRRS. B. The component-key target-disease interaction network. C. Protein-protein interaction (PPI) network of the core targets. D. KEGG pathway enrichment analysis. E. GO enrichment analysis displaying the top 10 enriched terms across the biological process (BP), cellular component (CC), and molecular function (MF) categories.

Table 4.

Core components of AC in the treatment of PRRS

No.	Compound	Degree value
1	Quercetin	37
2	Kaempferol	17
3	Pentahydroxyflavan	12
4	Luteolin	11
5	Calaliukiuenoside	10
6	7-O-galloyltricetifavan	10
7	Myricetin	10
8	Morin	9
9	(+)-Lyoniresinol	7
10	Linoleic acid	6

Table 5.

Core targets of AC in the treatment of PRRS

No.	Target	Degree value
1	TNF	65
2	IL-6	63
3	IL-1β	61
4	TLR4	57
5	IFN-γ	55
6	STAT3	54
7	IL-10	53
8	TP53	53
9	NF-κB1	53
10	JUN	53

AC attenuates PRRSV-induced inflammatory cascade in PAMs

To elucidate the anti-inflammatory effect of AC in PRRS, a time- and MOI-dependent PRRSV infection model was established in PAMs. Fig. 6A, B show that in PAMs infected with PRRSV-Li11, pro-inflammatory cytokine expression (e.g., TNF-α, IL-1β) at 24 hpi decreased with increasing MOI. At an MOI of 0.5, expression peaked, whereas at MOI of 2, it reached the lowest level (compared to 0 hpi). (Fig. 6C, D reveal that at MOI of 0.5, TNF-α and IL-1β expression in PAMs significantly peaked at 24 hpi compared to 6 and 12 hpi. Fig. 6E, F). demonstrate that in PAMs infected with PRRSV-Li11 (MOI = 0.5) for 24 h, AC exhibited a dose-dependent inhibition of N gene expression and reduced viral titers in cell supernatants. Fig. 6G-I show that all tested concentrations of AC significantly reduced TNF-α mRNA expression in PAMs versus the PRRSV-infected control group (p < 0.0001). At 60 µg/mL, AC significantly inhibited IL-1β (p = 0.0004) and IL-6 (p = 0.0002) gene expression in PAMs versus the PRRSV-infected control group. WB results (Fig. 6J) showed that AC significantly reduced TLR4 protein expression versus the PRRSV-infected control group (p = 0.0062), including down regulation of PRRSV-induced NF-κB protein expression (p< 0.0001) and decreased expression of phosphorylated NF-κB (p < 0.0001) and IκB-α versus the PRRSV-infected control group (p= 0.0035, p= 0.0031). ELISA results (Fig. 6K-M) demonstrated that 60 µg/mL AC significantly reduced TNF-α (p = 0.0008), IL-1β (p = 0.0039), and IL-6 (p = 0.0003) levels in PAMs supernatants versus the PRRSV-infected control group.

Fig. 6.

The anti-inflammatory activity of AC in a PRRSV-infected PAM model. A, B. mRNA levels of TNF-α and IL-1β were measured in PAMs infected with PRRSV-Li11 at MOIs of 0.5, 1, and 2 at 24 hpi. C, D. Time-dependent changes in TNF-α and IL-1β mRNA expression at 6, 12, and 24 hpi. E. Effect of AC on N gene expression at 24 hpi. F. Quantification of viral titers in cell culture supernatants at 24 hpi. G-I. mRNA expression of TNF-α, IL-1β, and IL-6 at 24 hpi. J. Western blot analysis of N, IκB-α, NF-κB, p-NF-κB, and TLR4 protein levels. K-M. Quantification of TNF-α, IL-1β, and IL-6 via ELISA. Values represent mean ± SD (n = 3). Statistical analysis was carried out using Student’s t-test or one-way ANOVA. ^#, p < 0.05; ^##, p < 0.01; ^###, p < 0.001; ^####, p < 0.0001 versus the mock-infected group; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 versus the PRRSV-infected control group; ns, not significant.

Screening of the primary components of AC for Anti-PRRSV activity

To evaluate the anti-PRRSV activity of the highly abundant compounds identified in the quantitative analysis of AC, an EGFP viral reporter system was utilized. Chromatographic separation yielded the 7-GTDF monomer and a mixture of 7,4’-di-O-galloyltricetiflavan and 7,3’-di-O-galloyltricetiflavan, with the identification details provided in Fig. S4. The antiviral activity profiles of these isolated compounds are illustrated in Fig. 7A-J. Based on fluorescence intensity of PRRSV-EGFP, 7-GTDF exhibited significant inhibitory activity at 12.5 µg/mL, equivalent to 28.28 µM. For both Marc-145 cells and PAMs, 7-GTDF exhibited CC₅₀ values exceeding 400 µM (Fig. 7K, L). Against the PRRSV-Li11 and PRRSV-CHR6 strains, the EC₅₀ values were 16.08 µM and 22.84 µM, respectively (Fig. 7M, N). In PAMs (Fig. 7O, P), 7-GTDF downregulated the N protein expression as the concentration increased. At a concentration of 20 µM, 7-GTDF significantly reduced the N protein expression in PAMs infected with either PRRSV-Li11 (p < 0.0001) or PRRSV-CHR6 (p = 0.0003) versus the PRRSV-infected control group.

Fig. 7.

The anti-PRRSV activity of major compounds in AC. A-J. Inhibition of PRRSV-EGFP at a dose of 100 TCID₅₀ by compounds at gradient concentrations in Marc-145 cells. The evaluated compounds included gallic acid, gallocatechin, catechin, epigallocatechin, myricitrin, hyperoside, quercitrin, a di-O-galloyltricetifllavan mixture comprising 7,4’-di-O-galloyltricetiflavan and 7,3’-di-O-galloyltricetiflavan, 7-GTDF, and 5,7,3’,4’,5’-pentahydroxyflavan. K, L. Cytotoxicity of 7-GTDF in Marc-145 cells (K) and PAMs (L). M, N. Determination of EC₅₀ values of 7-GTDF against PRRSV-Li11(M) and PRRSV-CHR6 (N) within Marc-145 cells at 48 hpi. O, P. N protein expression levels in PAMs infected with PRRSV-Li11 (O) or PRRSV-CHR6 (P) at an MOI of 0.5 and treated with 7-GTDF at 24 hpi. Values represent the mean ± SD (n = 3). Statistical significance was analyzed using Student’s t-test or one-way ANOVA. ^####, p < 0.0001 versus the mock-infected group; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 versus the PRRSV-infected control group; ns indicates not significant. Scale bar = 200 μm.

7-GTDF interacts with CD163 receptor to potentially inhibit PRRSV entry

To elucidate the interaction between 7-GTDF and CD163, comprehensive in silico analyses including molecular docking, MD simulations, and binding free energy calculations were performed. Molecular docking predicted key binding sites on CD163, revealing that the residues LEU-553, ARG-169, GLU-517, and SER-554 formed stable intermolecular hydrogen bonds with 7-GTDF (Fig. 8B). Upon binding to CD163 receptor, 7-GTDF adopted two stable conformations, yielding a docking binding energy of -7.0 kcal/mol (Table S3). Subsequent MD simulations revealed that 7-GTDF remained stably bound to CD163 receptor throughout the simulation period. The RMSD values consistently remained below 2 Å, which indicates robust structural stability of the complex (Fig. 8C). Furthermore, the RMSF values were maintained below 1 Å, reflecting high residue rigidity (Fig. 8 D), while an average of approximately one intermolecular hydrogen bond per frame was continuously observed (Fig. 8E). Additionally, the solvent-accessible surface area of the complex remained remarkably constant following ligand binding, suggesting minimal perturbation of the overall protein architecture (Fig. 8F).To further evaluate thermodynamic stability, a free energy landscape (FEL) plot was constructed to visualize the free energy distribution based on RMSD values during the simulations (Fig. 8G, H). This analysis demonstrated a concentrated energy minimum, with the color gradient transitioning from red representing high energy states to blue indicating the lowest energy conformational basin. Quantitative assessment utilizing the molecular mechanics Poisson-Boltzmann surface area method calculated a total binding free energy of -6.82 kcal/mol, which confirms a thermodynamically favorable binding interaction (Fig. 8I). A detailed residue contribution analysis identified ARG-169 and SER-554 as the most critical residues for binding (Fig. 8J). To experimentally validate this direct interaction in a cellular context, a cellular thermal shift assay was conducted. The results demonstrated that 7-GTDF treatment induced a notable shift in the thermal denaturation profile of CD163 compared to the control group, significantly reducing its thermal stability across a temperature gradient from 37 °C to 70 °C (Fig. 8K). Moreover, an isothermal dose-response evaluation revealed that 7-GTDF progressively reduced the abundance of intact CD163 protein as the treatment concentration increased from 0.5 to 5000 μM, definitively confirming a concentration-dependent target engagement (Fig. 8L).Collectively, these comprehensive computational models and cell-based biophysical results strongly suggest that 7-GTDF engages with the CD163 receptor.

Fig. 8.

The binding interaction between 7-GTDF and the CD163 receptor. A. Molecular structure of 7-GTDF. B. Molecular docking model of 7-GTDF bound to CD163 receptor based on the PDB 5HRJ structure template. C. RMSD values (Å) of the protein-ligand complex over time. D. RMSF values (Å) of the protein-ligand complex. E. H-bonds between the protein and ligand over time. F. SASA values of the protein-ligand complex over time. G. Rg values of the protein-ligand complex over time. H. FEL plot of the complex. I-J. MM/PBSA binding free energy calculations. K. CETSA analysis of protein-ligand interactions across a temperature gradient. L. Isothermal dose-response (ITDR) analysis of 7-GTDF concentration-dependent effects. Values represent mean ± SD (n = 3). Statistical significance was analyzed using Student's t-test. *, p < 0.05; ****, p < 0.0001 versus the DMSO control group.

Discussion

To identify novel antiviral agents, ethanol extracts from 30 traditional Chinese medicines were screened for anti-PRRSV activity. Many of these extracts are traditionally characterized by their "heat-clearing-detoxifying" and "spleen-fortifying and dampness-resolving" properties, alongside well-documented immunomodulatory effects. Several of these have been reported to inhibit PRRSV in vitro. For example, extracts from Ilex rotunda Thunb. and Taraxacum mongolicum Hand.-Mazz. show potent activity [19]. Furthermore, a combined extract of Curcuma zedoaria-Astragalus membranaceus formulated at a 1:4 ratio successfully enhanced piglet weight gain and antibody titers as a PRRSV vaccine adjuvant [20], while Houttuynia cordata Thunb. extract improved in vivo immune responses to PRRSV-1 MLV vaccine [21]. Isatis r oot polysaccharides (IRPS) inhibit PRRSV by regulating TLR4 signaling and cytokine storms [22]. Among the 30 evaluated extracts, AC exhibited the most robust anti-PRRSV activity. AC, a medium-sized tree endemic to subtropical China, is traditionally used to treat upper respiratory infections and gastroenteritis [23]. Phytochemical studies reveal the presence of various bioactive polyphenols, flavonoids, and terpenoids within this plant [24]. Among these components, flavan derivatives such as 7-GTDF have demonstrated remarkable broad-spectrum antiviral activity. Specifically, compounds like 7-O-galloylplumbocatechin A and (-)-5,3',4',5'-tetrahydroxyflavan-7-gallate have been shown to inhibit HIV-1, while 7-GTDF and 7,4'-di-O-galloyltricetiflavan effectively suppress multiple viruses, including RSV, H1N1, CVB3, and HSV-1 [17]. Furthermore, molecular docking analysis indicates that 7,4′-di-O-galloyltricetifavan can binds to the SARS-CoV-2 RdRp with a favorable binding energy of -8.17 kcal/mol [25]. These low-cytotoxicity flavan derivatives highlight AC’s potential as a source of next-generation antivirals.

PRRSV, a 50–72 nm enveloped virus with a 15 kb RNA genome, depends on its N protein for structural integrity and antigenicity [26]. The present findings demonstrated that AC inhibited N protein expression in both Marc-145 cells and PAMs, with consistent efficacy against two PRRSV-2 strains, namely the highly pathogenic Li11 and classical CHR6. This results confirm the broad-spectrum antiviral activity of AC, although further in vivo validation is required to fully assess its translational potential.

AC significantly suppressed PRRSV replication and blocked early infection events, including viral adsorption. PRRSV entry involves complex interactions between viral glycoproteins and host cell receptors [10], particularly the macrophage-specific CD163 receptor, which undergoes critical structural rearrangements during infection [27]. Given the abundance of phenolic compounds in AC, it is highly probable that these molecules directly interact with viral envelope proteins or host receptors via hydrogen bonding, thereby sterically hindering viral attachment and entry. Thus, characterizing AC’s chemical composition and identifying its specific active compounds are critical for elucidating the precise molecular mechanisms of this targeted antiviral engagement.

Guided by the network pharmacology predictions, which predominantly enriched inflammatory pathways, the anti-inflammatory effects of AC were further investigated. It is well-established that PRRSV activates the NF-κB and MAPK pathways to induce lung inflammation [28]. AC suppresses NF-κB by inhibiting IκB-α phosphorylation and p65 nuclear translocation, reducing pro-inflammatory cytokines. Additionally, it lowers reactive oxygen species (ROS) by 58.6% as measured via the DCFH-DA assay, and attenuates the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome [29]. Furthermore, TLR4 plays a key role in PRRSV-induced inflammation. Early activation of TLR4 upregulates TNF-α and IL-6 via the NF-κB pathway, while downregulating and promoting the shedding of the CD163 receptor. [30, 31]. The present PAM studies confirmed that AC reduces the expression of TLR4/NF-κB, IL-1β, and TNF-α, aligning with its potent anti-inflammatory and antiviral effects.

Current anti-PRRS strategies target CD163 via downregulation or small-molecule/monoclonal antibody (mAb) blockade [32]. Specifically, small molecules targeting the CD163-SRCR5 domain effectively block entry, with structure-activity relationship (SAR) identifying the critical moieties [33]. The present study identified 7-GTDF as a potent inhibitor. Molecular docking and MD simulations indicate a highly probable 7-GTDF-CD163 receptor interaction with a binding free energy of −6.82 kcal/mol, which is further supported by cell-based CETSA data revealing reduced CD163 receptor thermal stability upon ligand binding.

Flavonoids inhibit viruses by targeting viral entry or replication [34]. For instance, J10688, a gallocatechin-7-gallate isolated from Pithecellobium clypearia, exhibits anti-influenza activity [35]. Although J10688 and 7-GTDF target distinct viral entry pathways, their shared flavan scaffold suggests comparable bioavailability and target-binding properties, warranting further in vivo evaluation. Future structure-activity relationship analyses are essential to elucidate their antiviral mechanisms and guide structural optimizations. Ultimately, the present findings regarding the CD163-targeted action of 7-GTDF establish a robust foundation for developing novel CD163 inhibitors, underscoring the therapeutic potential of flavonoids.

Despite these promising findings, certain limitations of the current study are acknowledged. Direct biological validation of the 7-GTDF and CD163 interaction, such as co-immunoprecipitation (Co-IP) or in vitro surface plasmon resonance (SPR), was not performed. However, utilizing in vitro biophysical assays for CD163 typically necessitates recombinant, truncated soluble domains (e.g., solely the SRCR5 domain). Importantly, molecular docking data indicates that the binding pocket of 7-GTDF spans multiple domains, specifically interacting with ARG-169 in SRCR2 and SER-554 in SRCR5. Utilizing truncated fragments may disrupt this inter-domain binding cleft. Conversely, purifying the full-length transmembrane CD163 protein in vitro is technically demanding and often causes misfolding or false-negative artifacts. Therefore, CETSA was employed to validate target binding, which preserves the full-length CD163 receptor in its natural cellular environment. To definitively confirm this mechanism, future studies using gene-editing models or advanced structural biology are required.

Conclusions

Guided by traditional Chinese veterinary medicine principles, AC was evaluated as a promising therapeutic candidate for PRRS. Biological assays demonstrated that AC exerts direct antiviral activity, inhibits early viral entry, and suppressed PRRSV-induced inflammation by inhibiting the TLR4-NF-κB signaling pathway. Phytochemical analysis identified 40 bioactive components in AC, with the primary monomer 7-GTDF accounting for 37.59% of the total quantified constituents. Notably,7-GTDF exhibited potent anti-PRRSV activity. Computational and biophysical analyses indicate that 7-GTDF restricts viral entry by interacting with the CD163 receptor. Collectively, these findings elucidate the anti-PRRSV and anti-inflammatory mechanisms of AC and its active monomers, establishing a solid pharmacological basis for future preclinical development.

Abbreviations

EC₅₀

50% effective concentration

TCID₅₀

50% tissue culture infective dose

CC₅₀

50% toxic concentration

7-GTDF

7-O-galloyltricetifavan

AC

Archidendron clypearia (Jack) I.C. Nielsen

CETSA

Cellular thermal shift assay

CSF

Classical swine fever

CSFV-C

Classical swine fever virus strain C

CVB3

Coxsackie virus B3

CZ-AM

Curcuma zedoaria-Astragalus membranaceus

CR

Cyperi Rhizoma

CPE

Cytopathic effects

DAD

Diode array detector

DC

Dryopteris crassirhizoma

FBS

Fetal bovine serum

FEL

Free energy landscape

GO

Gene ontology

GP

Granati Pericarpium

HSV-1

Herpes simplex virus type 1

HPLC

High-performance liquid chromatography

HRP

Horseradish peroxidase

hpi

Hour post-infection

HC

Houttuynia cordata Thunb. extract

H-bonds

Hydrogen bonds

H1N1

Influenza A virus subtype H1N1

IFN

Interferon

IFN-α/β

Interferon alpha/beta

IRPS

Isatis root polysaccharides

KEGG

Kyoto Encyclopedia of Genes and Genomes

LC-ESI-Q-TOF-MS

Liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry

MAPK1

Mitogen-activated protein kinase 1

MLV

Modified live virus

MD

Molecular dynamics

MM/PBSA

Molecular mechanics Poisson-Boltzmann surface area

mAbs

Monoclonal antibodies

MOI

Multiplicity of infection

MF

Mori Folium

NF-κB

Nuclear factor κB

OD

Optical density

PCs

Perillae Caulis

PMSF

Phenylmethylsulfonyl fluoride

PMOs

Phosphorothioate modified oligonucleotides

PC

Polygoni Chinensis

PVDF

Polyvinylidene fluoride

PAMs

Porcine alveolar macrophages

PRRS

Porcine reproductive and respiratory syndrome

PRRSV

Porcine reproductive and respiratory syndrome virus

PPI

Protein-protein interaction

qPCR

Quantitative real-time polymerase chain reaction

Rg

Radius of gyration

ROS

Reactive oxygen species

RSV

Respiratory syncytial virus

RESP

Restrained electro static potential

RNAi

RNA interference

RdRp

RNA-dependent RNA polymerase

RMSD

Root-mean-square deviation

RMSF

Root-mean-square fluctuation

SI

Selection index

SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2

STAT1/IRF7

Signal transducer and activator of transcription 1 / interferon regulatory aactor 7

siRNAs

Small interfering RNAs

SASA

Solvent-accessible surface area

SRC

Src proto-oncogene, non-receptor tyrosine kinase

SEM

Standard error of the mean

TLR

Toll-like receptor

TLR4

Toll-like receptor 4

TCM

Traditional Chinese medicine

WB

Western blotting

Authors’ contributions

JY: Writing – original draft, Validation. XZ: Methodology. HW: Resources, Project administration. WP: Methodology, Project administration. WS: Review & Editing, Resources, Project administration. PL: Methodology, Review & Editing. All the authors have read and agreed to the published version of the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

All data generated or analyzed during this study are included in this published article. The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.