The permissive cell line Marc-145 was maintained in the Veterinary Medicine Laboratory of Guangxi University. Porcine alveolar macrophages (PAMs) were aseptically isolated from PRRSV-negative healthy piglets. The cells were cultured at 37°C in a 5% CO₂ incubator and grown in Dulbecco's modified Eagle's medium (DMEM, Wisent) supplemented with 10% fetal bovine serum (FBS, Gibco). This study utilized two PRRSV strains preserved in the aforementioned laboratory: the currently prevalent NADC30 strain and a high-pathogenicity PRRSV (HP-PRRSV) strain engineered to express the green fluorescent protein (GFP) gene (designated HP-PRRSV-GFP). Both NADC30 and HP-PRRSV-GFP strains were propagated in PAMs and Marc-145 cells. Viral amplification and titration were performed in Marc-145 cells, yielding titers of 10^5.1 TCID₅₀/mL for the NADC30 strain and 10^4.8 TCID₅₀/mL for the HP-PRRSV strain. The TCID₅₀ represents the viral dilution required to infect 50% of inoculated cell cultures. This quantitative virological analysis measures infectious virus titers in tissue culture systems using the Reed-Muench method.

Mogroside V (MV) was obtained from Chengdu Mansite Biotechnology Co., Ltd. (China) with a certified purity of ≥98%. The lyophilized MV powder was stored at −20°C under desiccation and reconstituted in sterile double-distilled water (ddH₂O) immediately prior to experimentation. To preserve stability, working solutions were aliquoted to minimize repeated freeze-thaw cycles.

The cytotoxicity of MV was assessed using the Cell Counting Kit-8 (CCK-8) assay to determine cell viability. Porcine alveolar macrophages (PAMs) and Marc-145 cells were seeded in 96-well plates and treated with varying concentrations of MV (0, 100, 200, 400 or 800 μM) for 24, 48, 72, and 96 h. Following the manufacturer's protocol, CCK-8 reagent was added to each well, and cells were incubated for 2 h at 37°C under 5% CO₂. Absorbance was measured at 450 nm using a microplate reader. Cell viability (%) was calculated using the formula:Cell viability (%) = [OD (sample) – OD (blank)/OD (control) – OD (blank)] × 100%, where OD (blank) represents the background absorbance of cell-free medium, and OD (control) denotes untreated cells.

To investigate the stage-specific antiviral effects of mogroside V (MV) and evaluate its ability to inhibit PRRSV replication in Marc-145 and PAM cells, three distinct treatment groups were established for in vitro evaluation: pre-treatment group, post-treatment group, and co-treatment group. Viral replication was assessed through multiple approaches: quantification of viral mRNA copies by RT-qPCR, determination of viral titers using the TCID₅₀ method, and analysis of PRRSV N protein expression via immunofluorescence assay (IFA). The experimental design was as follows:

Pre-treatment group: to investigate the preventive effect of MV prior to PRRSV infection, Marc-145 or PAM cells were pretreated with MV before viral challenge. Cells in good growth condition were seeded in 6-well plates and cultured until monolayer formation. After removing the old medium, cells were washed twice with PBS, followed by incubation with varying concentrations of MV for 24 h. The drug was then removed, and cells were washed twice with PBS before infection with PRRSV (100 TCID₅₀) for 48 h. Subsequently, viral mRNA copies, viral titers, and PRRSV N protein fluorescence expression were measured to calculate viral inhibition rates.

Post-treatment group: In order to investigate the effect of MV on PRRSV infection, MV was added after Marc-145 or PAM cells were infected with PRRSV. The cells with good growth status were inoculated in the 6-well cell culture plate. After the cells grew to monolayer, the old medium was discarded and the cells were cleaned twice with PBS. PRRSV (100 TCID₅₀) was inoculated and incubated for 1 h, during which the virus could be shaken repeatedly to fully interact with cells. The virus venom was removed, washed twice with PBS, and incubated with different concentrations of MV for 48 h. After that, the viral mRNA copies and viral titer were detected, the viral inhibition rate was calculated, and the fluorescence expression of PRRSV N protein was observed.

Co-treatment group: In order to study the direct effect of MV on PRRSV, PRRSV and MV were simultaneously inoculated in Marc-145 or PAM cells for treatment. The cells with good growth status were inoculated in the 6-well cell culture plate. After the cells grew to monolayer, the old medium was discarded and the cells were cleaned twice with PBS. MV at different concentrations was mixed with PRRSV (100 TCID₅₀) and inoculated into Marc-145 and PAM cells for 48 h. After that, the viral mRNA copies and viral titer were detected, viral inhibition rate was calculated, and the fluorescence expression of PRRSV N protein was observed.

The half-maximal inhibitory concentration (IC₅₀) value for MV-mediated cytotoxicity was calculated through nonlinear regression fitting of dose-response curves.

Real-time quantitative PCR (RT-qPCR) was performed to determine PRRSV gene expression levels. Viral RNA was extracted from cell culture supernatants using a commercial viral DNA/RNA extraction kit according to the manufacturer's protocol, with subsequent storage at −80°C until further analysis. RT-qPCR amplification were conducted using TransScript Green One-Step qRT-PCR SuperMix kit. The relative expression levels of cellular cytokines were analyzed by RT-qPCR with primer sequences listed in Table 1.

The supernatant from 6-well culture plates was collected into 1.5 mL microcentrifuge tubes and stored at −80°C for subsequent analysis. Cells were washed twice with PBS and fixed with ice-cold methanol for 15 min at room temperature. Non-specific binding sites were blocked with 1% BSA containing 0.05% Tween 20 for 30 min at ambient temperature. Cells were then incubated with anti-PRRSV N protein monoclonal antibody either for 2 h at 37°C or overnight at 4°C. Following primary antibody incubation, cells were treated with fluorophore-conjugated secondary antibody (goat anti-mouse IgG, Alexa Fluor 488) for 1 h at 37°C. Nuclei were counterstained with DAPI for 10 min at room temperature protected from light. Fluorescence microscopy was performed to examine the subcellular distribution and expression levels of PRRSV N protein. Stained cell monolayers were visualized under 4 × , 10 × , and 20 × objective lenses using an epifluorescence microscope. Representative digital images were acquired using camera integrated with the microscopy system.

All experimental data are presented as mean ± standard deviation (SD). Raw data were collated using Microsoft Excel and subjected to statistical analysis using SPSS 29.0 software. Intergroup differences were evaluated by one-way analysis of variance (ANOVA) followed by appropriate post-hoc tests. The threshold for statistical significance was set at P < 0.05. Significant differences among groups are denoted by lowercase superscript letters (a, b, c), where: Groups sharing the same letter indicate no statistically significant difference (P > 0.05), Groups with different letters demonstrate significant differences (P < 0.05). Graphical representations were generated using GraphPad Prism 8.0 software with error bars representing SD. All experiments were performed with a minimum of three biological replicates.

The cytotoxicity of MV was evaluated using the CCK-8 assay. Marc-145 and PAM cells were treated with varying concentrations of MV (0, 100, 200, 400, and 800 μM), alongside a negative control group (untreated cells) and a blank control group (cell-free medium), followed by incubation for 24, 48, 72, and 96 h. The half-maximal inhibitory concentration (IC₅₀) of MV in Marc-145 and PAM cells was determined. At 24 h, the IC₅₀ values were 685.8 μM and 577.2 μM (Figure 1A), respectively; at 48 h, they were 756.5 μM and 598.8 μM (Figure 1B); at 72 h, they were 755.9 μM and 703.7μM (Figure 1C); and at 96 h, they were 759.6 μM and 582.2 μM (Figure 1D). The IC₅₀ values were all ≥ 577.2 μM. The results demonstrated that at an MV concentration of 400 μM, the viability of both PAM and Marc-145 cells exceeded 75%, indicating no significant cytotoxicity. Therefore, 400 μM was established as the maximum safe drug concentration for subsequent antiviral studies.

In this study, Marc-145 and PAM cells were treated with escalating concentrations of MV (0, 100, 200, and 400 μM) prior to infection with two PRRSV strains (NADC30 and HP-PRRSV-GFP) at a standardized inoculum of 100 TCID₅₀. Viral replication was assessed 48 h post-infection (hpi) through integrated virological and molecular analyses. MV exhibited suppression of viral protein expression and replication across both PRRSV subtypes, as validated by TCID₅₀ determine, RT-qPCR, and IFA (Figure 2).

Notably, 400 μM MV significantly reduced viral titers and PRRSV mRNA levels in both cell types compared to untreated controls (P < 0.01; Figures 2A–H). In Marc-145 cells, HP-PRRSV-GFP titers decreased from 10^4.7 to 10^3.9 TCID₅₀/mL (Figure 2B), while NADC30 titers declined from 10^5.2 to 10^2.8 TCID₅₀/mL (Figure 2F). Similar trends were observed in PAMs: HP-PRRSV-GFP titers dropped from 10^3.6 to 10^2.6 TCID₅₀/mL (Figure 2D), and NADC30 titers decreased from 10^5.1 to 10^3.5 TCID₅₀/mL (Figure 2H). At 200 μM, MV significantly suppressed HP-PRRSV-GFP mRNA copies in PAMs (P < 0.05; Figure 2C), though no significant reduction in viral titers was observed (P >0.05; Figure 2D). IFA corroborated these findings, in Marc-145 cells, high-dose MV (400 μM) markedly diminished viral fluorescence intensity (P < 0.01; Figure 2I). Similarly, PAMs treated with 400 μM MV exhibited significantly reduced PRRSV N protein expression (P < 0.01; Figure 2J), whereas lower concentrations (100 and 200 μM) showed no statistically significant effects. Collectively, these data establish MV as a potent inhibitor of PRRSV replication in both Marc-145 and PAM cell models, with efficacy related to the concentration of MV.

To gain a deeper understanding of the mechanism underlying MV-mediated PRRSV inhibition, we systematically compared two therapeutic strategies: post-treatment (V–M) and co-treatment (M+V), relative to pre-treatment (M–V). Marc-145 cells and PAMs were exposed to 100 TCID₅₀/mL of NADC30 or HP-PRRSV-GFP strains with concurrent or sequential MV administration (0, 100, 200, 400 μM) (Figure 1E). Following 48 h of drug treatment, viral supernatants were harvested, and viral titers as well as viral mRNA copies were quantified using TCID₅₀ assay and RT-qPCR, respectively. Concurrently, IFA was performed on the cells for viral validation.

The results demonstrated that treatment with MV at varying concentrations initiated 1 h post-PRRSV infection led to a concentration-dependent suppression of viral replication. Specifically, at 400μM MV, viral mRNA copies were significantly reduced (P < 0.01), and viral titers decreased to undetectable levels, indicating complete inhibition of infectious viral replication under these experimental conditions (Figures 3A–H). Similarly, when MV was co-administered with PRRSV across a range of concentrations, the 400 μM MV treatment markedly suppressed viral replication in cells (P < 0.01) (Figures 3I–P). Specifically, in HP-PRRSV-GFP-infected Marc-145 and PAM cells, as well as NADC30-infected Marc-145 cells, viral titers dropped below the detection threshold (Figures 3J, L, N). In NADC30-infected PAM cells, viral titers approached the lower detection limit (CPE observed in only two replicate wells in TCID₅₀ assays), with minimal residual infectivity (Figure 3P). Notably, immunofluorescence assay (IFA) revealed no detectable PRRSV N protein expression in either Marc-145 or PAM cells treated with 400 μM MV under both post-infection and co-treatment regimens (Figures 3Q, R). These findings indicate that both MV-mediated blockade (post-infection treatment) and co-treatment strategies exhibited comparable efficacy in suppressing PRRSV replication, effectively inactivating the virus at sufficient concentrations. Furthermore, both strategies demonstrated superior inhibitory efficacy compared to prophylactic treatment (M–V). Collectively, the study validates that MV inhibits PRRSV replication in a treatment modality-dependent manner.

In this study, three concentrations of MV (100, 200, and 400 μM) were applied to Marc-145 and PAM cells under three treatment regimens (M–V, V–M, M+V). Viral inhibition rates for both viral titers (TCID₅₀) and viral mRNA copies (RT-qPCR) were calculated using the formula: Inhibition rate = [1 – (Viral load in MV-treated group/Viral load in control group)] × 100%.

For HP-PRRSV-GFP-infected Marc-145 and PAM cells, treatment with MV at concentrations ≤ 200 μM resulted in average mRNA inhibition rates below 42% and viral titer inhibition rates below 43%. In contrast, the 400 μM MV treatment achieved average mRNA and viral titer inhibition rates exceeding 95%. Notably, the high-dose group (400 μM) exhibited a 53-percentage-point increase in mRNA inhibition and a 52-percentage-point increase in viral titer inhibition compared to the medium-dose group (200 μM) (Figures 4A–C). Similarly, in NADC30-infected Marc-145 and PAM cells, MV concentrations ≤ 200 μM yielded average mRNA inhibition rates below 45% and viral titer inhibition rates below 55%. However, the 400 μM MV treatment elevated these values to >94% and >98%, respectively. The high-dose group demonstrated a 49-percentage-point enhancement in mRNA inhibition and a 43-percentage-point improvement in viral titer inhibition relative to the medium-dose group (Figures 4D–F).

Critically, across both viral strains (HP-PRRSV-GFP and NADC30), treatment with 400 μM MV consistently suppressed viral mRNA and viral titers in Marc-145 and PAM cells, with average inhibition rates surpassing 94%. These findings unequivocally demonstrate that MV inhibits PRRSV replication in a dose-dependent manner, with maximal efficacy achieved at the highest concentration tested.

Our findings demonstrate that MV significantly upregulates the gene expression of key immunomodulatory factors, including IL-1, IL-2, IL-8, IL-18, and IL-1β (Figure 5). Beyond its direct antiviral activity against PRRSV replication, MV enhances host defense mechanisms by activating immune response pathways and upregulating critical immunoregulatory factors. This dual mode of action—combining viral suppression and immune potentiation—likely facilitates early cellular recognition of viral invasion, thereby augmenting the cell's intrinsic antiviral capacity (Ruan et al., 2024).