The porcine intestinal epithelial cell line (IPEC-J2) were obtained from Dr. Li Wang at the Institute of Animal Science, Guangdong Academy of Agricultural Sciences, China. The cells were cultured in Roswell Park Memorial Institute (RPMI) Medium 1640 supplemented with 10% fetal bovine serum (FBS). Vero cells were stored in our laboratory at the Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou, China, were used for propagation of the GD/HZ/2016 (GenBank Accession: OP191700.1) strain. The vero cells were cultured in DMEM containing 15 μg/ml trypsin. The L. mucosae G01 strain uploaded with NCBI sequence number OR783178.

The Lactobacillus strains utilized in this research were obtained from recently collected fecal samples of Guangxi Bama scented pigs. These samples were isolated from five sows in the early gestation stage and were collected over a span of three consecutive days. A total of 15 pig fecal samples were collected using sterile swabs. Subsequently, the samples were transferred into 1.5 mL centrifuge tubes containing PBS and promptly placed in ice boxes for transportation to the laboratory.

The procedures for isolating Lactobacillus strains from swine fecal samples were conducted following the methodology outlined by Halimi and Mirsalehian (2016). The collected fecal samples were introduced into de Man, Rogosa, and Sharpe (MRS) broth and incubated at a temperature of 37°C for a duration of 15 h. Subsequently, the samples were diluted and streaked onto MRS agar plates in triplicate. To amplify the 16S rRNA genes, the universal primers (27 F and 1492 R) were employed: 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′- GGTTACCTTGTTACGACTT-3′). The PCR products obtained were ligated into the pMD19T vector and subsequently subjected to sequencing analysis using the automated sequencing service offered by Sangon Bioengineering (Guangzhou) Co., Ltd. The 16S rRNA sequences obtained were subjected to comparison using the MUSCLE algorithm in Mega 11 v11.0.13 (Callaway and Singh-Cundy, 2019). Subsequently, a phylogenetic tree was constructed employing the maximum Neighbor Joining (NJ) algorithm to elucidate the evolutionary relationships among the isolated strains. The resulting files were subsequently uploaded to iTOL¹ (Shi et al., 2018) for the purpose of visualizing the evolutionary tree, which included species information for each strain.

The cell viability assay of Lactobacillus strains on IPEC-J2 cells was conducted following modifications to the method described by EL-Adawi (El-Adawi, 2012). IPEC-J2 cell suspensions were prepared at a concentration of 5×10⁴ cells/ml and inoculated into 96-well plates, followed by incubation at 37°C for 12 h. Simultaneously, Lactobacillus strains were incubated overnight, washed three times with sterile PBS buffer, and subsequently centrifuged at 6,000 × g for 10 min. Subsequently, 10, 1, and 0.1 MOI of the Lactobacillus strains were added to each sample, which were then incubated for 24 h. The CCK-8 reagent was added and incubated at 37°C for 1 h. The absorbance at a wavelength of 450 nm was quantified utilizing the Multiskan Sky instrument manufactured by Thermo Scientific. The result of the calculation is: Cell viability (%) = (ODt / ODc) × 100%, where ODt and ODc are the absorbance of treated and control cells, respectively (Kurpe et al., 2020).

The experimental methodology employed to evaluate the therapeutic efficacy of Lactobacillus strains on IPEC-J2 cells infected with the PEDV strain GD/HZ/2016 was derived from the work of Chang-Liao et al. (2020). To assess the anti-PEDV effect, the IPEC-J2 cells were subjected to both prestimulation and poststimulation using Lactobacillus strains. For the prestimulation phase, the IPEC-J2 cells were cultured in 12-well plates and exposed to either 1 or 0.1 MOI of Lactobacillus strains. Following a 2 h incubation period, the cells were rinsed thrice with sterile PBS and subsequently infected with 0.1 MOI of PEDV. Post-stimulation, the IPEC-J2 cells were exposed to 0.1 MOI of PEDV, followed by three washes with sterile PBS. Subsequently, the cells were treated with either 1 or 0.1 MOI of Lactobacillus strains for a duration of 2 h. After another wash with sterile PBS, the cultures were incubated at 37°C in a 5% CO₂ environment and harvested 24 h post-infection with PEDV. Cell samples were collected for qRT-PCR analyses to evaluate the impact of Lactobacillus strains on virus replication.

The TaKaRa MiniBEST Universal RNA Extraction Kit, manufactured in China by TaKaRa, was utilized to extract total RNA from IPEC-J2 cells subjected to various experimental conditions. For qRT-PCR analysis, the HiScript^® II one Step qRT-PCR SYBR Green Kit, produced by Vazyme in China, was employed. The primer sequences employed for gene detection can be found in Table 1.

The experimental procedure was performed according to the methodology outlined by Shen et al. (2016). In brief, IPEC-J2 cells were lysed using 150 μL of lysis buffer obtained from Thermo Fisher Scientific, China. The resulting proteins were then separated and transferred onto a polyvinylidene fluoride membrane. Subsequently, the membrane was incubated for 1 h with 5% non-fat milk, followed by a 2 h incubation with primary antibodies against PEDV N-protein (Medgene Labs, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ABclonal, China), synthetic phosphorylated peptide around interferon regulatory Factor 3 (pIRF3) (ABclonal, China), and interferon regulatory Factor 3 (IRF3) (ABclonal, China), followed by three washes with PBST. The PVDF membranes were subsequently subjected to incubation with horseradish peroxidase-conjugated goat anti-mouse and rabbit anti-lgG(H + L) secondary antibodies (Bioworld, China). The visualization of immunity protein bands was achieved by employing an ECL Kit (Millipore, China).

Refer to Zhang W’s method (Zhang et al., 2023) as follows. The cells were fixed using a 4% paraformaldehyde solution (Beyotime, China) and permeabilized with a PBS solution containing 0.1% Triton X-100. Subsequently, the cells were blocked with a 1% bovine serum albumin solution (BBI Life Sciences, China) for 1 h. Following this, the cells were incubated overnight at 4°C with a mouse anti-PEDV N IgG antibody, and then with Alexa Fluor™ 594 goat anti-mouse IgG (Invitrogen, China) for 1 h at 37°C. After washing with PBS three times, the cells were incubated with 4,6-diamidino-2-phenylindole (DAPI, Beyotime) for 5 min. Finally, the cells were examined using a differential fluorescence microscope.

Refer to Zhang et al. (2023) method as follows. In order to ascertain the virus titers, Vero cells were cultivated in 96-well plates until reaching a confluence of 70–80%. Subsequently, the cells were infected with 10-fold serial dilutions of PEDV, with a total of four replicates. Following an incubation period of 72 h, the cells were evaluated using the immunofluorescence assay (IFA). The Reed-Muench method was employed to determine the virus titers, which were quantified as TCID₅₀ per milliliter.

The data analysis was performed utilizing GraphPad Prism v8.0 software, employing an ANOVA to evaluate the statistical significance of differences in mean values. The mean values for each group were reported as the mean ± standard deviation (SD). Statistical analyses were conducted to ascertain the presence of significant differences between the treatment and control groups. One-way analysis of variance followed by the Tukey-Kramer post-test was used. A p < 0.05 was considered statistically significant.

A total of 70 Lactobacillus strains were selected for polymerase chain reaction (PCR) using the 27F/1492R common primer specific for bacterial 16S rRNA. This PCR amplification resulted in a bright band at approximately 1500 base pairs, indicating successful amplification of the 16S rRNA product (Figure 1A). The PCR products were subsequently sequenced and compared to the National Center for Biotechnology Information (NCBI) database for further analysis of the isolated Lactobacillus species. The analysis revealed that 28 strains potentially belonged to Limosilactobacillus reuteri DSM 20016 (L. reuteri DSM20016), 3 strains to Weissella thailandensis FS61-1, and 4 strains to Limosilactobacillus vaginalis ATCC 49540 (L. vaginalis ATCC49540). Seventeen strains were identified as Limosilactobacillus mucosae G01 (L. mucosae G01), twelve as Limosilactobacillus johnsonii CIP103620 (L. johnsonii CIP103620), and 6 strains as Ligilactobacillus salivarius JCM1231 (L. salivarius JCM1231) (Figure 1B). The construction of a phylogenetic tree utilizing the 16S rRNA gene sequences revealed a range of Lactobacillus species that were obtainable from Bama pigs fecal (Figure 1C).

To assess the cytotoxic effects of various Lactobacillus strains on IPEC-J2 cells, a CCK-8 assay was conducted using three different bacterial concentrations (10, 1, and 0.1 MOI). As illustrated in Figure 2, the L. mucosae G01 strain was observed to promote the proliferation of IPEC-J2 cells, as evidenced by the optical density value (OD₄₅₀) detected using the CCK-8 reagent. At a concentration of 0.1 MOI, L. johnsonii CIP103620, L. salivarius JCM1231, and the L. mucosae G01 strain exhibited no cytotoxicity toward IPEC-J2 cells. Conversely, Weissella thailandensis FS61-1 and L. vaginalis ATCC49540 demonstrated cytotoxic effects on IPEC-J2 cells.

The inhibitory effects of L. reuteri DSM20016, L. mucosae G01, L. johnsonii CIP103620, and L. salivarius JCM1231 on PEDV infection in IPEC-J2 cells were assessed through prestimulation and poststimulation (Figure 3). Analysis using qRT-PCR revealed a significant reduction in PEDV N mRNA levels with L. mucosae G01 treatment. Furthermore, L. reuteri DSM20016 and L. mucosae G01 demonstrated inhibition of viral replication in the poststimulation mode, while the other strains did not exhibit a significant effect. Considering the antiviral properties observed, L. mucosae G01 was chosen for subsequent investigation.

In order to assess the anti-PEDV efficacy of various L. mucosae G01 strains, the L. mucosae DSM13345 strain (obtained from Minebea) was utilized as a control. IPEC-J2 cells infected with PEDV were subjected to pre-stimulation with L. mucosae at a 1 MOI concentration, and the expression levels of PEDV N mRNA were determined using qPCR. The L. mucosae G01 strain isolated from the intestine demonstrated a significant inhibitory effect on PEDV replication in comparison to the L. mucosae DSM13345 strain (Figure 4). The L. mucosae G01 significantly reduced (p < 0.05) PEDV N mRNA expression compared to L. mucosae DSM13345 (Figure 4A), and supernatant viral titers were shown to be consistent with mRNA levels as well (Figure 4C). The findings from the figure strongly suggest that L. mucosae G01 possesses the potential to counteract PEDV infection in IPEC-J2 cells.

To ascertain the potential correlation between the concentration of L. mucosae G01 and the potency of antiviral activity, our findings indicate that pre-stimulating IPEC-J2 cells with 0.1 and 1 MOI of L. mucosae G01 for a duration of 24 h resulted in a notable reduction in PEDV N mRNA levels (p < 0.05) (Figure 5A). Furthermore, a dose-dependent decline in PEDV N protein expression was observed (p < 0.05) (Figures 5B, E). These outcomes strongly imply that L. mucosae G01 effectively impedes PEDV production, with inhibition rates ranging from 21.90 to 68.10%. Furthermore, our study revealed a significant decrease in PEDV titers in a dose-dependent manner (p < 0.05), as evidenced by a 1.53-log reduction in progeny virus levels, indicating the inhibition of PEDV replication (Figures 5C, F). However, no statistically significant differences were observed in the expression levels of PEDV N mRNA in poststimulation IPEC-J2 cells following PEDV infection at 0.1 MOI L. mucosae G01 (Figure 5D). These findings suggest that L. mucosae G01 exhibits anti-PEDV activities, which hold potential for the development of effective interventions against this pathogen.

In the present study, the investigation focused on assessing the levels of interferons (IFN α, IFN β, IFN λ1, and IFN λ3) in IPEC-J2 cells before and after stimulation with the L. mucosae G01 strain. The obtained results revealed a significant elevation in the expression of IFN types of interferons in IPEC-J2 cells inoculated with L. mucosae G01 compared to the control group. Furthermore, both pre- and post-stimulation with L. mucosae G01 were found to significantly enhance the expression of IFN α, IFN β, IFN λ1, and IFN λ3 (Figures 6A–D). These findings suggest that L. mucosae G01 has the potential to augment the antiviral response by inducing the expression of interferons in IPEC-J2 cells.

When comparing the IPEC-J2 cells after stimulation with L. mucosae G01 to the PEDV cells, it was observed that both prestimulation and poststimulation resulted in a significant increase in MX2 expression (p < 0.001) (Figure 6G). Furthermore, the levels of OAS1, MX1, and ZAP expression in the prestimulated and post stimulated IPEC-J2 cells were found to be significantly different following L. mucosae G01 infection (p < 0.05) (Figures 6E, F, H). However, L. mucosae G01 did not cause changes in ISG15 (Figure 6I).

PEDV binds to the TLR3 receptor in IPEC-J2 cells, triggering an intracellular signaling cascade that results in the phosphorylation of interferon regulatory factor 3 (IRF3). Subsequently, the activated IRF3 translocates to the nucleus and stimulates the transcription of interferons (Stanifer et al., 2019).

To investigate the impact of L. mucosae G01 on IRF3 phosphorylation and IFN signaling, we conducted an assessment of the pIRF3/IRF3 ratio in IPEC-J2 cells subjected to L. mucosae G01 treatment. The outcomes demonstrate that exposure to 1 MOI and 0.1 MOI L. mucosae G01 strain resulted in a dose-dependent stimulation of IRF3 phosphorylation in IPEC-J2 cells (Figure 7). These findings imply that L. mucosae G01 possesses the ability to induce IRF3 phosphorylation in IPEC-J2 cells, potentially contributing to its anti-PEDV properties. The quantification of p-IRF3/IRF3 levels was accomplished through western blot analysis of lysed cellular proteins. The results of this study indicate that L. mucosae G01 potentially contributes to the activation of IFN signaling and the induction of IRF3 phosphorylation in IPEC-J2 cells.