This work was conducted in accordance with the guidelines approved by the Quality Supervision, Inspection, and Quarantine of the People's Republic of China (GB/T 17236–2008). The animal experimental proposals were approved by the Animal Welfare Committee of China Agricultural University (permit number: DK996) and performed in accordance with the Guidelines for Experimental Animals of the Ministry of Science and Technology (Beijing, China).

The piglets in this study came from a commercial breeding farm in Shandong Province (Binzhou, China). All the piglets were offspring of Landrace and Yorkshire pigs. The sows were fed corn and soybean meal based commercial diets under the same conditions. When the sows gave birth within a week, multiple piglets had diarrhea symptoms in farrowing house. Four litters of adjacent sows were chosen that gave birth at similar times in the farrowing house with diarrhea symptoms, and two piglets with diarrhea symptoms and two piglets without diarrhea symptoms in each litter were chosen. A total of 16 piglets (Landrace-Yorkshire hybrids) were selected. All piglets were 7–9 days old and weighed about two kilograms during the normal lactation process. The selected suckling piglets were slaughtered, and samples of blood, jejunum chyme and jejunum mucosa were collected from each piglet within 30 min under aseptic conditions. All samples were collected in sterile tubes and snap frozen in liquid nitrogen.

To confirm the status of PEDV infection in each piglet, quantitative real-time PCR (qPCR) was used to detect PEDV. First, total virus RNA was extracted from the jejunal mucosa leach supernatant using the QIAamp viral RNA MINI kit (QIAGEN, Valencia, CA). Reverse transcription was performed using the PrimeScript®RT reagent kit with a gDNA eraser (Takara, Japan). Viral quantification was carried out using the absolute quantification method. To construct the recombinant plasmid, a 170bp fragment of the PEDV CV777 strain was amplified and cloned into the pTZ57R/T vector (2886bp, Takara, Japan). The concentration of recombinant plasmids was measured using Nanodrop 2000 (Thermal Fisher, USA), and the plasmid copy number was measured using the formula: number of copies of PEDV = (concentration in ng ×6.022 ×10²³) / (genome length ×10⁹ ×650). To generate the standard curve for SYBR Green I real time qPCR, 10-fold serial dilutions of plasmids DNA from 1 ×10⁸copies/μLto 1 ×10³ copies/μL were produced. Thereafter, the number of viral copies was determined for these assays. Specific primer sequences (forward: 5′-GGTTCTATTCCCGTTGATGAGGT-3′; reverse: 5′ -AACACAAGAGGCCAAAGTATCCAT-3′) were used for the PEDV CV777 detection, as previously described (26). SYBR Green I real time qPCR was performed using a Roche LightCycler480 instrument with a total reaction volume of 20 μL (comprising 10 μL SYBR-Green I Master Mix, 1 μL upstream primer, 1 μL downstream primer, 1 μL cDNA template, and ddH₂O) (Roche Applied Science). All qPCR reactions were performed in 96-well plates, and each sample was run in triplicate.

Based on viral copy number and phenotype, piglets were classified into uninfected [U group, n = 8, −1.0904 ± 0.2029 log(copies)], low viral load [L group, n = 3, 0.3713 ± 0.4093 log(copies)], and high viral load [H group, n = 5, 3.0170 ± 0.522 log(copies)]. Both L and H groups were composed of piglets suffering from diarrhea, but their viral load significantly differed (Kruskal–Wallis test, p < 0.01) (Supplementary Figure 1).

Microbial genomic DNA was extracted from jejunal mucosa and jejunal digesta samples and purified using a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. Thereafter, the DNA concentration was measured using a UV-Vis spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA, USA), and the quality was confirmed with 1% agarose gel electrophoresis.

The V4 region of the 16S rRNA gene from jejunal mucosa samples was PCR-amplified using 515F-806R universal primers combined with adapter and barcode sequences (forward primer: 5′ -GTGCCAGCMGCCGCGGTAA-3′; reverse primer: 5′ -GGACTACHVGGGTWTCTAAT-3′). For jejunal digesta samples, the V3-V4 region of the 16S rRNA gene was amplified with 314F-608R universal primers (forward primer:5′-CCTAYGGGRBGCASCAG-3′;reverse primer: 5′ -GGACTACNNGGGTATCTAAT-3′) (27). The total reaction volume was 30 μL, containing 15 μL Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA), 0.2 μM forward and reverse primers, and 10 ng template DNA. The thermocycling protocol was as follows: 98°C for 1 min, followed by 30 cycles of 98°C for 10 s, 50°C for 30 s, and 72°C for 60 s; and a final extension at 72°C for 5 min. The PCR products were purified using the GeneJET Gel Extraction Kit (Thermo Fisher Scientific) following the manufacturer's protocol. Thereafter, the constructed library was sequenced using the Illumina Hiseq 2500 platform (2 ×250 paired ends). The data were deposited in the National Center for Biotechnology Information's Short Read Archive under the accession number SRP334838.

To minimize the effects of random sequencing errors, raw FASTQ files were demultiplexed, quality-filtered using Trimmomatic (version 0.33), and merged using FLASH version 1.2.7 (28, 29). To obtain high-quality tag sequences, initial base sites with Phred score < 20 were truncated; tags were filtered if their continuous, high-quality base length was less than three-quarters of the whole sequence; chimeric sequences were removed using UCHIME (version 4.2) (30). Sequences with ≥ 97% similarity were assigned to the same operational taxonomic units (OTUs) using USEARCH (version 10.0) (31). A representative sequence for each OTU was screened and taxonomically analyzed against the SILVA database (https://www.arb-silva.de/) using Ribosomal Database Project Classifier (version 2.2) (32). The composition of each sample community was determined at the phylum, class, order, family, genus, and species level.

The alpha diversity indices were evaluated using Mothur (version 1.30) (33). The beta diversity per group was calculated using principal coordinate analysis (PCoA) based on weighted UniFrac distances (34, 35) in QIIME (version 1.9.1). Linear discriminant analysis (LDA) effect size (LEfSe) was performed to identify biomarkers that differed significantly between groups (35). Statistical significance was set at an LDA score of 3.

The porcine intestine epithelial cell J2 (IPEC-J2) cell line was maintained in our laboratory. Vero E6 cells and PEDV CV777 strain were kindly provided by Dr. Jun Han at the College of veterinary Medicine, China Agricultural University (Beijing, China). IPEC-J2 and Vero E6 cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) without penicillin-streptomycin. The cells were cultured in a humidified atmosphere at 37°C and 5% CO₂. The Lactobacillus salivarius JCM strain (Genebank: AB289296) was obtained from the China General Microbiological Culture Collection Center (CGMCC). L. salivarius cells were grown in MRS broth (Oxoid) at 37°C for 18 h. The number of CFU/mL was measured after incubation at 37°C for 18 h via serial fold dilutions and planting on MRS agar (Oxoid). Vero E6 cells were seeded in 96-well plates, inoculated with the PEDV CV777 strain at a multiplicity of infection (MOI) of 0.1, and cultured in serum-free DMEM for 60 h at 37°C and 5% CO₂ in a humidified environment. The tilter of the progeny virions was measured using the TCID50 method.

To determine the reasonable duration of virus infection, the PEDV CV777 was inoculated into IPEC-J2 cells at four different time points. IPEC-J2 cells were seeded in six-well plates and treated with the PEDV CV777 strain at a MOI of 0.1. After 0.5, 1, 2, and 3 h of incubation for at 37°C and 5% CO₂, the cells were washed three times with PBS to remove unattached virions. Thereafter, internalized viruses were released through three freeze-thaw cycles and quantified by qPCR, as mentioned above.

After estimating the best time point, we tested whether L. salivarius strain can provide protection against PEDV by incubating IPEC-J2 cells with different concentrations of L. salivarius before PEDV infection. First, IPEC-J2 cells were seeded in six-well plates and pretreated with either antibiotic-free DMEM or L. salivarius at multiplicity of bacteria (MOB) of 1, 10, or 100, respectively. After 2 h of incubation, the unbound L. salivarius cells were washed three times with PBS. Thereafter, IPEC-J2 cells were infected with PEDV CV777 strain at a MOI of 0.1. Cells were cultured at 37°C and 5% CO₂ for 1 h and 2 h, in separate culture vessels. Finally, the unattached virions were removed, and IPEC-J2 cells were washed with PBS three times before being overlaid with 1 mL DMEM. To release the internalized viruses, the cells were subjected to three freeze-thaw cycles. The supernatants were collected and the total numbers of infected cells were quantified via qPCR for absolute quantification. The most appropriate L. salivarius dose was determined to be one corresponding to the lowest quantity of PEDV-infected cells.

Based on the system established above, IPEC-J2 cells were seeded in six-well plates and pretreated with either antibiotic-free DMEM or L. salivarius at a MOB of 1 for 2 h. Thereafter, unbound bacteria were removed by washed the cells with PBS three times. Subsequently, IPEC-J2 cells were infected with CV777 at a MOI of 0.1 and cultured at 37°C and 5% CO₂ for 2 h. Then, the expression levels of candidate genes in IPEC-J2 cells were quantified via qPCR at 2, 8, and 16 h post-infection.

All experiments were performed in triplicates and repeated three times. Data are presented as the means ± standard error. Differences in the PEDV copy number between the groups were analyzed using the Kruskal-Wallis test, followed by Bonferroni post hoc adjustment. Differences in the expression levels of genes between the two groups were compared using Student's t-test. A p value below 0.05 and 0.01 were considered significant and very significant, respectively. Statistical analysis was performed using the R package.

As PEDV invades the host via the jejunum, we focused on the jejunal microbiota rather than fecal microbiota. In addition, this study aimed to distinguish “transient microbes” from “resident microbes.” Therefore, microbes attached to the jejunal mucosa were sequenced and referred to as jejunal mucosal microbiota. The microbiota in the jejunal digesta, which we referred to as the jejunal content microbiota, was also sequenced. The mean number of taxon tags in jejunal mucosa samples was 92,311, ranging from 54,926 to 126,987 reads, whereas the average of taxon tags of jejunal digesta samples was 90,448, ranging from 61,510 to102,058. The coverage of jejunal mucosa samples and jejunal digesta samples was 98.472 and 99.995%, respectively, indicating reliable sequencing accuracy.

Different measures of the alpha diversity index were calculated to characterize the microbial diversity within individuals. Although a different trend of alpha diversity index was observed between piglets with diverse infection status, the difference was not statistically significant (p > 0.05). The alpha diversity index of the U group was higher than that of the H group for both sample types (Supplementary Table 1). The PCoA scatterplot revealed clear segregation of gut bacterial communities between piglets in different groups based on beta diversity; however, no clear segregation was observed between different sample types (Figure 1).

Thereafter, the taxonomic analysis indicated that four phyla, namely, Firmicutes, Bacteroidetes, Proteobacteria, and Fusobacteria, accounted for 90% of the bacteria in each group (Figure 2). At phylum level, Firmicutes (48.03%), Proteobacteria (26.99%), Bacteroidetes (9.54%), Fusobacteria (5.69%) and Actinobacteria (1.90%) were identified as the dominant phyla in the bacterial communities of all mucosa samples, among which Firmicutes and Proteobacteria accounted for over 65% of the bacteria. The abundance of Firmicutes in the U group was the highest (61.47%), followed by 46.21% in the L group and 27.64% in the H group. The H group had a higher abundance of Proteobacteria (56.47%) than the L group (21.83%) and U group (10.51%), whereas the abundance of Bacteroidetes and Fusobacteria in the L group was higher than that in the H and U group (Figure 2A). In jejunal content samples, the dominant phyla were the same as those of jejunal mucosa, whereas the abundance of Firmicutes (52.83%) and Proteobacteria (35.22%) was higher than that in jejunal mucosa samples (Firmicutes, 48.03%; Proteobacteria, 26.99%) (Figure 2B).

At the genus level, Actinobacillus, Lactobacillus, Escherichia-Shigella, Clostridium_sensu_stricto_1, Fusobacterium and Bacteroides were the dominant genera in each group in jejunal mucosa (Figure 3A). In the U group, the abundance of Lactobacillus was 27.74%, which was higher than that in the L (6.30%) and H groups (4.74%). The H group had a higher abundance of Actinobacillus (36.61%) than the U group (2.45%) and the L group (2.15%). In contrast, the L group showed a higher abundance of Bacteroides (9.36%) and Fusobacterium (9.00%). The abundance of Escherichia-Shigella in infected groups (17.18% and 17.14% in the H and L groups, respectively) was higher than that in the U group (2.15% in the U group). In the jejunal contents, Terrisporobacter was the dominant microbe (Figure 3B).

LEfSe analysis was performed to identify potential biomarkers distinguishing between groups. In jejunal mucosa samples, 33 terms of differential bacteria belonging to different taxonomic level were found in the three groups (Figure 4A). There were 37 significantly enriched differential bacteria in the jejunal digesta samples (Figure 4B). At the species level, seven significant bacteria were found in jejunal mucosa samples, compared to four significant bacteria in jejunal digesta samples. Actinobacillus pleuropneumoniae was enriched in all samples of the infected group. L. salivarius, Lactobacillus reuteri, Streptococcus suis, Clostridium pernser, Rothia nasimurium and Clostridium sp ND2 were significantly enriched in the mucosa of uninfected piglets.

Members of Lactobacillus have been regarded as probiotics; therefore, we were particularly interested in determining whether the enrichment of Lactobacillus spp. such as L. salivarius in uninfected piglets could provide effective protection against PEDV infection. We established an in vitro model using IPEC-J2 cells, the PEDV CV777 strain, and the L. salivarius JCM strain to simulate different infection statuses. First, virus replication kinetics experiments were performed to determine in time point at which PEDV fully entered the cell. IPEC-J2 cells were inoculated with PEDV at 0.1 MOI and harvested at 0.5, 1, 2, and 3 h post-infection. Our results showed that the number of viral copies in the cells increased with time. The difference in the number of viral RNA copies between cells harvested at 3 h [3.379027 ± 0.1831912 log(copies)] and 2 h [3.234857 ± 0.1096953 log(copies)] post-infection was not significant, whereas the number of viral RNA copies in cells at 0.5 h [3.052131 ± 0.1327514 log(copies)] and 1 h [3.188493 ± 0.1515467 log(copies)] post-infection was significantly lower than that in cells harvested at 3 h post-infection. The results indicated that PEDV entry occurred between 1 and 2 h post-infection (Figure 5A).

To confirm the role of L. salivarius in the inhibition of PEDV infection, cells were first incubated with different concentrations of L. salivarius (0, 1, 10, and 100 MOI) for 2 h, and then incubated with PEDV for 1 or 2 h at a concentration of 0.1 MOI (Figure 5B). Viral RNA was consistently lower in L. salivarius-pretreated cells than in untreated cells at both 1 and 2 h post-infection. This incubated that L. salivarius has a protective effect on IPEC-J2 cells. When incubated with PEDV for 1 h, L. salivarius-treated cells were infected with significantly less PEDV various than untreated cells (p < 0.01). When IPEC-J2 cells were treated with 1 MOI L. salivarius for 2 h followed by a 2 h incubation with PEDV, PEDV infection was observed to decrease to the minimum level compared with other groups (p < 0.01). The results indicated that L. salivarius treatment at a concentration of 1 MOI exhibited a more robust activity against PEDV infection than treatments at other concentrations (Figure 5B). Therefore, we assumed that treatment with 1 MOI L. salivarius for 2 h, before 0.1 MOI PEDV inoculation to simulate the PEDV infection, in healthy piglets in vitro.

To investigate the effect of L. salivarius on host genes at the transcriptional level, the mRNA expression of some important genes inIPEC-J2 cells was quantified at 2, 8, and 16 h post PEDV-infection via qPCR. Glucose-regulating protein 78 (GRP78) is a chaperone protein of the endoplasmic reticulum (ER), and up-regulation of GRP78 expression is a sign of ER stress. To determine whether PEDV infection causes ER stress, the expression of GRP78 in IPEC-J2 cells was evaluated at different time points post-PEDV infection by qPCR. Indeed, the expression level of GRP78 increased gradually with the increase in viral infection time (Figure 6A). Furthermore, we examined whether pretreatment with L. salivarius would reduce ER stress. Compared with untreated cells, the 1 MOI L. salivarius-treated group showed a gradual decrease in the expression of GRP78, which was significantly lower than that in untreated cells at 8 and 16 h post-PEDV infection (Figure 6A).

The PI3K-Akt signaling pathway plays an important role in PEDV infection. The focal adhesion signaling pathway is also involved in the process of PEDV infection. Therefore, we quantified the mRNA expression levels of ITGA1, ITGA5, ITGB5, FAK, PIK3R1, PIK3CA and AKT1 in IPEC-J2 at 2, 8, and 16 h post-PEDV infection (Figures 6B–H). The expression levels of ITGA1, ITGA5, FAK, PIK3R1, PIK3CA and AKT1 were significantly higher in the 1 MOI L. salivarius-treated group, than in untreated cells at 2, 8, and 16 h post-PEDV infection. Although there was no significant difference in the expression of ITGB5 between the two groups at 2 h, the expression levels of ITGB5 were significantly higher in the 1 MOI L. salivarius-treated group than in untreated cells at 8 and 16 h post-PEDV infection. The results indicated that L. salivarius may prevent PEDV infection in IPEC-J2 cells by up-regulating the FAK/PI3K/Akt signaling pathway.