Correction: Lactobacillus reuteri-derived HDCA suppresses PEDV replication while alleviating virus-triggered inflammation in piglets

PEDV is an extremely contagious coronavirus that not only infects its primary host, pigs but also has the potential to cross species barriers and is capable of infecting cells from a variety of mammals, including humans. 1,2 PEDV spreads mainly through fecal-oral contact and airborne transmission, targeting and invading small intestinal epithelial cells for replication and dissemination. 3 This process directly disrupts intestinal barrier function, triggering severe inflammatory responses such as diarrhea and vomiting, making PEDV a major pathogen responsible for high morbidity and mortality rates in pig populations. 4 The critical lack of effective antiviral drugs or effective vaccines against PEDV has led to mortality rates approaching 100% in neonatal piglets, thus triggering substantial scientific and veterinary concern regarding its pandemic potential and economic impact. 5,6 In recent years, frequent mutations in PEDV strains have posed substantial challenges to the efficacy of existing vaccines, including issues such as immune escape due to antigenic variation and insufficient cross-protection between different genotypes. 7,8 Consequently, the development of novel and effective prevention and control strategies has become a critical focus of current research. Studies have shown that transplanting feces from healthy adult pigs to piglets can increase the survival rate of piglets infected with PEDV by regulating the homeostasis of the gut microbiota. 9 These findings indicate that the gut microbiota is essential for PEDV prevention and management.With the development of multiomics sequencing technology, the influence and regulation of microorganisms on the host have been gradually revealed. In particular, the effects of commensals on the treatment and prevention of intestinal diseases have received extensive attention. Commensals not only maintain intestinal health by modulating the composition of the gut microbiota but also combat pathogenic invasions through the transformation and synthesis of their metabolites. [10][11][12][13][14] Emerging evidence highlights the crucial role of secondary bile acids, microbial-derived metabolites, in regulating host pathogenesis by activating immune responses and preserving intestinal structural integrity. However, current research predominantly focuses on host-microbe interactions while overlooking the direct mechanistic interplay between the gut microbiota/metabolites and the pathogens themselves. 9,[15][16][17][18] We postulate that the gut microbiota and its metabolism may inhibit PEDV replication. To test this hypothesis, we employed 16S rRNA gene amplicon and metagenomic sequencing to identify core microbial taxa associated with PEDV resistance. These core strains were subsequently transplanted into a PEDV-challenged model. Multiomics approaches-including targeted/untargeted metabolomics, transcriptomic profiling, and proteomic analysis-were systematically integrated to delineate the antiviral effects of these core commensals and their metabolites. This comprehensive strategy aims to elucidate the mechanistic interplay between gut-derived microbes and their metabolic repertoire in suppressing PEDV pathogenesis.The PEDV strain GDqy2017 was isolated in 2017 and stored in the laboratory. L. reuteri GZ-1 was isolated in 2022 via De Man, Rogosa and Sharpe (MRS) medium at 37°C under anaerobic conditions.Long Line Continuous -Pig Kidney 1 (LLC-PK1) andIntestinal Porcine Epithelial Cell -Jejunum 2 (IPEC-J2) cells (ATCC)were maintained in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) or Minimum Essential Medium (MEM) supplemented with 10% heat-inactivated FBS (Gibco).Design 1: Investigation of gut microbiota differences between healthy and PEDV-infected piglets. This experiment aimed to compare the gut microbiota of healthy and PEDV-infected piglets. To this end, seven-day-old Landrace piglets were screened to ensure they were Piglets exhibiting severe clinical signs (watery diarrhea, inability to stand, or dyspnea) were euthanized immediately to preclude suffering.Euthanasia was performed via intravenous injection of pentobarbital sodium salt (100 mg/kg) into the marginal ear vein. Death was confirmed by absence of corneal reflex and cessation of cardiac activity for >5 minutes. No anesthesia was administered, as all sampling procedures were conducted postmortem. All procedures adhered to AVMA Guidelines for the Euthanasia of Animals (2020).At 80% confluence, IPEC-J2 monolayers were supplemented with 100 μM HDCA, 20 μM TGR5 antagonist (SBI-115), 20 μM TGR5 Receptor Agonist (CCDC), or DMSO (the culture medium for the mock and PEDV groups was replaced with basal DMEM). After 2 h of incubation, the cells were harvested for measurement of G Protein-Coupled Bile AcidReceptor 1 (TGR5).In a parallel experiment, after 2 h of incubation, the medium from each well was aspirated and stored for later use. All the wells (except those in the mock group) were inoculated with PEDV at an multiplicity of infection (MOI) of 0.1 for 2 h. The supernatant was then discarded, and the cells were replenished with the previously collected conditioned medium to maintain the original treatment environment, with fresh bovine trypsin added to a final concentration of 3.75 μg/mL. After 12 h, cell samples were obtained to assess the Interferon-β (IFN-β) and Interferon-Stimulated Gene 15 (ISG15) levels. In another parallel experiment, after 24 hours of incubation, the supernatants were collected for PEDV copy number and titre determination, while the cells were harvested for western blot analysis of PEDV-N. All treatments included triplicate wells, and experiments were repeated independently three times.IPEC-J2 cells were counted and seeded at 1×10⁶ cells per well. After washing with PBS to remove impurities, cells were suspended in DMEM supplemented with 100 μM HDCA, 20 μM SBI-115, 20 μM CCDC, or DMSO (control groups received basal DMEM). Following 1.5 h incubation, all cells were collected into EP tubes and subjected to three freeze-thaw cycles at -80°C to fully release intracellular Cyclic Adenosine Monophosphate (cAMP). After centrifugation at 1,500 × g for 10 min, supernatants were collected. cAMP concentrations were measured using a commercial ELISA kit (Elabscience, Cat# E-EL-0056) strictly following the manufacturer's instructions.PEDV titres were determined via indirect immunofluorescence assay (IFA). Briefly, following standard processing (4% PFA fixation, 0.1% Triton X-100 permeabilization), the cells were blocked with 5% skim milk (1 h). After primary antibody treatment (1:500, 4°C overnight), the samples were incubated with a FITC-conjugated secondary antibody(1:500) for 1 h at RT. Nuclei were counterstained with DAPI (5 min).Images were captured with a bioimaging instrument (EVOS FL AUTO, China).Upon reaching an OD600 of 0.3, L. reuteri cultures were treated with 100 µM Taurocholic Acid (TCA), Hyocholic Acid (HCA), or Taurochenodeoxycholic Acid (TCDCA). For the bile salt hydrolase (BSH) inhibition assays, additional wells were treated with 100 µM HCA + Gut restricted-7 (GR7). Both the negative control and MRS medium control groups were included. Cultures were terminated at 72 h by flash-freezing in liquid nitrogen.The cells were harvested and lysed in RIPA buffer (containing PMSF) on ice for 30 min. The mixture was subsequently centrifuged (12, After fixation, the jejunum was embedded in paraffin and sectioned. The sections were pretreated and stained with hematoxylin and eosin (H&E) for histological analysis. Additionally, Alcian blue-periodic acid Schiff (AB-PAS) staining was performed via a commercial kit (Servicebio, China). Following dehydration, tissue sections were coverslipped and subsequently imaged using a PANNORAMIC digital slide scanner (3DHISTECH, Hungary). Goblet cell counts and villus height-to-crypt depth ratios were quantified using CaseViewer 2.4 (3DHISTECH, Hungary) and Image-Pro Plus 6.0 software (Media Cybernetics, USA).For immunofluorescence staining of PEDV in the jejunum, tissue sections were blocked with 3% bovine serum albumin (BSA), followed by sequential incubation with PEDV-N primary antibodies and FITC-conjugated secondary antibodies (Servicebio, China). Following counterstaining with DAPI and quenching of tissue autofluorescence, the sections were mounted for microscopic imaging. The percentage of positive cells (number of positive cells / total number of cells) was statistically analyzed from images captured at 20x magnification using Aipathwell software.The Cells were seeded in a 96-well plate at a density of 5 × 10³ cells/well and incubated for 24 h. Following treatment with serially diluted test samples in DMEM for 24 h in a CO₂ incubator, the medium was aspirated and Genomic DNA was extracted from bacterial cultures using a Bacterial DNA Kit (Omega Bio-tek). PCR amplification of the BSH gene was performed using the primer pair BSH-F and BSH-R (Table 1). Each 25 µL reaction contained approximately 50 ng of template DNA, 0.5 µM of each primer, 2 × Taq Plus Master Mix Ⅱ (Vazyme). Amplification was carried out in a thermal cycler under the following conditions: initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 sec, primer annealing at 57°C for 30 sec, and extension at 72°C for 45 sec;followed by a final extension at 72°C for 10 min. PCR products were analyzed by electrophoresis on a 1% agarose gel stained with Super GelBlue, alongside a DNA molecular weight marker. Negative controls (MRS medium) were included in each run.Total RNA was extracted from the jejunum using a Total RNA Kit (Omega Biotek, USA) according to the manufacturer's instructions.PEDV RNA was isolated from cell culture supernatants via a Viral RNA Kit (Omega Biotek, USA). After DNase treatment and cDNA synthesis, SYBR Green or TaqMan probe qPCR was performed. PEDV load, key bile acid metabolism regulators, and TGR5 and IFN-β mRNA levels were quantified via SYBR Green assays. Relative gene expression was calculated via the 2 -ΔΔCt method, with GAPDH used as the reference gene.For absolute quantification of PEDV RNA in the cell culture supernatants, TaqMan probe-based qPCR (Vazyme, China) was employed.Viral copy numbers were determined via a standard curve generated from serial dilutions of a PEDV-specific recombinant plasmid.Total cholesterol levels in blood and liver tissues were quantified using the Beyotime Amplex Red Cholesterol Assay Kit (S0211S). Liver samples (20 mg) were homogenized in 200μL BeyoLysis™ Buffer A for Metabolic Assay per 20 mg tissue, followed by centrifugation at 12,000 × g for 5 min at 4°C. The supernatant was collected for analysis. A 5-fold diluted sample (50 μL) was combined with 50 μL Total Cholesterol Working Solution (containing cholesterol esterase, enzyme mix, and Amplex Red) in 96-well plates. After incubation at 37°C for 30 min, absorbance was measured at 570 nm. Cholesterol concentrations were calculated against a 0-500 μM standard curve according to the manufacturer's protocol.Genomic DNA was extracted via the Mag-Bind® system DNA Kit (Omega Biotek, USA), followed by quality assessment (1% agarose gel electrophoresis) and fragmentation (Covaris M220; 400 bp target size).PE libraries were constructed via NEXTFLEX Rapid DNA-Seq (Bioo Scientific, USA), which involves adapter ligation, magnetic bead purification, PCR enrichment, and final library validation. The Illumina NovaSeq platform was used for sequencing. Quality-filtered reads (fastp v0.20.0: removal of adapters, reads