Background: Enterovirus G (EV-G) is a significant causative agent of diarrhea in piglets, complicating differential diagnosis and impeding effective control of porcine diarrheal diseases. Enhanced clinical surveillance and pathogenicity assessment of EVG are essential to inform disease prevention strategies. Methods: The EV-G strain was isolated and identified in MA-104 cells using plaque assay, immunofluorescence staining, viral replication kinetics analysis, and transmission electron microscopy. The complete genomic sequence was obtained for phylogenetic and recombination analysis. Five-day-old piglets were orally challenged with the prevalent EV-G strain, and pathogenicity was assessed based on diarrhea scoring, viral load quantification in feces and tissues, and histopathological examination. Gut microbiota dynamics were profiled via high-throughput 16S rRNA sequencing pre- and post-infection. Results: A recombinant G1 genotype EV-G strain, designated EVG-SH-2024, was successfully isolated. Phylogenetic analysis indicated its closest amino acid identity (94.7%) with KF985175, while recombination analysis suggested origin from genetic exchange between KF985175 and MN734577. Inoculation with EVG-SH-2024 induced mild diarrhea and persistent viral shedding in piglets, accompanied by significant tissue damage including neuronal vacuolation in the brain, pulmonary consolidation, and intestinal inflammatory infiltration. 16S rRNA sequencing revealed substantial gut microbiota dysbiosis post-infection, marked by an increase in pathogenic bacteria and a decline in beneficial microbes. Notably, the top ten differentially abundant species were predominantly short-chain fatty acid (SCFA)-producing bacteria, whose abundance showed a significant Spearman’s rank correlation with pathogenicity indicators. Conclusion: This study reports the first isolation and characterization of a G1 subtype EV-G strain in Shanghai, China. Integrated pathogenicity and microbiota analyses demonstrated that EV-G infection reduces the abundance of SCFA-producing bacteria, with this reduction significantly correlated with disease severity. These findings suggest a potential role of SCFAs in EV-G pathogenesis and provide a basis for developing microbiota-targeted interventions against EV-G infection.

Porcine epidemic diarrhea virus (PEDV) severely threatens the global swine industry and causes massive economic losses due to high morbidity and mortality in neonatal piglets. To date, key host receptors and molecular determinants regulating PEDV entry remain poorly characterized. Here, we found that overexpression of porcine interferon-induced transmembrane protein 1 (pIFITM1) in LLC-PK1, IPEC-J2, and Huh7 cells enhanced PEDV-HM infection. Conversely, knockout of endogenous pIFITM1 suppressed PEDV-HM infection, and this phenotype was rescued by pIFITM1 reconstitution. Additionally, pIFITM1 interacted with the PEDV spike, envelope, and membrane proteins and promoted PEDV attachment and internalization. As PEDV invades host cells via caveolae-mediated endocytosis, we further confirmed that pIFITM1 interacted with caveolin-1 (CAV1) and modulated plasma membrane cholesterol distribution to promote PEDV entry through this pathway. Mechanistically, deletion of the conserved intracellular loop (CIL) abolished the proviral effect of pIFITM1. Notably, site-directed mutagenesis of lysine residues K62 and K83 within the CIL abolished its proviral function, suggesting that these two residues are essential for pIFITM1-mediated promotion of PEDV entry. In summary, our study demonstrates that pIFITM1 acts as a critical proviral factor that facilitates PEDV entry by binding viral structural proteins and regulating the cholesterol–CAV1 complex. This study also identifies pIFITM1 as a promising target for developing anti-PEDV interventions.

Porcine epidemic diarrhea virus (PEDV) is a major enteric coronavirus that causes severe economic losses to the global swine industry. Current vaccines suffer from weak immunogenicity, insufficient mucosal immunity, and a short duration of protection. Nanoparticle delivery systems have emerged as a promising strategy for next-generation vaccine development. In the present study, we constructed an S1-Ferritin nanoparticle vaccine using the SpyTag-SpyCatcher modular conjugation system. The morphology, particle size, and uniformity of the nanoparticle vaccine were systematically characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). After two immunizations, the S-Ferritin nanoparticle vaccine elicited potent humoral and cellular immune responses in both mice and piglets. In piglets, at 2 weeks post booster vaccination, PEDV-specific serum IgG endpoint titers peaked at 1:2560, virus-neutralizing antibody titers reached 1:256 against the JS-2/2015 strain, and serum IFN-γ levels reached 274 pg/mL. All these immunological indicators were significantly higher than those observed in the PEDV-inactivated whole-virus vaccine group. Furthermore, challenge tests showed that the S1-Ferritin nanoparticle vaccine provided complete protection against PEDV infection and significantly reduced the severity of diarrhea and intestinal damage in piglets after challenge. These findings suggest that the S1-Ferritin nanoparticle vaccine constitutes a promising candidate against PEDV infection, though our study is only a preliminary step and subsequent field trials in pigs are still required.