Extracellular Vesicles from Probiotic and Beneficial Escherichia coli Strains Exert Multifaceted Protective Effects Against Rotavirus Infection in Intestinal Epithelial Cells

<b>Background/Objectives:</b> Rotavirus remains a major cause of severe acute gastroenteritis in infants worldwide. The suboptimal efficacy of current vaccines underscores the need for alternative microbiome-based interventions, including postbiotics. Extracellular vesicles (EVs) from probiotic and commensal <i>E. coli</i> strains have been shown to mitigate diarrhea and enhance immune responses in a suckling-rat model of rotavirus infection. Here, we investigate the regulatory mechanisms activated by EVs in rotavirus-infected enterocytes. <b>Methods:</b> Polarized Caco-2 monolayers were used as a model of mature enterocytes. Cells were pre-incubated with EVs from the probiotic <i>E. coli</i> Nissle 1917 (EcN) or the commensal EcoR12 strain before rotavirus infection. Intracellular Ca²⁺ concentration, ROS levels, and the expression of immune- and barrier-related genes and proteins were assessed at multiple time points post-infection. <b>Results:</b> EVs from both strains exerted broad protective effects against rotavirus-induced cellular dysregulation, with several responses being strain-specific. EVs interfered with viral replication by counteracting host cellular processes essential for rotavirus propagation. Specifically, EV treatment significantly reduced rotavirus-induced intracellular Ca²⁺ mobilization, ROS production, and COX-2 expression. In addition, both EV types reduced virus-induced mucin secretion and preserved tight junction organization, thereby limiting viral access to basolateral coreceptors. Additionally, EVs enhanced innate antiviral defenses via distinct, strain-dependent pathways: EcN EVs amplified IL-8-mediated responses, whereas EcoR12 EVs preserved the expression of interferon-related signaling genes. <b>Conclusions:</b> EVs from EcN and EcoR12 act through multiple complementary mechanisms to restrict rotavirus replication, spread, and immune evasion. These findings support their potential as effective postbiotic candidates for preventing or treating rotavirus infection.