Establishment of an oral enterovirus 71 (EV71) infection model in immunocompetent mice for antiviral therapy evaluation.

Enterovirus 71 (EV71) is one of the major pathogens responsible for hand-foot-and-mouth disease in children and is associated with severe neurological complications, including death. Despite the fact that EV71 naturally infects humans through the oral route, the absence of a simple and reliable oral infection model has significantly impeded investigations into its pathogenesis and the development of antiviral interventions. In this study, we established a robust oral EV71 infection model in widely used immunocompetent BALB/c mice, employing a clinical isolate devoid of adaptive mutations. Mice infected via the oral route developed disease progression and clinical, as well as pathological features that closely recapitulate those observed in human infection. We further identified marked differences in viral dissemination and host immune responses between oral and intraperitoneal inoculation, underscoring the importance of physiologically relevant oral infection models for elucidating EV71 pathogenesis. In addition, the anti-EV71 candidate drug rupintrivir showed reduced efficacy in the oral infection model compared with the intraperitoneal model. Notably, this oral model enables evaluation of therapies designed to block intestinal viral infection-a capability lacking in intraperitoneal models. Using this platform, we assessed an intestinal probiotic blockade therapy for the first time. Our findings demonstrate that the probiotic formulation effectively suppresses viral replication and alleviates pathological damage in the spleen, lungs, kidneys, and other organs, providing a scientific basis for developing probiotic-based therapeutic strategies against viral infections. IMPORTANCE: The restricted host tropism of enterovirus 71 (EV71) has limited the development of animal models that accurately recapitulate human disease. Here, we demonstrate for the first time that the clinically isolated EV71-GZCII strain successfully infects 14-day-old immunocompetent BALB/c mice via oral gavage, establishing a physiologically relevant model that addresses this critical gap. Orally infected mice displayed distinct viral dissemination patterns and host immune responses compared with intraperitoneal models, highlighting the critical role of infection route in EV71 pathogenesis. Using this system, we evaluated the