Construction of a suckling mouse model based on an EV-C96 clinical isolate and its application in drug screening and vaccine development

Enterovirus C96 (EV-C96) is a novel human enterovirus associated with diseases such as hand, foot, and mouth disease and acute flaccid paralysis (AFP). In recent years, the global detection rate of EV-C96 has risen markedly, underscoring its epidemic potential. However, there is currently a lack of suitable animal models for studying its pathogenic mechanisms and antiviral research. This study utilized the clinical isolate AFP149/GD/CHN/2014 of EV-C96 and, through screening of infection doses, mouse strains, age, and inoculation routes, established the first EV-C96 suckling mouse infection model. The experimental results showed that intracranial injection of a viral dose of 103.425 TCID50 caused limb paralysis, weight loss, and 100% mortality in 1-day-old ICR suckling mice. Histopathological analysis indicated that the virus primarily targeted the central nervous system (brain and spinal cord) and myocardial tissue, a distribution characteristic that corresponds with the typical clinical manifestations of EV-C96 infections. Using this model to evaluate antiviral drugs, we found that treatment with IFN-α2a (1000 U) and IFN-γ (1000 U) increased the survival rate of infected suckling mice to 75% and 67%, respectively. In vaccine evaluation experiments, the survival rate of the EV-C96 inactivated vaccine immunization group was 100%. Pathological examinations showed that the myocardial and spinal cord tissue structures of the treatment and immunization groups were intact, and the degree of brain tissue lesions was significantly reduced. The EV-C96 suckling mouse model provides a platform for investigating viral pathogenesis and supports preclinical evaluation of antiviral drugs and vaccines.