A neonatal gnotobiotic pig model of human enterovirus 71 infection and associatedimmune responses

摘要

Vaccine development and pathogenesis studies for human enterovirus 71 are limited by a lack of suitable animal models. Here, we report the development of a novel neonatal gnotobiotic pig model using the non-pig-adapted neurovirulent human enterovirus 71 strain BJ110, which has a C4 genotype. Porcine small intestinal epithelial cells, peripheral blood mononuclear cells and neural cells were infected in vitro. Oral and combined oral–nasal infection of 5-day-old neonatal gnotobiotic pigs with 5×10⁸ fluorescence forming units (FFU) resulted in shedding up to 18 days post-infection, with viral titers in rectal swab samples peaking at 2.22×10⁸ viral RNA copies/mL. Viral capsid proteins were detected in enterocytes within the small intestines on post-infection days (PIDs) 7 and 14. Additionally, viral RNA was detected in intestinal and extra-intestinal tissues, including the central nervous system, the lung and cardiac muscle. The infected neonatal gnotobiotic pigs developed fever, forelimb weakness, rapid breathing and some hand, foot and mouth disease symptoms. Flow cytometry analysis revealed increased frequencies of both CD4⁺ and CD8⁺ IFN-γ-producing T cells in the brain and the blood on PID 14, but reduced frequencies were observed in the lung. Furthermore, high titers of serum virus-neutralizing antibodies were generated in both orally andcombined oral–nasally infected pigs on PIDs 7, 14, 21 and 28. Together, theseresults demonstrate that neonatal gnotobiotic pigs represent a novel animal model forevaluating vaccines for human enterovirus 71 and for understanding the pathogenesis ofthis virus and the associated immune responses.