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

Abstract

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 and combined oral–nasally infected pigs on PIDs 7, 14, 21 and 28. Together, these results demonstrate that neonatal gnotobiotic pigs represent a novel animal model for evaluating vaccines for human enterovirus 71 and for understanding the pathogenesis of this virus and the associated immune responses.

Lactobacillus reuteri ZJ617 maintains intestinal integrity via regulating tight junction, autophagy and apoptosis in mice challenged with lipopolysaccharide

“…Ileum tissues were fixed in formalin, embedded in paraffin, and cut into 4 μm sections according to previous methods [ 40 ]. Deparaffinized and rehydrated sections were blocked with 10 % normal goat serum for 1 h. Sections were incubated with the rabbit anti- occludin (1:5000), anti-claudin3 (1:5000), anti-caspase-3 (1:5000), anti-LC3 (1:1000) and anti-mTOR (1:1000) overnight at 4 °C, then with HRP-conjugated secondary antibody for 1 h. The Diaminobenzidine-HRP detection system was added, sections were then counterstained with hematoxylin, dehydrated and cover-slipped.…”

Tissue-specific mRNA expression profiles of porcine Toll-like receptors at different ages in germ-free and conventional pigs

“…This suggests that the murine model may not be adequate for studies of human TLRs and highlights the need for alternative animal models. Pigs are being increasingly recognized and used as a relevant model for studies of infectious disease and human immunity (Fairbairn et al, 2011; Gonzalez et al, 2010; Yang et al, 2014; Yang and Yuan, 2014; Yuan et al, 1996; Q. Zhang et al, 2013); however, knowledge of the porcine TLR expression and function is limited in comparison to mice and humans.…”

Modelling person-to-person transmission in an Enterovirus A71 orally infected hamster model of hand-foot-and-mouth disease and encephalomyelitis

“… 29 In a pig model involving oronasal and orally infected gnotobiotic, only very mild fever has been observed, and rectal swabs from infected animals have been found to be positive for virus. 30 A promising SCARB2 (a well-recognized viral receptor) transgenic mouse model for EV-A71 infection has been reported but unfortunately cannot be consistently orally infected. 24 …”