Figures & data
Table 1 Summary of virus shedding in neonatal gnotobiotic pigs infected with the human enterovirus 71 BJ110 strain
Figure 1 The human EV71 BJ110 strain infects and replicates in pig intestinal epithelial cells, PBMCs and neural cell culture in vitro. (A) Identification of virus inoculum. (a) RT-PCR detection of the EV71 BJ110 strain. The positive PCR products were purified and sequenced. The sequence shares 100% identity with the published EV71 BJ110 strain VP1 gene sequence. (b) Mock-infected Vero cell culture. (c) CPE in Vero cells 72 h post-inoculation with the EV71 BJ110 strain at a MOI of 10. (d) EV71-infected Vero cells were detected using CCIF. (B) Porcine cell cultures can be infected by the EV71 BJ110 strain. EV71 was detected in IPEC-J2 cells, PBMCs and neural cells using CCIF. (C) Growth curves for the EV71 BJ110 strain in Vero and IPEC-J2 cells suggest that EV71 infects and replicates efficiently in IPEC-J2 cells.
Figure 2 EV71 BJ110 strain fecal shedding, tissue distribution and dynamics in infected neonatal gnotobiotic pigs. (A) RT-PCR detection of EV71 in rectal swab samples. Left, RT-PCR detection of EV71 viral RNA in one orally infected pig (M, marker; lane 1, positive control; lanes 2–10, PID 0–8). Middle, RT-PCR detection of EV71 viral RNA in one oral–nasally infected pig (M, marker; lanes 1–8, PID 0–7). Right, RT-PCR detection of EV71 viral RNA in a mock control pig (lanes 1–8, PID 0–7; M, marker). (B) Taqman real-time PCR detection of virus shedding in rectal swab samples from different treatment groups. The mean viral RNA titer for each group at a specific time point is presented. The error bar indicates the standard error of the mean. No virus shedding was detected for any mock control group pigs at any time point; therefore, no bars are visible. (C) Detection of viral antigen on PID 7 and PID 14 in the ileum of gnotobiotic pigs infected with the EV71 BJ110 strain through the oral–nasal route at a dose of 5×108 FFU by immunofluorescence staining. A mouse anti-EV71 capsid protein VP1 monoclonal antibody (Abcam) was used as the primary antibody, and a goat anti-mouse IgG1 antibody labeled with fluorescein isothiocyanate (Sigma-Aldrich) was used as the secondary antibody. Nuclei were stained red by propidium iodide (Invitrogen). (D) Taqman real-time PCR detection of EV71 viral RNA in tissues of infected gnotobiotic pigs at PID 7 or PID 14. The route of inoculation and the euthanasia time (in parentheses) of the pigs are marked in the legends. Viral titers are presented as the mean of two replicates for the same sample. The negative samples are shown as blank on the bar graph. All data are representative of at least two independent experiments. BG, basal ganglia; CCC, caudal cerebral cortex; Duo, duodenum; Jej, jejunum; MLN, mesenteric lymph nodes; OB, olfactory bulb; RCC, rostral cerebral cortex; SC-C, spinal cord-cervical; SC-L, spinal cord-lumbar; SC-T, spinal cord-thoracic; SC-S, spinal cord-sacral.
Figure 3 Fever, limb paralysis, vesicles and lung lesions in EV71-infected neonatal gnotobiotic pigs. (A) Body temperature in the EV71-infected neonatal gnotobiotic pigs. Body temperature was measured using subcutaneously implanted microchips posterior to the ear. The body temperature at each time point represents an average of three measurements. The normal core body temperature of pigs ranges from 38 to 40 °C, with an average of 38.8 °C. A temperature higher than 40 °C is considered a fever. (B) Vesicles (indicated by black arrow) on the snouts of EV71-infected neonatal gnotobiotic pigs. (C) Forelimb weakness in neonatal gnotobiotic pigs infected with the EV71 BJ110 strain. (D) An age-matched mock-infected control neonatal gnotobiotic pig. B (E) Multifocal mottling with petechial hemorrhages (indicated by the circle) in the lung was observed in an oral-nasally inoculated gnotobiotic pig on PID 21. (F) Normal lung from a mock-infected gnotobiotic pig. *P<0.05, as indicated by the Kruskal–Wallis test.
Table 2 Clinical signs in neonatal gnotobiotic pigs infected with the human enterovirus 71 BJ110 strain
Figure 4 Microscopic lesions in neonatal gnotobiotic pigs infected with the EV71 BJ110 strain. (A) The upper panel shows a section of the lung of an orally infected gnotobiotic pig and a mock-infected control on PID 21. The infected pig has peribronchial and perivascular hemorrhage (indicated by the arrow). An adjacent alveolus contains scattered erythrocytes and macrophages (indicated by the black triangle). (B) The lower panel shows the small intestine of an oral-nasally infected gnotobiotic pig on PID 7, with a prominent presence of immune cells in the lamina propria and a significantly increased number of Peyer's patches (indicated by the asterisk). Lung tissues were stained with H&E; small intestinal tissues were sections of resin-embedded tissue stained with toluidine blue. H&E, hematoxylin and eosin.
Figure 5 Immune responses during EV71 infection in neonatal gnotobiotic pigs. (A) Representative dot plots showing the frequency of CD3+CD4+IFN-γ+ and CD3+CD8+IFN-γ+ T lymphocytes among the total CD3+ mononuclear cells in the blood. MNCs were stimulated with semi-purified whole EV71 antigen or control medium for 17 h prior to staining. (B) The frequency of IFN-γ-producing CD3+CD4+ and CD3+CD8+ T cells among the CD3+ mononuclear cells in the ileum, spleen, blood, brain and lung on PID 7 and PID 14 after EV71 BJ110 infection via the oral–nasal route in neonatal gnotobiotic pigs. The mean frequencies were calculated by subtracting the mean frequency value of medium/mock stimulated cells from the mean frequency value of virus-stimulated cells. A positive mean frequency value indicates that IFN-γ production was upregulated upon virus stimulation, whereas a negative mean frequency value indicates that IFN-γ production was downregulated upon virus stimulation. The mean value for the EV71-infected group is indicated by the solid line, and the mean value for the mock control group is indicated by the dashed line. (C) Serum neutralizing antibody response in EV71-infected neonatal gnotobiotic pigs. Different uppercase letters (i.e., A, B and C) indicate significant differences between different treatment groups and different time points within the same treatment group. Shared uppercase letters or no letters indicate that no significant differences were observed. (Kruskal–Wallis test, P<0.05; n=4). MNC, mononuclear cell.
