Fig 1.
Experimental infection of cynomolgus monkeys with EV71 strains.
(A) Scheme of cDNA-derived PSGL-1-binding (PB) and nonbinding (non-PB) strains of EV71 with two amino acid substitutions at VP1-98 and VP1-145. (B) Experimental schedule. Four monkeys were intravenously inoculated with 1 ml containing 106.3 CCID50 of EV71-02363-KE (non-PB) or EV71-02363-EG (PB) strain at Day 0 and the clinical manifestations were observed daily for 10 days. Under anesthesia, clinical samples were collected on indicated days and postmortem tissue samples were collected at 10 days postinfection.
Fig 2.
Time-course of clinical observations.
Four monkeys (#5061, #5132, #5133, and #5137) were inoculated with the EV71-02363-KE (non-PB) strain and four monkeys (#5131, #5134, #5135, and #5136) were inoculated with the EV71-02363-EG (PB) strain. One monkey (#5061) exhibited decreased limb grip strength for 5–10 days postinfection (p.i.), without apparent paralysis, tremor, or ataxia.
Fig 3.
Kinetics of lymphocytes in peripheral blood.
Number of (A) CD4+ T-lymphocytes, (B) CD8+ T-lymphocytes, (C) CD16+ lymphocytes (NK cells), and (D) CD20+ lymphocyte (B cells) in peripheral blood collected from monkeys inoculated with EV71-02363-KE (non-PB; left) or EV71-02363-EG (PB; right) were analyzed by flow cytometry. Pre-inoculation lymphocyte numbers in all eight monkeys are shown at Day 0 and the average was used as the uninfected control. Numbers of lymphocytes in peripheral blood, collected at 3, 7, and 10 days postinfection from the 02363-KE-iniculated group are indicated in blue and those from the 02363-EG-inoculated group are indicated in orange. Averages of lymphocyte numbers are indicated as black solid line plot. Significant differences between the preinfection control (eight monkeys at Day 0) and postinfection (four monkeys) at indicated days were determined using Student's t-test and P values < 0.05 are marked by asterisks Significant differences in lymphocyte numbers between 02363-KE- and 02363-EG-inoculated groups at indicated days are shown in S2 Fig.
Fig 4.
Serum cytokine level of the inoculated monkeys.
Serum levels of cytokines, (A) IL-1β, (B) TNFα, (C) IL-6, (D) G-CSF, (E) IL-1RA, and (F) IFN-γ collected from the monkeys inoculated with EV71-02363-KE (non-PB) or EV71-02363-EG (PB) were analyzed using Luminex 200. Cytokine levels of all eight preinoculation monkeys are shown at Day 0 and the average was used as the uninfected control. Serum cytokine levels, collected at 3, 7, and 10 days postinfection from the 02363-KE-inoculated group are indicated in blue and those from the 02363-EG-inoculated group was indicated in orange. ND (not detected) indicates that serum cytokine levels of all samples are below the limit of detection (dashed lines). Average of cytokine concentrations are indicated as black solid line plot. Significant differences between the preinoculation control (eight monkeys at Day 0) and postinfection (four monkeys) at indicated days were determined using Student’s t-test and P values < 0.05 are marked by asterisks. Significant differences in cytokine levels between 02363-KE- and 02363-EG-inoculated groups at indicated days are shown in supplemental S3 Fig.
Table 1.
Amino acid residues at VP1-98 and VP1-145 of EV71 variants in clinical samples.
Table 2.
Amino acid residues at VP1-98 and VP1-145 of EV71 variants in postmortem tissue samples.
Fig 5.
Schematic illustration of the detection of non-PB and PB variants of EV71.
Virus detection and genetic variants of EV71 from clinical samples (Table 1) and postmortem tissue samples (Table 2) are summarized. Positive samples by molecular detection (++ or + in Tables 1 and 2) for EV71 are indicated and classified into PB (VP1-145G) or non-PB (VP1-145E) variants according to the amino acid residue at VP1-145 from the partial VP1 sequences determined by direct RT-PCR from samples. PB and non-PB variants are shaded in orange and blue, respectively.
Fig 6.
Distribution of EV71-induced lesions in CNS tissues.
Postmortem CNS tissues at 10 days postinfection from four monkeys inoculated with either (A) EV71-02363-KE (non-PB) or (B) EV71-02363-EG (PB) strain. Rows, from top to bottom, indicate the cerebrum/diencephalon, midbrain, pons/cerebellum, medulla oblongata and spinal cord, respectively. Filled triangle indicates neuronal damage and the gray areas show inflammation in the parenchyma and the meninges of the central nervous system.
Fig 7.
Histopathological spinal cord findings.
Representative spinal cord sections from monkeys inoculated with EV71-02363-KE (non-PB; #5133) or EV71-02363-EG (PB; #5135) strain, stained with hematoxylin and eosin (H&E). Typical histopathological changes are shown in the spinal cord from both samples. Upper panels: Prominent inflammatory infiltrations including mononuclear cuffing (*) and gliosis were seen in the anterior horn of the spinal cords of both monkeys. Neuronal degeneration (arrows) was observed within the lesion. Scale bar; 500 μm. Lower panels: perivascular cuffing with mononuclear cells was observed within the lesion (*) with higher magnification of the squared area of upper panel. Lower left panel: Loss of nissle granules within some motor neurons (central chromatolysis, arrow). Lower right panel: neuronophagia observed within the lesion (arrow). Scale bar; 100 μm.