A mouse-adapted enterovirus 71 strain causes neurological disease in mice after oral infection

Abstract

A mouse-adapted enterovirus 71 (EV71) strain with increased virulence in mice, MP4, was generated after four serial passages of the parental EV71 strain 4643 in mice. Strain MP4 exhibited a larger plaque size, grew more rapidly, and was more cytotoxic in vitro than strain 4643. Although strains 4643 and MP4 both induced apoptosis of SK-N-SH human neuroblastoma cells, MP4 was more virulent than 4643 in 1-day-old mice (50% lethal doses, 10(2) and 10(4) PFU/mouse, respectively). Strain MP4 (5 x 10(6) PFU/mouse), but not 4643, could orally infect 7-day-old mice, resulting in rear-limb paralysis followed by death 5 to 9 days after inoculation with the virus. Histopathologically, neuronal loss and apoptosis were evident in the spinal cords as well as the brain stems of the infected mice. The limb muscles displayed massive necrosis. There was early and transient virus replication in the intestines, whereas the spinal cord, brain, and muscle became the sites of viral replication during the late phase of the infection. Virus transmission occurred among infected and noninfected cagemates, as demonstrated by the occurrence of seroconversion and the presence of viable viruses in the stool samples of the latter. Protection against EV71 challenge was demonstrated following administration of hyperimmune serum 1 day after inoculation with the virus. Nucleotide sequence analysis of the genome of EV71 strain MP4 revealed four nucleotide changes on the 5' untranslated region, three on the VP2 region, and eight on the 2C region, resulting in one and four amino acid substitutions in the VP2 and 2C proteins, respectively.

FIG. 1.

Mouse-adapted EV71 grows more rapidly and is more cytotoxic than the parental strain. (A) One-step growth curves of the parental EV71 strain Tainan/4643/98 and the mouse-adapted strain MP4 in RD, SK-N-SH, and Caco-2 cells were determined as described in Materials and Methods. (B) SK-N-SH cells (2 × 10⁴) grown in 96-well plates were infected with 4643 or MP4, and cell viability was determined at the times indicated. Data represent the means ± SEM of results of three independent experiments performed in triplicate (A) and of two experiments with eight duplicates (B). *, P < 0.05.

FIG. 2.

EV71 induces apoptosis of Caco-2 and SK-N-SH cells. (A) Caco-2 or SK-N-SH cells were infected with the parental (Tainan/4643/98) or mouse-adapted (MP4) EV71 strain at an MOI of 1. The expressions of both the proform and the cleavage form of caspase 9 in whole-cell lysates were determined by Western blotting. (B) Apoptosis of EV71-infected cells was determined by flow cytometry after staining with Annexin V. Values are the means ± SEM of results of three independent experiments. *, P value of <0.05 compared with the results for the same cell type infected with 4643.

FIG. 3.

The mouse-adapted EV71 strain is more virulent than the parental strain in 1-day-old mice. One-day-old ICR mice were inoculated i.p. with the parental (Tainan/4643/98) or mouse-adapted (MP4) EV71 strain at 3 × 10⁶ PFU/mouse (A) or 10¹ to 10⁷ PFU/mouse (B). Survival was then monitored daily after infection. Control mice were given cell lysate instead of virus suspension. Each group contained six to eight mice.

FIG. 4.

Mouse-adapted EV71 induces death in mice after oral inoculation. Seven-day-old ICR mice were orally inoculated with the parental (Tainan/4643/98) or mouse-adapted (MP4) EV71 strain at 1 × 10⁴ to 5 × 10⁶ PFU/mouse (A) or 5 × 10⁶ PFU/mouse (B), both at 200 μl, with or without i.p. administration of mouse anti-EV71 serum (50 μl). Survival was then monitored daily after infection. Control mice were given cell lysate instead of virus suspension. Each group contained six to eight mice.

FIG. 5.

Representative histology of various tissues of EV71-infected mice. Seven-day-old ICR mice were orally inoculated with cell lysate (mock control) or the mouse-adapted EV71 strain (MP4) (5 × 10⁶ PFU/mouse), and tissues were examined histopathologically at 6 dpi as described in Materials and Methods. Typical lesions seen in MP4-infected mice include neuronal loss in ventral horns of the spinal cord (A and B), severe necrotizing myositis (C and D), decreased hematopoiesis in the liver (E and F), splenic atrophy (G and H), and villous blunting in the small intestine (I and J; 1 dpi). Hematoxylin and eosin stain. Magnifications, ×400 (A, B, I, and J), ×100 (C to F), and ×40 (G and H).

FIG. 6.

Neuronal loss, apoptosis, and VP-1 expression in EV71-infected neural tissues. Seven-day-old mice were orally inoculated with cell lysate (mock control) or the mouse-adapted EV71 strain (MP4) as described in the legend to Fig. 5. Representative sections are shown, demonstrating neuronal loss (indicated by arrowheads) and apoptosis as detected in the ventral horns of the thoracic spinal cord by Nissl staining (A and B) and TUNEL (C and D), respectively, following inoculation with EV71. The immunohistochemical expression and localization of VP-1 (indicated by arrows) are shown for tissues from the brains (E and F) and spinal cords (G and H) of control and MP4-infected mice. Magnifications, ×100 (A to D) and ×400 (E to H).

FIG. 7.

Tissue viral titers in EV71-infected mice. Seven-day-old mice were inoculated with the parental (Tainan/4643/98) or mouse-adapted (MP4) EV71 strain (5 × 10⁶ PFU/mouse, 200 μl). Viral titers were assessed by plaque assays of tissues collected at the times indicated. Results represent the mean virus titer (log₁₀ PFU) per milligram of tissue or per milliliter of blood ± SEM (six mice per group).

FIG. 8.

Alignment of the nucleotide sequences of the 5′ UTRs of EV71 strains reveals six mutation hot spots. The first two isolates, Tainan/4643/98 (GenBank accession number AF304458) and Tainan/6356/98, are associated with encephalomyelitis, and the Tainan/5746/98 (accession number AF304457) strain is associated with HFMD. Only the areas with nucleotide variations are shown.