Figure 1.
21-day-old gerbils infected with EV71 intraperitoneally (IP) at a dose of 1×104.0 TCID50.
(A) Photograph was representative picture of hind limb paralysis caused by 58301 at 6 dpi. (B) Photograph was representative picture of normal 21-day-old gerbils.
Figure 2.
21-day-old gerbil infected IP with ten-fold serial dilution of EV71.
Control animals were given uninfected Vero cell lysate. Survival (A) and clinical scores (B) were monitored daily after inoculation. Clinical scores were defined as following: 0,healthy; 1,ruffled hair, hunchbacked or reduced mobility; 2,limb weakness; 3,paralysis in 1 limb; 4,paralysis in both limbs; 5,death. Each group contained seven or eight gerbils. One representative of two independent experiments was shown.
Figure 3.
Pathologic changes in various tissues of EV71-infected gerbils.
21-day-old gerbils were inoculated with EV71 (1×105.0 TCID/gerbil) IP or Vero cell lysate (mock control) and tissues were examined histopathologically at 4–5 dpi. Pathologic lesions could be seen in infected gerbils. (B) Spinal cord presented as a sieve-like change and nerve cells arranged disorder and some were swelling (solid arrow) with decrease of Nissl bodies. (C)Nerve cells became disintegrated, necrotic and disappearance of the nucleus (solid arrows). (D)Multiple foci of microglial accompanied by neuronophagia (solid arrows) were in anterior horn cells. (F)In brainstem, neuronophagia (solid arrows), neuronal swelling (solid arrow-heads) and microglial (open arrows) were seen. (G)The myelin sheath expansion (solid arrows) was occasionally observed. Severe necrotizing myositis in the skeletal muscle (I) and splenic atrophy (K). Normal tissues were shown (A, E, H and J). (A–G) Nissl stain, (H–K) Hematoxylin and eosin stain.
Figure 4.
Immunohistochemistry of tissues in EV71- infected gerbils.
21-day-old gerbils were inoculated IP with 1×105.0 TCID/gerbil of EV71 at 4–5 dpi. The neuron and adjacent sections were positive for EV71 antigen in the spinal cord which was mainly located in gray matter (B). The neuron was viral antigen-positive in the brainstem (D). Viral antigen was detected in the skeletal muscle fibers (F). Mock controls were shown (A, C and E). Immunohistochemistry with DAB chromogen and counterstain with hematoxylin.
Table 1.
Clinical signs, pathological changes and virus replication in EV71-infected gerbils.
Table 2.
The pathological types of CNS in EV71-infected gerbils.
Figure 5.
Virus replication in various tissues of EV71-infected gerbils.
Twelve 21-day-old gerbils were inoculated EV71 IP with a dose of 1×105.0TCID50 and tissue samples were collected at the time points indicated (3 gerbils/each). Virus titer in culture (solid bars) was expressed as TCID50 per milliliter (blood) or per gram (tissue). Calculated virus titer (hatched bars) was based on standard curve of real-time RT-PCR obtained with 10-fold serial dilutions of virus strain 58301. The data shown here are the mean virus titers ±standard errors (n = 3 each). Results are representatives of two independent experiments. The data between the virus titer in culture and calculated virus titer were no statistically significant by student’s t-test.
Figure 6.
Survival and clinical score of immunized gerbils after IP challenge with lethal doses of EV71.
Experiment groups (n = 8) were immunized with the EV71 inactive whole-virus vaccine at birth and boosted a week later. The control groups (n = 8) were immunized with normal saline. All gerbils were challenged by the IP route with lethal doses (100×LD50) of EV71 at day 21. The survival rates and clinical scores were monitored daily after challenge. Clinical scores were graded as described in the legend of Figure 2.