Transgenic mouse model for the study of enterovirus 71 neuropathogenesis

Abstract

Enterovirus 71 (EV71) typically causes mild hand-foot-and-mouth disease in children, but it can also cause severe neurological disease. Recently, epidemic outbreaks of EV71 with significant mortality have been reported in the Asia-Pacific region, and EV71 infection has become a serious public health concern worldwide. However, there is little information available concerning EV71 neuropathogenesis, and no vaccines or anti-EV71 drugs have been developed. Previous studies of this disease have used monkeys and neonatal mice that are susceptible to some EV71 strains as models. The monkey model is problematic for ethical and economical reasons, and mice that are more than a few weeks old lose their susceptibility to EV71. Thus, the development of an appropriate small animal model would greatly contribute to the study of this disease. Mice lack EV71 susceptibility due to the absence of a receptor for this virus. Previously, we identified the human scavenger receptor class B, member 2 (hSCARB2) as a cellular receptor for EV71. In the current study, we generated a transgenic (Tg) mouse expressing hSCARB2 with an expression profile similar to that in humans. Tg mice infected with EV71 exhibited ataxia, paralysis, and death. The most severely affected cells were neurons in the spinal cord, brainstem, cerebellum, hypothalamus, thalamus, and cerebrum. The pathological features in these Tg mice were generally similar to those of EV71 encephalomyelitis in humans and experimentally infected monkeys. These results suggest that this Tg mouse could represent a useful animal model for the study of EV71 infection.

Keywords: neurotropism; picornavirus; viral receptor.

Fig. 1.

Expression profiles of hSCARB2 in human and hSCARB2-Tg10 mouse tissues. (A) Expression of hSCARB2 in various human tissues detected using an anti-hSCARB2 antibody. (B) Expression of hSCARB2 in various tissues in Tg and non-Tg mice. Tissues were homogenized and subjected to SDS/PAGE, and hSCARB2 was detected using an anti-hSCARB2 antibody. Arrowheads indicate the positions of hSCARB2 and mScarb2. Asterisks indicate nonspecific bands. (C) Immunohistochemical analysis of hSCARB2 expression in human, Tg, and non-Tg mouse CNS tissues stained with an anti-human SCARB2 antibody (brown) and counterstained with hematoxylin. (Scale bars, 50 and 100 μm in human and mouse tissue images, respectively.)

Fig. 2.

Replication of EV71 in the CNS of hSCARB2-Tg10 mice. (A) Viral titers in the brain and spinal cord. After i.v. inoculation of the EV71 Isehara strain at a dose of 1 × 10⁶ TCID₅₀ in 3-wk-old animals (non-Tg; n = 4 in each time point, Tg; n = 4 at day 1 and day 3, n = 6 at day 5, n = 5 at day 7), the mice were euthanized at the indicated times, the tissues were collected, and viral titers were measured using the microtiter method. The open and closed circles show the viral titers in individual non-Tg and Tg mice, respectively. The viral titer detection limit is less than 10³ TCID₅₀/g (B) Histopathological changes and viral antigen expression in the CNS of 3-wk-old Tg mice inoculated i.v. with the EV71 Isehara strain (Table 1; Table S2). The pons, medulla, lumbar spinal cord, and cerebellar nuclei are shown (Upper, H&E staining; Lower, immunohistochemistry). Open arrowheads, closed arrowheads, and asterisks indicate degenerated neurons, neuronophagia, and gliosis, respectively.

Fig. 3.

EV71 infection in hSCARB2-expressing neurons in Tg mice. The medulla and spinal cord sections from a Tg mouse showing neurological signs (Table S2) were double-stained with an anti-EV71 antibody (green) and anti-hSCARB2 antibody (red). Nuclei were stained with DAPI (blue).