A new animal model containing human SCARB2 and lacking stat-1 is highly susceptible to EV71

Abstract

Enterovirus 71 (EV71) is a major threat to children worldwide. Children infected with EV71 could develop subclinical infection and hand-foot-and -mouth disease (HFMD). In severe cases, patients could develop encephalitis, paralysis, pulmonary edema, and death. A more user-friendly and robust animal model is essential to investigating EV71 pathogenesis. Here, we established a hybrid (hSCARB2(+/+)/stat-1(-/-)) mouse strain from crossbreeding SCARB2 transgenic and stat-1 KO mice, and compared the susceptibilities to EV71 infection and pathogenesis between parental and hybrid mice. Virus-encoded VP1 protein can be detected in the streaking nerve fibers in brain and spinal cord. This hybrid mouse strain at 2-week-old age can still be infected with different genotypes of EV71 at 1000-fold lower titer via an ip route. Infected hybrid mice developed earlier onset of CNS disease, paralysis, and death at a higher incidence. These advantages of this novel model meet the urgent need from the scientific community in basic and preclinical research in therapeutics and pathogenesis.

Figure 1. Generation of a hybrid mouse model SCARB2/stat-1 KO by crossbreeding hSCARB2 transgenic mice and stat-1 KO mice.

(a) Human SCARB2 (hSCARB2) cDNA was cloned under its own native promoter in an SV40 expression vector. (b) The generation of homozygous hSCARB2^+/+ transgenic mice was as detailed in M&M. The heterozygote strain of hSCARB2^+/−/stat-1^+/− mice were generated by crossing the stat-1^−/− and the hSCARB2^+/+ parental mice. The hybrid strain hSCARB2^+/+/stat-1^−/− was generated by crossing the heterozygote mice hSCARB2^+/−/stat-1^+/− to each other. (c) Genotyping of parental and hybrid mouse strains was performed by PCR assay using genomic DNAs extracted from mouse tail. The transgene of hSCARB2 was screened by detection of a 369 bp PCR product using primers specific for hSCARB2. For the stat-1^+/− heterozygote mice, 320 bp and 150 bp PCR products were used as markers for screening. The former indicates a mutant stat-1 allele, while the latter indicates a wild type stat-1 allele. (d) The expressions of hSCARB2 protein were compared among parental and hybrid mouse strains in brain, spinal cord, spleen and muscle by immunoblot via an anti-SCARB2 antibody. The weaker signals of the SCARB2 protein detected in stat-1 KO mice reflect cross reactivity between human and mouse SCARB2 proteins to the anti-SCARB2 antibody.

Figure 2. The hybrid mouse strain hSCARB2^+/+/stat-1^−/− is most susceptible to infection and pathogenesis.

One-week-old newborns of parental stat-1^−/−, parental hSCARB2^+/+ transgenic, and hybrid hSCARB2^+/+/stat-1^−/− mice were i.p. infected with EV71 (genotype C2) at high titer (10⁸ pfu/mouse) and low titer (10⁶ pfu/mouse). Clinical scores, paralysis rates, and survival rates were compared among three different mouse strains. Disease manifestation and death were monitored daily post-injection. Clinical scores were defined as follows: 0, healthy; 1, hair loss, wasting, or ruffled hair; 2, limb weakness; 3, paralysis in only 1 limb; 4, paralysis in 2 to 4 limbs; 5, death. The hybrid strain exhibited the highest clinical score (a) paralysis rate (b) and the lowest survival rate (c). Numbers of infected mice were as summarized in Table 1.

Figure 3. Young age is critical for the clinical score and survival rate in the hybrid mouse model infected with EV71.

Clinical score (a) and survival rate (b) were compared in hybrid mice i.p. infected with EV71 (10⁷ pfu/mouse) at one-, two-, and three-week-old ages. Three-week-old hybrid mice showed no clinical symptoms and death. Numbers of infected mice were as summarized in Table 1.

Figure 4. Histopathological examination of CNS sections in three different mouse models.

(a) One-week-old of parental stat-1^−/−, parental hSCARB2^+/+ transgenic, and hybrid hSCARB2^+/+/stat-1^−/− mice were in vivo infected with 10⁸ pfu/mouse of EV71 (genotype C2) by i.p. route, respectively. Moribund mice were sacrificed. Paraffin-embedded sections of brain and spinal cord were examined with H&E stain at x100 and x200 magnifications, respectively (left panel). IHC staining detected the strongest expression of viral VP1 protein in brain and spinal cord in the hybrid mice (right panel). Black arrowheads in IHC of the transgenic hSCARB2^+/+ mice indicate the VP1 positive signals. (b) Upper panel: Parallel streakings of VP1-positive nerve fibers and neuron cells in the mid-brain were detected by IHC staining in 2-week-old hybrid mice infected with EV71. Lower panel: A section from brain stem displayed VP1 positive signals. (c) Left panel: VP1 signal can be detected in lumbar section. Right panel: Streaking of nerve fibers was also detected in spinal cord sections (x400). Black arrowheads highlight the streaking nerve fibers. (d) Spinal cord sections were examined by Nissl Staining at 400x and 800x magnifications. Upper panel: EV71-infected hybrid strain mouse. Black arrowheads highlight abnormal neurons with extensive vaccuoles. Lower panel: PBS injected control mouse. (e) A microglial marker IBA1 was detected in spinal cord of infected hybrid strain mouse by immunohistochemistry (magnification at 400x in left panel). A cell proliferation marker Ki67 was strongly increased in infected spinal cord of hybrid strain mouse (magnification at 400x in right panel). (f) The expression of IBA1 protein in spinal cord appeared to correlate with the disease severity by immunoblot analysis. Each lane represents one spinal cord sample from each infected mouse with different clinical scores.

Figure 5. Expression profiles of EV71 specific RNA, protein and cellular cytokines in CNS of infected hybrid strain mice before, during, and after onset of limb paralysis.

Two-week-old hybrid strain mice were infected with EV71 (genotype C2) at 10⁷ pfu/mouse by i.p. route. Brain, spinal cord, spleen and muscle were collected on dpi 4 (before-onset), dpi 7 (onset), and dpi 9 (after-onset). Extractions of viral RNA and protein were as described in Materials and Methods. (a) VP1 specific RNA in brain and spinal cord was both significantly increased at disease onset by RT-qPCR analysis. The results were normalized by GAPDH. (b) Viral proteins in brain (3D protein) and spinal cord (VP1 protein) peaked after disease-onset by immunoblot analysis. In contrast, no viral protein VP1 in spleen and muscle was ever detected throughout the entire time course. Lysates of EV71-infected RD cells were included as a positive control. (c) Expression of IFNB (IFN-beta) mRNA in brain and spinal cord was significantly increased at disease onset by RT-qPCR analysis. (d) Expression of IL-6 mRNA in spinal cord, but not in brain, was also increased at disease onset (Student’s t test). The results in (c,d) were normalized by GAPDH.