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. 2018 Dec 5;7(1):205.
doi: 10.1038/s41426-018-0201-3.

Severity of enterovirus A71 infection in a human SCARB2 knock-in mouse model is dependent on infectious strain and route

Junping Zhu  1 , Ning Chen  1 , Shuya Zhou  2 , Kai Zheng  1 , Lin Sun  1 , Yuxiao Zhang  1 , Lina Cao  1 , Xiaoyan Zhang  1 , Qiaoyan Xiang  1 , Zhiyun Chen  1 , Chenfei Wang  2 , Changfa Fan  3 , Qiushui He  4   5
Affiliations

Severity of enterovirus A71 infection in a human SCARB2 knock-in mouse model is dependent on infectious strain and route

Junping Zhu et al. Emerg Microbes Infect. .

Abstract

Enterovirus A71 (EV-A71) is a major etiological agent of human hand, foot and mouth disease, and it can cause severe neurological complications. Although several genotypes of EV-A71 strains are prevalent in different regions of the world, the genotype C4 has circulated in mainland China for more than 20 years. The pathogenicity of different EV-A71 clinical isolates varies and needs to be explored. In this study, hSCARB2 knock-in mice (N = 181) with a wide range of ages were tested for their susceptibility to two EV-A71 strains with the subgenotypes C4 and C2, and two infection routes (intracranial and venous) were compared. The clinical manifestations and pathology and their relationship to the measured viral loads in different tissues were monitored. We observed that 3 weeks is a crucial age, as mice younger than 3-week-old that were infected became extremely ill. However, mice older than 3 weeks displayed diverse clinical symptoms. Significant differences were observed in the pathogenicity of the two strains with respect to clinical signs, disease incidence, survival rate, and body weight change. We concluded that hSCARB2 knock-in mice are a sensitive model for investigating the clinical outcomes resulting from infection by different EV-A71 strains. The intracranial infection model appears to be suitable for evaluating EV-A71 neurovirulence, whereas the venous infection model is appropriate for studying the pathogenicity of EV-A71.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Characterization of CMU4232 and CDV-Isehara.
a Cytopathic effects at 1dpi displayed on RD cells infected with CMU4232 or CDV-Isehara. b Detection of viral structural proteins VP0 and VP2 of CMU4232 and CDV-Isehara by western blotting with antibody specific for EV-A71. β-actin was used as a internal control. c Growth curves of CMU4232 and CDV-Isehara in RD cells. RD cells were inoculated with CMU4232 or CDV-Isehara as indicated at either MOI of 1 or 0.1. Samples were collected at the times indicated and titrated by PFU assay. All assays were performed in triplicate. At each time point, titer values are means of three samples; error bars represent SEM. dpi days post infection, PFU plaque forming unit, SEM standard error of mean
Fig. 2
Fig. 2. Variation of the VP1 amino acid sequences between CMU4232 and CDV-Isehara strains.
In total, there were 297 amino acids in the VP1 protein of EV-A71, and five residue variations (22 H/Q, 145 A/E, 237 N/T, 249 V/I, and 289 A/T) were found in the VP1 amino acid sequence between CMU4232 and CDV-Isehara strains
Fig. 3
Fig. 3. Phylogenetic relationship among 30 worldwide EV-A71 strains based on the complete VP1 gene.
CMU4232, CDV-Isehara, 18 representative CHN strains, and 10 international representative strains of different subgenotypes are included in this dendrogram. The 18 representative CHN strains were selected from different isolation places in mainland China from 1998 to 2017 according to available data on internet. Details of all the EV-A71 strains included in the dendrogram are provided in Table S1. CHN Chinese
Fig. 4
Fig. 4. Susceptibility of hSCARB2 KI mice to CDV-Isehara infection.
The 1, 2, 3, 4, and 6-week-old hSCARB2 Tg mice were infected intracerebral (I.C.) with CDV-Isehara strain at a dose of 4.8 × 106 pfu and monitored daily after infection. a The CNS-like hindlimb paralysis, ruffled fur, and shrinking. b The clinical symptoms (asymptomatic, mild, severe, and dead), followed by the criteria described in the materials and methods section (c) asymptomatic (%) and (d) survival (%). Significant difference of asymptomatic (%) and survival (%) with different weeks old Tg mice infected with CDV-Isehara was shown as ****p,0.0001. pfu plaque forming unit, CNS central nervous system, I.C. intracerebral
Fig. 5
Fig. 5. The clinical symptoms, asymptomatic (%), survival (%), and body weight change (%) of different EV-A71 Tg mouse models.
Three-week-old hSCARB2 KI mice were inoculated I.C. with DMEM/ EV-A71 (a) or inoculated intravenous (I.V.) with DMEM/EV-A71 (b) respectively. Three-week-old hSCARB2 KI mice were inoculated with CMU4232 (c) /CDV-Isehara (d) strain via I.C. or I.V. route respectively. (1) The clinical symptoms, (2) asymptomatic (%), (3) survival (%), and (4) body weight change (%) of the Tg mice were observed and recorded daily for 2 weeks after viral infection. ***p,0.000. I.C. intracerebral, I.V. intravenous
Fig. 6
Fig. 6. Virus replication in some tissues of hSCARB2 KI mice by EV-A71 infection.
Three-week-old hSCARB2 KI mice were inoculated with CMU4232/ CDV-Isehara via I.C. or I.V. route. EV-A71 genome copy numbers in the brain, heart, lung, intestine, and muscle of EV-A71-infected Tg mice were determined at several time points by quantitative RT-PCR. Mouse β-actin gene expression in each tissue was used as the internal control. Results represent the mean of three to five samples; error bars represent STDEV. a CMU4232/I.C. (N = 3); b CDV-Isehara/I.C. (N = 4); c CMU4232 /I.V. (N = 5); d CDV-Isehara/I.V. (N = 4)
Fig. 7
Fig. 7. Pathological analysis of brain and the skeletal muscle of EV-A71-infected mouse models.
Sporadic neuronal necrosis and neuronophagia (arrowhead) in the brain (1) and Inflammatory cell infiltration (arrowhead) in the skeletal muscle (5) of mouse model with CDV-Isehara/I.C.; glial nodule (arrow) and neuronophagia (arrowhead) in the brain (2) and severe necrotizing myositis with a mass of inflammatory cell infiltration (arrow) in the skeletal muscle (6) of mouse model with CDV-Isehara/I.V.; perivascular cuffing (arrowhead), partial neuronal necrosis and neuronophagia in the brain (3) and degeneration and necrosis in muscle bundles with inflammatory cell infiltration (arrowhead) (7) in mouse model with CMU4232/I.V.; the brain (4) and the skeletal muscle (8) of MOCK mouse with DMEM/I.V. (magnification: 200 × ). Representative samples are shown for each group
Fig. 8
Fig. 8. VP2 expression in the brain and the skeletal muscle of EV-A71-infected mouse models.
The brain (1) and the skeletal muscle (5) of mouse model with Isehara/I.C.; the brain (2) and the skeletal muscle (6) of mouse model with CDV-Isehara/I.V.; the brain (3) and the skeletal muscle (7) of mouse model with CMU0804/I.V.; the brain (4) and the skeletal muscle (8) of MOCK mouse with DMEM/I.V. Arrowheads indicate EV-A71-positive cells (IHC staining against EV-A71 VP2, magnification: 200 × ). Representative samples are shown for each group
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