Development and characterization of hNCL-Tg mice

Modified BAC was injected into the pronucleus of fertilized mouse eggs to create transgenic mice. A Stable hNCL expressing transgenic mice strain was obtained after three breeding passages. We mated hNCL heterozygous mice (hNCL-Tg mice) with WT mice. Genotypes of the neonate mice were investigated at 7 days old. Fusion red protein expressed by hNCL-Tg mice could be observed from the tail using a fluorescence microscope. (Fig. 1A, mice No. 1, 2, 3, 5, 7, and 8). The mRNA of fusion red and actin were analyzed after PCR in which the hNCL-Tg mice showed two bands (199 base pairs for fusion red and 254 base pairs for actin, mice No. 1, 2, 3, 5, 7, and 8) and WT mice showed one band (245 base pairs for actin, mice No. 4 and 6) (Fig. 1B). The expression of the Flag tagged hNCL (100 kDa) determined by western blotting was consistent with PCR and fluorescent microscope results (Fig. 1C). Because the anti-nucleolin antibody cross-reacted with human and mouse nucleolin.

Fig. 1

Characterization of hNCL-Tg mice. hNCL-Tg mice were characterized by fluorescence microscope, genotyping, and immunoblotting. (A) The co-expressed Fusion Red was observed by fluorescence microscopy in hNCL-Tg mice. (B) The genomic DNA was extracted from the tail, and the hNCL genome was detected by PCR. The mRNA of fusion red and actin were analyzed after PCR in which the hNCL-Tg mice showed two bands (199 base pairs for fusion red and 254 base pairs for actin, mice No. 1, 2, 3, 5, 7, and 8) and WT mice showed one band (245 base pairs for actin, mice No. 4 and 6). (C) The protein was extracted from the tail, and flag-tagged hNCL was determined by Western blot. (D) The clinical scores and (E) survival rates of EV-A71-infected hNCL-Tg and WT mice. The 7-day-old hNCL-Tg and WT mice were intraperitoneally infected with the EV-A71 strain (5 × 104 pfu/mouse). The clinical score and survival rate were measured everyday post-infection. (Clinical scores: 0, healthy; 1, reduced motility; 2, limb weakness; 3, one limb paralysis; 4, two limb paralysis; 5, moribund and death) The data were expressed as the mean ± SD of each group. **, p < 0.01 and ***, p < 0.001

In addition, the expression of human nucleolin in the spinal cord and skeletal muscle was detected by Western blot. The overall nucleolin expression was observed because the anti-nucleolin antibody cross-reacted with human and mouse nucleolin. The expression of hNCL in both tissues was higher in hNCL-Tg mice than in WT mice (Supplementary Fig. 2, upper). The relative intensities of hNCL in the spinal cord and the skeletal muscle were 5.5/3.9 and 8.3/7.1, respectively. We analyzed the flag-tagged human nucleolin using an anti-flag antibody to determine the expressed hNCL in mice (Supplementary Fig. 2, middle). The relative intensities of flag-tagged hNCL in the spinal cord and the skeletal muscle were 1.0/1.5 and 12.3/18.1, respectively. The results indicated that the skeletal muscle expressed much higher human nucleolin than the spinal cord. Besides, hNCL-Tg mice showed normal body weight and appearance compared to WT mice (Supplementary Fig. 3).

Clinical scores and survival rates of EV-A71-infected hNCL-Tg and WT mice

To evaluate the effect of EV-A71 infection, 7-day-old hNCL-Tg and WT mice were intraperitoneally inoculated with virus. We found that the clinical scores of both WT and hNCL-Tg mice were elevated three days post-infection. The hNCL-Tg mice began to develop paralysis four days post-infection (Supplementary Fig. 4A). Both hind limbs of the hNCL-Tg mouse were paralyzed. The mouse also showed seizure symptoms such as myoclonic jerks. Some of the mice had hind limb skin desquamation and hemorrhage of the fingertips, like what occurs in HFMD (Supplementary Fig. 4B and 4 C). Skin rashes observed in hNCL-Tg mice correspond to rashes (lesions) on the palms and soles of EV-A71-infected children. After 7 days, the clinical scores of hNCL-Tg mice significantly increased and finally died (Fig. 1D). In contrast, those of WT mice decreased and finally recovered (Fig. 1D). 90% of hNCL-Tg mice died 10 days post-infection, but WT mice survived (Fig. 1E). Although the clinical score dropped to 0 two weeks after infection, the size of the recovered mice was smaller than that of the uninfected mice. These findings suggest that the disease progression in the hNCL-Tg mice was faster than that in the WT mice.

Viral titers in the skeletal muscle, spinal cord, and brain stem of EV-A71-infected hNCL-Tg and WT mice

To investigate the viral load in organs of mice after EV-A71 infection, 7-day-old hNCL-Tg and WT mice were inoculated with 5 × 104 Pfu/ 50µL/mouse EV-A71 intraperitoneally. The mice were sacrificed at 2, 4, and 6 days post-infection. The brain, spinal cord, and muscle of the mice were collected, followed by viral load determination using plaque assay. We found that the viral titers in the brain stem, spinal cord, and skeletal muscle showed no significant differences between EV-A71 infected hNCL-Tg and WT mice at 2 and 4 days post-infection (Fig. 2A and B). The viral titers of hNCL-Tg mice were significantly higher than those of WT mice in the spinal cord and skeletal muscle at 6 days post-infection (Fig. 2C).

Fig. 2

The viral load in skeletal muscle, spinal cord, and brain stem of EV-A71 infected hNCL-Tg and WT mice. The 7-day-old WT and hNCL-Tg mice were intraperitoneally injected with 5 × 104 pfu/mouse of EV-A71. The virus titers were determined by plaque assay. (A) The WT (n = 5, blue) and Tg mice (n = 4, red) were sacrificed at 2 days post-infection. (B) The WT (n = 5) and Tg mice (n = 4) were sacrificed at 4 days post-infection. (C) The WT (n = 6) and Tg mice (n = 3) were sacrificed at 6 days post-infection. Dashed lines indicated the Mean of the virus titers (log10 pfu/g). *, p < 0.05 and **, p < 0.01

H&E and IHC stains in the organs of EV-A71-infected hNCL-Tg and WT mice

To evaluate disease progression in EV-A71 infection, 7-day-old hNCL-Tg and WT mice were inoculated with EV-A71 intraperitoneally. Mock: the WT mice were inoculated with 50 µL of 2% DMEM. The mice were sacrificed on days 4 and 6 post-infection. The organs (including the brain, spinal cord, and skeletal muscle) were collected, followed by formalin fixation, paraffin embedding, and H&E or IHC staining. The skeletal muscle fibers exhibit structural disruption (Fig. 3A, upper). The dense clusters of dark-staining nuclei within the muscle tissue represent inflammatory cell infiltration (Fig. 3A, upper). The widened spaces between muscle fibers may indicate edema. The pale or fragmented cytoplasm was consistent with necrosis caused by viral infection and inflammation-mediated damage (Fig. 3A, upper). Severe viral infection was also observed in skeletal muscle IHC stains of both mice groups (Fig. 3B, upper).

Fig. 3

Pathological and molecular characterization of EV-A71 infected hNCL-Tg and WT mice. Mock: the WT mice inoculated with 50 µL, 2% DMEM. 7-day-old hNCL-Tg mice and WT mice were infected with 5 × 104 pfu/mouse EV-A71 by i.p. route. On 4 and 6 days post-infection, the mice were sacrificed, and paraffin-embedded sections of skeletal muscle, spinal cord, and brain were examined with (A) H&E stain and (B) IHC stain, respectively. The IHC stain detected the strongest expression of viral VP1 protein in the skeletal muscle, spinal cord, and brain tissue of the hNCL-Tg mice (B)

In the spinal cord images, some prominent vacuolated areas within the neuropil were observed in the H&E stain images in both mice groups, indicating spongiform changes caused by neuronal damage (Fig. 3A, middle). The neurons appeared to have disrupted structures and showed shrunken cytoplasm, suggesting necrosis or apoptosis induced by the viral infection (Fig. 3A, middle). Viral antigens were observed in IHC stain images in both mice groups, which indicated minor viral infection in the spinal cord (Fig. 3B, middle).

The neurons in the brain stem H&E stain of EV-A71 infected WT mice appear relatively intact with identifiable nuclei and cytoplasm (Fig. 3A, down). The tissue exhibited an organized structure, and fewer visible vacuoles or disrupted regions were observed. The absence of viral antigens in the IHC image also indicated that EV-A71 did not infect the brain stem of WT mice (Fig. 3A, down). Compared with brain stem H&E stain of EV-A71 infected hNCL-Tg mice, darker nuclei and loss of clear cytoplasmic borders were observed, which suggested signs of degeneration in neurons (Fig. 3A, down). Some neuron fibers were disrupted with increased vacuolation and showed a spongiform appearance. Combined with the IHC images in which the viral antigens were observed, these findings supported the neuronal damage caused by EV-A71 infection in hNCL-Tg mice (Fig. 3A, down).

The cytokine expression in skeletal muscle of EV-A71 infected hNCL-Tg and WT mice

Severe EV-A71-associated cases exhibit high levels of pro-inflammatory cytokines, chemokines, and cytokines, suggesting that inflammatory responses are essential in the immune response to EV-A71 [7, 21]. To investigate whether the current mice model exhibited the same phenomenon, we quantified the levels of chemokines (MCP-1 and IP-10), pro-inflammatory cytokines (IL-1β, IL-6, IL-22, and TNF-α), immunoregulators (IL-5 and IFN-γ), and anti-inflammatory cytokines (IL-4, IL-10, and IL-13) in the skeletal muscle of hNCL-Tg and WT mice (Fig. 4A and K).

Fig. 4

Cytokine expression in the skeletal muscle of EV-A71-infected hNCL-Tg mice and WT mice. Mock: the WT mice inoculated with 50 µL, 2% DMEM. WT (black blocks) and hNCL-Tg (white blocks) mice were infected intraperitoneally with 5 × 104 pfu/50 µL/mouse EV71. Mice were sacrificed at 2, 4, and 6 days post-infection, and the skeletal muscles were collected and analyzed. Cytokine concentrations were presented as pg/g of the organ. (A) IL-1β, (B) IL-4, (C) IL-5, (D) IL-6, (E) IL-10, (F) IL-13, (G) IL-22, (H) MCP-1, (I) IFN-γ, (J) IP-10, and (K) TNF-α. The nonparametric Mann-Whitney U test determined each group’s statistical significance, the mean ± SD. *, p < 0.05, **, p < 0.01. Mice number: 3 to 9

The expression of most chemokines or cytokines showed no significant difference between the hNCL-Tg and WT mice, except IL-1β, IL-5, and IL-22. The pro-inflammatory cytokine IL-1β exhibited a significantly higher level in the skeletal muscle of hNCL-Tg mice than WT mice at 6 DPI (Fig. 4A). However, another pro-inflammatory cytokine, IL-22, showed a significantly lower level in the skeletal muscle of hNCL-Tg mice than WT mice at 4 DPI (Fig. 4G). The same phenomenon was observed in immunoregulator IL-5, in which the expression was significantly lower in the skeletal muscle of hNCL-Tg mice than in WT mice at 4 DPI (Fig. 4C). In addition, the expression of IL-1β, IL-5, MCP-1, IFN-γ, IP10, and TNF-α was higher after EV-A71 infection in both mice groups compared with mock (Fig. 4A, C, H, I and J, and 4K).

The cytokine expression in the spinal cord of EV-A71 infected hNCL-Tg and wild-type mice

The characteristics of cytokine responses in patient blood and cerebrospinal fluid (CSF) have been associated with EV-A71-associated brainstem encephalitis, ANS dysregulation, and pulmonary edema in clinical settings [7]. In this study, cytokine responses in the spinal cord tissue covered part of the cerebrospinal fluid of EV-A71-infected hNCL-Tg and WT mice, suggesting a central nervous system (CNS) immune response after EV-A71 infection. Although statistical significance was observed after EV-A71 infection in the spinal cord samples of both mice groups compared with mock (Fig. 5A, D, F, H, I and J, and 5K), the expression levels of all chemokines and cytokines showed no significant difference between the hNCL-Tg and WT mice (Fig. 5B, C and E, and 5G).

Fig. 5

Cytokine expression in the spinal cord of EV-A71 hNCL-Tg mice and WT mice. Mock: the WT mice inoculated with 50 µL, 2% DMEM. WT (black blocks) and hNCL-Tg (white blocks) mice were infected intraperitoneally with 5 × 104 pfu/50 µL/mouse EV71. Mice were sacrificed at 2, 4, and 6 days post-infection, and the spinal cord were collected and analyzed. Cytokine concentrations were presented as pg/g of the organ. (A) IL-1β, (B) IL-4, (C) IL-5, (D) IL-6, (E) IL-10, (F) IL-13, (G) IL-22, (H) MCP-1, (I) IFN-γ, (J) IP-10, and (K) TNF-α. The nonparametric Mann-Whitney U test determined each group’s statistical significance, the mean ± SD. *, p < 0.05, **, p < 0.01. Mice number: 3 to 9

The cytokine expression in the serum of EV-A71 infected hNCL-Tg and wild-type mice

The serum is the most commonly used sample for EV-A71-infected patients in clinical studies. It can reflect the total immunity against the virus. Here, we determined the cytokine response in the serum of hNCL-Tg mice, and a further comparison was made with the expression of clinical patients to assess the suitability of mice for enterovirus studies. The expression levels of immunoregulator IL-5 and pro-inflammatory cytokine IL-22 in the serum of WT mice were significantly higher than those of hNCL-Tg mice at 2 DPI (Fig. 6C and G). In contrast, the expression levels of pro-inflammatory cytokine IL-6 and anti-inflammatory cytokine IL-13 were significantly higher in the serum of hNCL-Tg than WT mice at 6 and 2 DPI, respectively (Fig. 6D and F). Besides, most chemokines and cytokines showed higher expression levels after EV-A71 infection in both mice groups than in mock at different DPIs, except IL-5 (Fig. 6A and K).

Fig. 6

Cytokine expression in the serum of EV-A71 hNCL-Tg mice and WT mice. Mock: the WT mice inoculated with 50 µL, 2% DMEM. WT (black blocks) and hNCL-Tg (white blocks) mice were infected intraperitoneally with 5 × 104 pfu/50 µL/mouse EV71. Mice were sacrificed at 2, 4, and 6 days post-infection, and the serum was collected and analyzed. Cytokine concentrations were presented as pg/mL of the organ. (A) IL-1β, (B) IL-4, (C) IL-5, (D) IL-6, (E) IL-10, (F) IL-13, (G) IL-22, (H) MCP-1, (I) IFN-γ, (J) IP-10, and (K) TNF-α. The nonparametric Mann-Whitney U test determined each group’s statistical significance, the mean ± SD. *, p < 0.05, **, p < 0.01. Mice number: 3 to 9

The effects of EV-A71 infection in the lung tissue of hNCL-Tg and WT mice

To assess the symptoms of neurogenic pulmonary edema, lung tissue from uninfected mice, EV-A71-infected WT mice at clinical scores 1 and 4, and hNCL-Tg mice at clinical scores 4 and 5 were investigated. The mock group, the control group without EV-A71 infection, was inoculated with 50 µL of 2% DMEM. Generally, the lung-to-body weight ratio was not significantly different between EV-A71 infected hNCL-Tg and WT mice. The lung tissue of mock mice appeared normal, with a smooth surface, uniform pink coloration, and no signs of hemorrhage, congestion, or swelling (Fig. 7A). The lung tissue of WT mice at clinical score 1 exhibited slight congestion, darkened regions, subtle vascular abnormalities, and intact structure, suggesting early-stage vascular involvement or edema (Fig. 7B). Severe pathology with evident hemorrhage, congestion, and darker coloration throughout the tissue was observed in EV-A71 infected WT mice at clinical score 4 (Fig. 7D). Compared to Fig. 7A and B, it looked inflamed, with clear signs of damage indicative of advanced disease. The lung tissue of EV-A71-infected hNCL-Tg mice displayed severe congestion and inflammation. The lung surface appeared swollen, and hemorrhage induced significant dark red regions (Fig. 7C). It is similar in severity to WT mice at clinical score 4 (Fig. 7D). Still, the pattern of damage may reflect differences in disease progression between WT and hNCL-Tg mice. Extensive hemorrhage and severe tissue damage were observed in the lung tissue of EV-A71-infected hNCL-Tg mice (Fig. 7E). There were large dark red areas with an irregular surface, indicating widespread pulmonary edema and vascular leakage. Compared to Fig. 7C, the lung tissue of hNCL-Tg mice at clinical score 5 showed more pronounced pathology. Compared to Fig. 7D, hNCL-Tg mice at clinical score 5 exhibited more severe damage than WT mice at clinical score 4.

Fig. 7

Pathological characterization of the lung tissues of EV-A71 infected hNCL-Tg and WT mice. Mock: the WT mice inoculated with 50 µL, 2% DMEM. (A-E) The lung tissues of uninfected mice, EV-A71-infected WT mice at clinical scores 1 and 4, and hNCL-Tg mice at clinical scores 4 and 5 were investigated. (A) The lung tissue of mock mice appeared normal, with a smooth surface and uniform pink coloration. (B) The lung tissue of WT mice at clinical score 1 exhibited slight congestion, darkened regions, subtle vascular abnormalities, and intact structure, suggesting early-stage vascular involvement or edema. (C) The lung tissue of EV-A71-infected hNCL-Tg mice displayed severe congestion and inflammation. The lung surface appeared swollen, and hemorrhage induced significant dark red regions. (D) Severe pathology with evident hemorrhage, congestion, and darker coloration throughout the tissue was observed in EV-A71 infected WT mice at clinical score 4. (E) Extensive hemorrhage and severe tissue damage were observed in the lung tissue of EV-A71-infected hNCL-Tg mice. The lung section of uninfected mice, EV-A71-infected WT mice at clinical scores 4, and hNCL-Tg mice at clinical scores 4 were characterized. (F) The alveolar walls were thin, and the air spaces were large, clear, and unobstructed in the lung section of uninfected mice. no inflammatory cell infiltration is observed. (G) Moderate to severe alveolar wall thickening with persistent inflammatory cell infiltration was observed in EV-A71-infected WT mice at clinical score 4. Some red blood cells within the capillaries were observed. (H) The alveolar of EV-A71-infected hNCL-Tg mice showed a disrupted structure with noticeable thickening of the alveolar walls and infiltration of inflammatory cells, indicating severe acute lung injury. The alveolar air spaces were partially filled with cellular debris and exudate, consistent with pulmonary edema and inflammation

In addition, the lung section of uninfected mice, EV-A71-infected WT mice at clinical scores 4, and hNCL-Tg mice at clinical scores 4 were investigated, and the pathological features were characterized. The alveolar walls were thin, and the air spaces were large, clear, and unobstructed in the lung section of uninfected mice (mock, Fig. 7F). The structures appeared intact and well-preserved. Besides, no inflammatory cell infiltration is observed. Next, moderate to severe alveolar wall thickening with persistent inflammatory cell infiltration was observed in EV-A71-infected WT mice at clinical score 4 (Fig. 7G). The alveoli of EV-A71-infected hNCL-Tg mice showed a disrupted structure with noticeable thickening of the alveolar walls and infiltration of inflammatory cells (Fig. 7H). Red blood cells were observed within the alveolar spaces, suggesting hemorrhage. Compared to Fig. 7G, the lung tissue of hNCL-Tg mice at clinical score 4 exhibited more severe alveolar disruption, significant inflammatory cell infiltration, and more pronounced hemorrhage, suggesting a more aggressive disease progression in hNCL-Tg mice.