C57BL/6 mice were originally purchased from the Jackson Laboratory and maintained in the specific pathogen free facility at the Institute of Biomedical Sciences, Academia Sinica (Taipei, Taiwan). All animal experiments were approved by the Institutional Animal Care and Utilization Committee (IACUC) at Academia Sinica and performed in accordance with institutional guidelines.

Mouse-adapted EV71 virus (12) was intraperitoneally injected into mouse pups (11-12 days old) at a dose of 8 × 10⁵ PFU per mouse. After viral infection, mice were monitored daily for clinical signs of limb paralysis and scored from 1 to 5: score 1, weakness in one or two hindlimbs (wobbling); score 2, paralysis in one hindlimb; score 3, paralysis in both hindlimbs; score 4, paralysis in both forelimbs and hindlimbs; score 5, death.

The gastrocnemius and soleus muscles were collected from EV71-infected mice. Muscles were weighed and homogenized in PBS containing protease inhibitor cocktail (Merck). Muscle lysates were serially diluted in Dulbecco′s Modified Eagle′s Medium (DMEM) containing 2% fetal bovine serum (FBS) and added to a monolayer of RD (human rhabdomyosarcoma cell line; ATCC CCL-136) cells that were at a density of 5 × 10⁵ cells per well in 6-well plates. After 1 h incubation at 37°C, muscle lysates were removed, and RD cells were cultured in DMEM containing 2% FBS and 1% low melting agarose for 2 days. Cells were then fixed in 4% paraformaldehyde and stained with crystal violet solution. Viral plaques in each well were counted, and the plaque forming unit (PFU) was calculated.

Gastrocnemius and soleus muscles were collected, homogenized in Trizol solution (Invitrogen) using homogenizer (Kinematica Polytron PT3100) and subjected to total RNA extraction according to the manufacturer’s instructions. Total RNA was treated with RQ1 RNase-free DNase (Promega) and was reverse transcribed into cDNA by SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. The cDNA was subjected to quantitative PCR analysis on ABI QuantStudio^® 3 Real-Time PCR System (Applied Biosystems) using SYBR Green Master mix (Thermo Scientific). The thermal cycling protocol was 1 cycle at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The resultant PCR products were analyzed with ABI QuantStudio Design & Analysis Software v.1.4.3 (Applied Biosystems). The relative levels of gene expression were calculated using the ΔCt method with normalization to GAPDH. DNA sequences of the primer pairs used in this study are listed in Supplementary Table 1 .

EV71-infected mice were anesthetized and perfused with PBS. Gastrocnemius and soleus muscles were collected, cut into small pieces and digested with 1.6 mg/ml collagenase IV (Sigma) and 330 µg/ml DNase I (BioShop) at 37°C for 30 min. After removal of tissue debris, cells were washed and resuspended in DMEM containing 10% FBS. For surface marker staining, cells were washed with FACS buffer (HBSS with Ca²⁺/Mg²⁺, 1% FBS, 10 mM HEPES and 0.1% NaN₃) once, blocked with Fc blocker (clone 2.4G2, BD Pharmingen) on ice for 5-10 min, and then incubated with fluorochrome-conjugated antibodies against mouse CD45 (Brilliant Violet 785, clone: 30-F11, BioLegend), CD11b (PerCP/Cyanine5.5, clone: M1/70, BioLegend), Ly6C (Brilliant Violet 510, clone: HK1.4, BioLegend) and Ly6G (Brilliant Violet 650, clone: 1A8, BioLegend) on ice for 30 min. After two consecutive washes with HBSS, cells were stained with fixable viability dye (eFluor 780, eBioscience) on ice for 10 min. Cells were washed twice more and fixed in FACS buffer containing 0.8% paraformaldehyde. Intracellular staining was performed using a Fixation/Permeabilization solution kit (BD Biosciences), according to the manufacturer’s instructions. In short, cells were stained with antibodies to surface markers and viability dye, followed by fixation in Cytofix/Cytoperm solution (BD Biosciences). Cells were then washed twice with Fixation/Permeabilization solution (BD Biosciences) followed by incubation with normal rat serum at room temperature for 10 min. Next, cells were incubated with fluorochrome-conjugated antibody against mouse CD206 (Alexa Fluor 488, clone: C068C2, BioLegend) at room temperature for 30 min. Finally, cells were washed with Fixation/Permeabilization solution, resuspended in FACS buffer and subjected to FACS analysis. Cell populations were analyzed on a BD LSRII, and data were analyzed with FlowJo software (BD Biosciences).

Mice were anesthetized by inhalation of isoflurane vapor. Proper anesthetization was confirmed by a lack of deep tendon reflex. The gastrocnemius muscle was excised and fixed with 4% paraformaldehyde in PBS at 4°C for 24 h, followed by PBS washing. The muscle was embedded in OCT (Sakura Finetek) and longitudinally sectioned using a cryostat (Leica CM3050). For immunostaining, the muscle sections (10 µm) were incubated with blocking buffer (0.1% Triton X-100, 1% BSA, 10% normal goat serum in PBS) at room temperature for 1 h. The muscle sections were then incubated with primary antibodies in antibody diluent (0.1% Triton X-100, 1% BSA, 2% normal goat serum in PBS) overnight at 4°C. After washing in PBS for 30 min, the sections were incubated with secondary antibodies in the antibody diluent at room temperature for 2 h. After washing in PBS for 30 min, the muscle sections were counterstained with DAPI, mounted and observed under confocal microscopy (LSM800 with Airyscan, ZEISS or LSM700 stage, ZEISS). Antibodies used for immunofluorescence staining and other procedures are listed in Supplementary Table 2 .

To analyze the structures of NMJs in gastrocnemius muscle, tissue sections (50 µm) were incubated with blocking buffer (0.5% Triton X-100, 1% BSA, 10% normal goat serum in PBS) at room temperature for 2 h, followed by incubation with acetylcholine receptor (AChR) binding toxin, tetramethylrhodamine α-bungarotoxin (α-BTX, BIOTIUM), at room temperature for 2 h. After washing in PBS for 30 min, muscle sections were counterstained with DAPI, mounted and observed under confocal microscopy (LSM800 with Airyscan, ZEISS or LSM700 stage, ZEISS). Z-stack images of AChRs were obtained and illustrated with maximum intensity projection.

Muscles excised from mice were fixed in 4% paraformaldehyde/PBS at 4°C for 24 h, then dehydrated, embedded in paraffin and transversely sectioned at 5 µm. Sections were subjected to H&E staining for histological analysis or Alizarin Red S staining for muscle calcification. Images were scanned at 40× magnification using Pannoramic 250 Flash II (3D HISTECH Ltd.) and analyzed with Pannoramic Viewer software and ImageJ software (NIH Image). The image acquisition and analysis are shown on Supplementary Figure 1 .

The non-parametric one-way ANOVA (Kruskal-Wallis test) with Dunn’s multiple comparison test was used to evaluate the significant differences on different infection time points. P-values < 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism software version 8.

In severe cases, EV71 infection may cause limb paralysis in infants and young children (2). The EV71-induced limb paralysis is thought to result from damage to the CNS; however, the effects of EV71 on skeletal muscle are largely unknown. Thus, we examined the effect of EV71 infection on skeletal muscle in a mouse model. As with humans, adult mice are resistant to EV71 infection, and only mouse pups are susceptible to its effects. Mouse pups (11-12 days old) were infected with EV71 (8 × 10⁵ PFU), and limb paralysis was assessed with a clinical scoring system. We noted that mouse pups infected with EV71 exhibited exceptionally stiff limb muscles, which is more likely due to a musculoskeletal defect than neurological dysfunction (13, 14). The onset of disease was about 2-3 days post-infection (dpi), and the peak of disease was about 5-7 dpi. Mice then recovered from limb paralysis at about 20 dpi or later ( Figure 1A ). Analysis of virus titers in limb muscles (gastrocnemius and soleus muscles) revealed that virus loads peaked at 3 dpi and declined rapidly after 6 dpi and no virus was detected in the recovery phase ( Figure 1B ). We then examined whether EV71 targets muscle cells by performing immunofluorescence staining of muscle tissue sections with an antibody against viral protein, VP1, revealing that the viral protein could be readily detected in muscle tissues ( Figure 1C ). Since the viral load declined rapidly at 6 dpi, we speculated that muscle cells might induce anti-viral molecules to clear virus. We therefore examined the expression of anti-viral molecules after EV71 infection. Muscle tissues were collected from limbs at various times post-infection, and quantitative PCR was performed to measure the gene expression of pro-inflammatory cytokines, chemokines and anti-viral molecules. Several pro-inflammatory cytokines and chemokines (TNF-α, IL-1β, IL-6, CXCL10, CCL2) as well as anti-viral molecules (IFN-α, IFN-β, ISG15, Mx1) were induced upon EV71 infection. Notably, these molecules except TNF-α were induced as early as 16 hours post-infection, peaked at around 36 hours post-infection, and significantly declined at 6 dpi ( Figure 1D ). These results demonstrate that EV71 infects skeletal muscles and induces pro-inflammatory cytokines, chemokines and anti-viral molecules that are expected to promote viral clearance.

We next examined whether EV71 infection impaired muscle tissue structure, which might weaken the muscle and, at least in part, contribute to the loss of limb function and limb paralysis. Histopathological staining of gastrocnemius muscle sections revealed that EV71 infection resulted in severe myositis, which peaked at 5-7 dpi ( Figure 2 and Supplementary Figure 2 ). The muscle structure showed clear pathology, including leukocyte infiltration at 3 dpi ( Figure 2B ), and severe myositis with highly disrupted muscle fibers accompanied by severe leukocyte infiltration was observed at 5-7 dpi ( Figure 2C ). The myositis and infiltrating leukocytes gradually waned over the subsequent weeks, and muscle tissues were largely restored ( Figures 2D–G ). Of note, mouse pups infected with EV71 showed extensive abnormal white coloration in muscles of the body trunk and limbs ( Figure 2H ). Like the myositis, the white appearance also peaked at 6-7 dpi and then gradually disappeared. We next used Alizarin Red S staining to examine whether limb muscles with white appearance exhibited calcification; this stain is used to identify calcium deposits in tissue sections. The results revealed that the white appearance in the muscle tissues corresponded to positive Alizarin Red S staining, suggesting that the white appearance in the muscle tissue reflects calcification ( Figures 2J–O ). The percentage of calcification arears of muscles were calculated, which peaked at 6-7 dpi and then gradually waned ( Figure 2P ). This calcification of muscles may explain the stiffness of limb muscles we observed. Additionally, the severe myositis was concurrently observed with extensive calcification in muscles ( Figure 2 ), and the muscle inflammation and calcification gradually diminished in parallel as the muscle structure recovered ( Figure 2 and Supplementary Figure 2 ). Taken together, these results suggest that EV71 infection induces severe myositis correspondent with muscle calcification, which destroys muscle structure and may underlie the loss of limb function.

Although mice infected with EV71 developed severe myositis associated with extensive calcification at the peak of disease (5-7 dpi), the pathology gradually resolved as time went on ( Figure 2 ). We found this dynamic change in pathology of muscle tissues from severe myositis to regeneration of myofibers after EV71 infection to be quite intriguing, so we looked at other aspects of the tissue during this period. The major population of infiltrating leukocytes in muscle tissues with severe myositis appeared to be monocytes/macrophages, as shown by H&E staining ( Figures 2B–D ). We thus analyzed myeloid cell populations (CD45⁺CD11b⁺) in muscle tissues at various times post-infection using FACS analysis. The CD45⁺CD11b⁺ cells were identified as the myeloid cell population and were subjected to further analysis of Ly6G and Ly6C expression ( Figure 3A ). The Ly6G⁺Ly6C^int cells represented neutrophils ( Figure 3B , left panel), while CD45⁺CD11b⁺Ly6C^hi and CD45⁺CD11b⁺Ly6C^low/- were considered to be monocytes/macrophages since they were all positive for F4/80 (data not shown). Furthermore, Ly6C^hiLy6G^- cells were considered to be inflammatory macrophages, and Ly6C^low/-Ly6G^- cells were considered anti-inflammatory/restorative macrophages (15–18). The kinetics of neutrophil infiltration showed a peak at 3 dpi, followed by a gradual decline ( Figure 3B , left panel). Ly6C^hi macrophages are expected to be recruited from circulating monocytes to the tissue during tissue damage (19), and accordingly, the percentage of Ly6C^hi macrophages was significantly increased in muscle tissue as early as 36 hours post-infection. The high levels were sustained until 7 dpi and declined at 16 dpi ( Figure 3B , middle panel). On the other hand, Ly6C^low/- macrophages are typically resident tissue macrophages and/or anti-inflammatory/restorative macrophages, which function to resolve inflammation and promote regeneration of damaged tissues (15–18). The frequency of Ly6C^low/- macrophages was reduced upon EV71 infection as early as 36 hours post-infection, and it was further reduced at 3 and 7 dpi; the level was restored at 16 dpi ( Figure 3B , right panel). These results show that macrophages with different phenotypes appear at different times in the process of EV71 infection and resolution, as indicated by the ratio of Ly6C^hi/Ly6C^low/- macrophages ( Figure 3C ). The kinetics of Ly6C^hi and Ly6C^low/- macrophages in the muscle during EV71 infection were reciprocal, i.e., the increased frequency of Ly6C^hi macrophages (inflammatory macrophages) was accompanied by a decreased frequency of Ly6C^low/- macrophages (anti-inflammatory/restorative macrophages) during the early stage of EV71 infection, and a decreased frequency of Ly6C^hi macrophages was accompanied by an increased frequency of Ly6C^low/- macrophages during late stage of recovery from EV71 infection.

CD206, the mannose receptor, is a marker for tissue-resident and anti-inflammatory macrophages (20), and it is widely used to identify macrophage subsets that exhibit anti-inflammatory functions (21). Before infection, the major population of monocytes/macrophages was Ly6C^low/- macrophages, which also exhibited high expression of CD206 ( Figures 3A, D, E ). The frequency of CD206⁺ cells was gradually reduced upon EV71 infection, and it reached the lowest level at the peak of disease (7 dpi) with a rebound at the recovery phase (16 dpi) ( Figures 3D, E ). IFA staining revealed that at 3 dpi, damaged muscles were infiltrated with F4/80 macrophages, which were presumably inflammatory macrophages recruited to promote clearance of virus and cell debris. Some F4/80 macrophages were co-stained with different levels of CD206 ( Figure 3F ). At the later stage of infection, we observed macrophages around the periphery of damaged muscle fibers ( Figures 3G, H ), likely contributing to the removal of debris from tissues. This time corresponded to the resolution of inflammation and calcification as well as the regeneration of the muscle tissue. Of note, IFA staining showed that CD206⁺/F4/80^- cells increased with time in muscle tissues after EV71 infection ( Figures 3F–H ). These cells might be muscle satellite cells since CD206 is known to be expressed in that cell type (22). These results confirm that anti-inflammatory/restorative macrophages increase at the late stage of infection ( Figures 3A, B ) and suggest that anti-inflammatory/restorative macrophages may play an important role in repairing EV71-induced muscle damage and promoting regeneration ( Figures 3G, H ).

Since we suspected that anti-inflammatory/restorative macrophages might play a role in repairing tissue damage and regenerating muscles, we next examined gene expression for molecules produced by macrophages and known to be involved in tissue regeneration. The gene expression levels of TGF-β and IGF-1, which are produced by anti-inflammatory/restorative macrophages and promote repair and regeneration of damaged tissues (18, 23–25), were significantly increased at 36 h post-infection to at least 6 dpi and then declined in the recovery phase ( Figure 3I ). The gene expression level of osteopontin (OPN), which is known to regulate the regeneration of muscles following injury (26) and inhibit ectopic calcification (27), was also significantly increased at 3 dpi and further increased at 6 dpi and declined in the recovery phase ( Figure 3J ). We then further investigated the role of OPN in EV71-induced muscle calcification by immunofluorescence staining. We found that OPN was localized within the calcified area of muscle, likely to inhibit more calcification ( Figure 3K ); notably, macrophages surrounded the calcified area in the muscle tissue ( Figure 3K ). Altogether, the results from Figure 3 suggest that macrophages with Ly6C^hi in the muscle tissue at the early stage of EV71 infection are inflammatory macrophages responsible for the initiation of inflammation to clear virus and debris, while macrophages in the muscle tissue with Ly6C^low/- appear at the late stage of EV71 infection and are anti-inflammatory/restorative macrophages that facilitate the resolution of inflammation and calcification as well as the regeneration of muscles.

NMJs are specialized synapses between muscles and motor neurons, which control muscle movement. Given that EV71 infection induces weakness of limb muscles, severe myositis and muscle calcification, we rationalized that the NMJs in muscles may also be dysfunctional. Thus, we examined the structure of postsynaptic motor end plates in the muscle, specifically in the gastrocnemius muscle, by staining acetylcholine receptors (AChRs) in the postsynaptic membrane of the muscle fiber. It has been reported that the shapes of postsynaptic AChR clusters in young mice (2-3 weeks old) are quite different from those in adult mice. In mice, the shape of postsynaptic AChR clusters in NMJs is expected to change from plaque-like in young animals to pretzel-like in adult mice (28, 29). We identified innervated synapses by staining tissue sections of gastrocnemius muscle with α-bungarotoxin (α-BTX), which binds AChR (the marker for postsynaptic motor end plates). As expected, the structures of motor end plates in the muscle were plaque-like ( Figure 4A , left panel) in tissues from uninfected young mice (15 days old), while a pretzel-like structure ( Figure 4A , right panel) was observed in muscles from uninfected adult mice (34 days old). In virus treated mice, the structure of motor end plates from the muscle seemed to be unaffected at 16 hours post-infection, but the end plates became aberrant/fragmented at 3 dpi, when compared with uninfected 15-day-old mice. The motor end plates were gradually repaired and became more cohesive as time went on; the degree of repaired motor end plate structures was greatest at 51 dpi and progressively lower at 34, 21 and 14 dpi ( Figure 4B ). The motor end plates in infected mice did not appear to recover the pretzel-like structures seen in uninfected adult mice, even at 51 dpi ( Figure 4B , d51 vs. Figure 4A , right panel). The motor end plate structures seemed repaired macroscopically at day 51 dpi. However, when counting fragments within a AChR cluster, we found that almost all AChR clusters even at 51 dpi ( Supplementary Figure 3 ) showed five or more fragments per AChR cluster, which has been considered as an aberrant/fragmented AChR cluster (30). Of interest, the macroscopical structure of AChR clusters seemed improved along with the time, but the microscopical structure of an AChR cluster (aberrant/fragmented AChR cluster) seemed to stay impaired ( Supplementary Figure 3 ) even at 51 dpi. These results suggest that EV71 infection might drastically impair postsynaptic motor end plates of NMJs, resulting in incompletely innervated synapses. This structural damage would inevitably affect the transmission of signals from lower motor neurons to the limb muscle, leading to the loss of limb function.

The aforementioned results show that at late stage of infection, anti-inflammatory/restorative macrophages (Ly6C^low/-) are increased ( Figures 3B, C ), while molecules responsible for tissue repair (TGF-β, IGF-1, OPN) are upregulated ( Figures 3I, J ), and the postsynaptic motor end plates of NMJs are repaired ( Figure 4B ). Based on these results, it appeared that limb muscles damaged by EV71 infection underwent regeneration. Pax7 is a transcription factor expressed in muscle satellite cells that regulates cell survival, proliferation and differentiation (31). Moreover, MyoD and Myogenin are transcription factors involved in skeletal muscle development, myogenesis and repair (32, 33). Analyzing gene expression levels by quantitative PCR, we found that MyoD, Myogenin and Pax7 in limb muscles were all significantly increased at 3 dpi, further increased at 6 dpi, and declined in the recovery phase ( Figure 5 ). Taken together, these results suggest that limb muscles damaged by EV71 infection are indeed able to undergo muscle regeneration. However, whether EV71-infected mice can completely regenerate damaged muscles remains unclear since the muscle morphologies at days 51 post-infection were still not completely normal ( Figure 4B ).