Enteroviruses (EVs), belonging to the genus Enterovirus, are a group of RNA viruses classified within the Picornaviridae family. Enterovirus genus encompasses 15 distinct species. Among these, seven demonstrate human pathogenicity: four EV species (EV-A to EV-D) and three rhinovirus (RV) species (RV-A to RV-C) (Mbani et al., 2024). The EV group comprises highly contagious viruses that can lead to a wide spectrum of diseases, such as hand, foot, and mouth disease (HFMD), neurological disorders (e.g., encephalitis and aseptic meningitis), cardiac complications (e.g., myocarditis), ocular infections (e.g., acute hemorrhagic conjunctivitis), and respiratory and gastrointestinal infections. Although the majority of EV infections are subclinical or self-limiting, they may result in life-threatening complications in vulnerable populations including neonates, infants, and immunocompromised hosts (Khetsuriani et al., 2006; Xie et al., 2024).

EVs are non-enveloped viruses characterized by a positive-sense, single-stranded RNA genome enclosed within an icosahedral protein capsid (Rossmann, 1994). The EV capsid adopts an icosahedral symmetry, composed of 60 tightly packed protomers. Each protomer consists of four structural proteins: viral polypeptide 1 (VP1), VP2, VP3, and VP4. Among these, VP1-VP3 are surface-exposed, forming the outer capsid shell, while VP4 is internally positioned and functions as a structural stabilizer (Rossmann, 1994). The viral genome is a single-stranded RNA molecule ranging from 7,100 to 7,450 nucleotides (nt) in length (Kitamura et al., 1981). The genome features an open reading frame (ORF) bounded by structured 5’ and 3’ untranslated regions (UTRs), encoding a polyprotein processed into four structural (VP1-VP4) and seven nonstructural proteins (Wang et al., 2012). Recent genomic analyses have identified an additional open reading frame (ORF2) in certain EV strains, which encodes the ORF2p protein (Guo et al., 2019). This novel viral factor has been demonstrated to play a crucial role in facilitating viral replication within intestinal epithelial cells.

Through phylogenetic analysis of VP1 sequences, 116 distinct genotypes have been classified within the EV-A to EV-D groups. The distribution of these genotypes is as follows: EV-A contains 25 genotypes, EV-B encompasses 63 genotypes, EV-C includes 23 genotypes, and EV-D comprises 5 genotypes (Nix et al., 2006; Liu, 2017; Simmonds et al., 2020).

EVs infection produces pathogen-associated molecular patterns (PAMPs) that are detected by epithelial pattern recognition receptors (PRRs), including toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (Wei et al., 2024). Upon viral RNA recognition, TLRs activate immune responses through two distinct signaling cascades: the myeloid differentiation primary response protein 88 (MyD88)-mediated pathway and the toll/interleukin (IL)-1 receptor domain-containing adaptors inducing interferon (IFN)-β (TRIF)-dependent pathway (Takeda and Akira, 2015). TLR7/9 engagement initiates MyD88-dependent signaling through death domain-mediated recruitment of IL-1 receptor-associated kinase 4 (IRAK4), which phosphorylates IRAK1. Activated IRAK-1 subsequently binds tumor necrosis factor receptor-associated factor 6 (TRAF6), triggering downstream cascades that ultimately induce nuclear factor kappa-B (NF-κB) nuclear translocation and inflammatory gene expression (Takeda and Akira, 2004). Conversely, TLR3 activates a distinct TRIF-dependent pathway where TRIF recruits TRAF3 to scaffold TANK-binding kinase 1(TBK1)/inhibitor of κB kinase ϵ (IKKϵ) complexes. These non-canonical IκB kinases phosphorylate interferon (IFN) regulatory factors (IRF) 3/7, driving type I IFN production (Takeda and Akira, 2015; Ma et al., 2023). RLRs recruit mitochondrial antiviral-signaling protein (MAVS) (Hou et al., 2011), which then engages TRAF3 and TRAF6 via its proline-rich region domain. This interaction triggers the activation of both the TBK1 and IKK complexes, initiating downstream antiviral signaling (Ren et al., 2020).

The third major PRR family comprises NLRs, known to play a central role in mediating inflammatory responses against viral infections. NLRs primarily function as inflammasome sensors that detect both PAMPs and damage-associated molecular patterns (DAMPs) (Hu and Chai, 2023). This recognition triggers inflammasome assembly, leading to caspase-1 activation and subsequent maturation of IL-1β, IL-18, and gasdermin D (GSDMD), thereby driving inflammatory responses and pyroptotic cell death (Chou et al., 2023). The 2B protease (2B^pro) encoded by multiple EVs species directly interacts with NLR pyrin domain containing 3 (NLRP3), facilitating the recruitment and subsequent oligomerization of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD) adaptor protein (ASC). This interaction promotes NLRP3 inflammasome assembly and activation, ultimately inducing IL-1β maturation and secretion while initiating pyroptotic cell death (Wang et al., 2022). The 3C^pro of multiple EVs, including RV, coxsackievirus B3 (CV-B3), and EV-A71, specifically cleave human NLRP1, thereby activating the NLRP1 inflammasome and subsequently promoting the secretion of proinflammatory cytokines including IL-1β and IL-18 (Robinson et al., 2020; Tsu et al., 2021).

Cyclic GMP-AMP synthase (cGAS), a newly characterized PRR, detects cytoplasmic viral DNA and mitochondrial DNA, serving as a crucial mediator of innate antiviral immune responses (Sun et al., 2013). Notably, certain EVs, including EV-A71, EV-D68, and CV-A16, induce mitochondrial damage during infection. The resulting release of mitochondrial DNA (mtDNA) activates the cGAS-stimulator of IFN genes (STING) pathway, triggering IFN production (Zheng et al., 2023). Furthermore, TRAF3 has been identified as a critical mediator in this antiviral signaling cascade. A separate study indicates that the 2B^pro of EV-A71 and CV-A16 triggers mitochondrial permeability transition pore (mPTP) opening, leading to mtDNA release, which activates the cGAS-STING pathway and subsequently enhances type I IFN production, thereby exerting antiviral effects (Liu et al., 2023).

Functioning as key mediators of antiviral immunity, IFNs exert their protective effects via specific receptor complexes: type I (type I IFN receptor 1 (IFNAR1) and type I IFN receptor 2 (IFNAR2)), type II (IFN-γ receptor 1(IFNGR1) and IFN-γ receptor 2 (IFNGR2)), and type III (IFN-λ receptor 1 (IFNLR1) and IL-10 receptor 2 (IL-10R2)) (Schroder et al., 2004; De Weerd et al., 2007; Zhou et al., 2011). These receptors initiate Janus activated kinase (JAK)-signal transducers and activators of transcription (STAT) signaling cascades that ultimately induce the expression of hundreds of interferon-stimulated genes (ISGs), thereby establishing a multifaceted antiviral state (Wei et al., 2024). For example, the type I IFN induces protein kinase R (PKR) and oligoadenylate synthetase (OAS) expression, which collectively mediate antiviral defense through distinct mechanisms: PKR-mediated eukaryotic translation initiation factor 2 (eIF-2α) phosphorylation halts viral translation (Gao et al., 2022), while OAS-dependent ribonuclease L (RNase L) activation cleaves cytosolic viral RNA (Drappier and Michiels, 2015). The resulting viral RNA cleavage products activate melanoma differentiation-associated antigen 5 (MDA5) (a member of the RLR family), triggering IFN production (Chakrabarti et al., 2011).

NF-κB serves as a master regulator of virus-induced inflammation. The IKK complex—comprising catalytic subunits IKKα/IKKβ and the regulatory component NF-κB essential modulator (NEMO, also known as IKKγ)—precisely controls NF-κB activation through phosphorylation-dependent degradation of Inhibitors of NF-κB (IκB) (Barnabei et al., 2021). The 2C^pro of EV-A71, PV, CV-A16, and CV-B recruit protein phosphatase 1 (PP1) to form a ternary 2C-PP1-IKKβ inhibitory complex that suppresses NF-κB signaling through inhibiting IKKβ phosphorylation (Li et al., 2016). EV-A71 2C^pro exploits IKKβ as a scaffold to compartmentalize IKKα into viral inclusion bodies (IBs), thereby disrupting NF-κB signaling without direct IKKα interaction (Ji et al., 2021). The p65/p50 heterodimer represents the predominant and functionally critical NF-κB configuration. Viral 2C^pro from PV1, PV2, CV-B1, EV-D68, and EV-A71 allosterically disrupt heterodimer formation through specific interactions with the IPT domain of p65 (Du et al., 2015). EV-A71-mediated Sox4 expression inhibits IKKα/β kinase activity via TAD domain binding, resulting in decreased IκBα phosphorylation and delayed NF-κB nuclear translocation (Shang et al., 2021). Both EV-A71 and EV-D68 employ their 3C^pro to cleave IRF7, thereby suppressing interferon production (Lei et al., 2013; Xiang et al., 2016). However, their 3C^pro recognizes different cleavage sites on IRF7 ( Figure 1 ).

EVs employ diverse strategies to evade host antiviral responses, particularly through interference with IFN-mediated signaling. EV-A71 demonstrates multiple approaches to inhibit type I IFN-mediated signaling. One key mechanism involves the viral 2A^pro, which reduces IFNAR1 levels in a protease-dependent manner by upregulating LDL-receptor-related protein-associated protein 1 (LRPAP1), a ligand that binds IFNAR1’s extracellular domain, promoting its degradation and ubiquitination (Lu et al., 2012; Li et al., 2023). However, studies in human embryonic lung fibroblasts and rhabdomyosarcoma cells reveal an alternative pathway: EV-A71 infection suppresses IFN-mediated signaling by downregulating JAK1 independently of viral 2A^pro and 3C^pro or the cellular proteasome (Liu et al., 2014). The formation of the ISG Factor 3 (ISGF3) complex (comprising phosphorylated STAT1, STAT2, and IRF9) is critical for IFN signaling, but EV-A71 3C^pro cleaves IRF9, disrupting this complex (Hung et al., 2011; Nowicka et al., 2023). Additionally, STAT1 nuclear translocation relies on karyopherin-α1 (KPNA1), which EV-A71 degrades via caspase3 activation (Wang et al., 2017). While both 2A^pro and 3D^pol impair IFNγ signaling by blocking STAT1 nuclear transport, their mechanisms differ: 2A^pro reduces STAT1 expression, whereas 3D^pol diminishes its phosphorylation (Wang et al., 2015). Notably, other EVs like PV and EV-D68 share similar immune evasion strategies, employing 3C^pro to cleave STAT1 and block its nuclear translocation, thereby inhibiting JAK/STAT signaling (Li et al., 2024). Further modulating immune responses, EV-A71 exploits the suppressor of cytokine signaling (SOCS) proteins—endogenous inhibitors of JAK/STAT signaling. Early infection triggers SOCS1/3 expression via the NF-κB pathway, suppressing STAT3 phosphorylation and thereby dampening IFN-mediated antiviral defenses (Linossi and Nicholson, 2015; Gao et al., 2020). In contrast, EV-D68 has evolved an alternative mechanism involving upregulation of the transcriptional regulator regulatory factor X 7 (RFX7), which specifically enhances SOCS3 expression to inhibit STAT3 phosphorylation and subsequent IFN-β-induced ISG products (Zhang et al., 2023) ( Figure 2 ).

EVs have evolved sophisticated strategies to counteract the antiviral functions of ISG products, thereby enhancing their replication efficiency. One such ISG transcription protein, the zinc-finger antiviral protein (ZAP), demonstrates robust antiviral activity against EV-A71. However, EV-A71 effectively evades this defense mechanism by utilizing its 3C^pro to cleave ZAP in a protease-dependent manner. This cleavage generates non-functional fragments that no longer inhibit viral replication (Xie et al., 2018). Moreover, the 3C^pro of several EVs, including EV-A71, EV-D68, CV-B3, and CV-A6, but not CV-A16, mediates the proteolytic cleavage of OAS3. Specifically, EV-A71’s 3C^pro targets OAS3 at the Gln982-Gly983 site, thereby disrupting its antiviral function (Zhou et al., 2022). Similarly, EV-A71’s 3C^pro cleaves PKR at Gln188-Ser189, producing an N-terminal fragment that, counterintuitively, promotes viral replication (Chang et al., 2017). While PV also degrades PKR, the precise mechanism underlying this process remains to be elucidated (Black et al., 1989; Black et al., 1993). RNase L, an interferon-inducible antiviral effector, exists as an inactive monomer until viral infection triggers OAS-mediated 2’-5’ oligoadenylate (2-5A) production, which binds its ankyrin repeats to induce active dimerization and subsequent viral RNA degradation (Drappier and Michiels, 2015). Group C EVs utilize phylogenetically conserved RNA structural motifs that specifically impair RNase L’s catalytic function through competitive inhibition, while maintaining the enzyme’s ability to bind 2-5A (Townsend et al., 2008) ( Figure 2 ).

In the context of limited therapeutic options against the full spectrum of EV-induced diseases—from mild presentations to lethal cases—prophylactic vaccination emerges as the primary intervention for infection control. Polio vaccination efforts have achieved remarkable success in reducing the global disease burden. In 1988, approximately 350,000 cases were reported across 125 endemic countries. By 2012, the Americas, Western Pacific, and European regions had been certified as polio-free, with worldwide cases declining to just 650— representing a reduction exceeding 99% (M A, 2022). Currently, all remaining wild poliovirus cases globally are caused by serotype 1, while wild poliovirus types 2 and 3 have been officially declared eradicated (M A, 2022; Bandyopadhyay et al., 2024). Wild PV type 1 (WPV1) transmission persists in only a few endemic countries, predominantly Afghanistan and Pakistan. Surveillance data show these two nations reported 22 WPV1 polio cases in 2022, which declined to 12 cases in 2023 (Geiger et al., 2024). Polio-free nations must remain vigilant against potential resurgence. Systematic wastewater monitoring in five European nations (Finland, Germany, Poland, Spain, and UK) has identified poliovirus circulation since September 2024. While no paralytic cases have been reported, these environmental findings demonstrate the continued risk of poliovirus transmission worldwide (Esposito and Principi, 2018). The two polio vaccine formulations—oral polio vaccine (OPV, live-attenuated) and inactivated polio vaccine (IPV)—differ fundamentally in their protective mechanisms. OPV has a unique ability to replicate in the intestinal tract and induce superior mucosal immunity, making it significantly more effective than IPV at preventing wild-type virus transmission (Burns et al., 2014). However, OPV carries the risk of generating circulating vaccine-derived PVs (cVDPVs) through mutation and reversion to neurovirulent strains during intestinal replication (Burns et al., 2014). To address this issue, a novel oral type 2 polio vaccine (nOPV2) strain has been developed. This strain features targeted modifications to the Sabin genome, including structural optimization of the 5’-untranslated region (UTR) and fidelity-enhancing mutations in the viral 3D^pol (Yeh et al., 2020). These modifications collectively restrict viral evolutionary capacity while preventing reversion to neurovirulence (Yeh et al., 2020). HFMD is a highly prevalent communicable disease primarily caused by EV infections, notably EV-A71 and CV-A16, along with other human EV serotypes (Zhu et al., 2023). Epidemiological surveillance data consistently identify children under 5 years of age as the most vulnerable demographic group, exhibiting the highest disease susceptibility and clinical attack rates (Saguil et al., 2019). With its sophisticated surveillance network, China—the world’s most populous country—has maintained HFMD as a notifiable disease since 2008, accompanied by continuous pathogen surveillance (Esposito and Principi, 2018). Between May 2008 and June 2014, China reported a total of 10,717,283 HFMD cases with 3,046 fatalities, yielding a case fatality rate of 0.03% (Esposito and Principi, 2018). A comprehensive meta-analysis incorporating 23 epidemiological studies revealed that the average incidence rate of HFMD in China stands at 1.61 cases per 1000 population (Chen et al., 2021). In addition to PV vaccines, multiple EV-A71 inactivated vaccine candidates targeting diverse subtypes have progressed through clinical development, with Singapore’s Inviragen (B2 genotype) and Taiwan’s National Health Research Institutes (NHRI) (B4 genotype) advancing their formulations to clinical trials (Chang et al., 2012; Hwa et al., 2013), while three Chinese-developed vaccines from Sinovac Beijing, Vigoo Beijing, and the Chinese Academy of Medical Science (CAMS) (all C4 genotype) have already obtained market approval in China (Lu, 2014; Zhu et al., 2014; Guan et al., 2020; Liu et al., 2021). The Pichia pastoris-expressed EV-A71 virus-like particles (VLP) vaccine maintains authentic viral conformation without genetic material, exhibiting strong immunogenic potential in preclinical evaluations (Wang et al., 2021). The EV-A71 live-attenuated vaccine, engineered through VP1 codon deoptimization combined with high-fidelity 3D^pol substitutions, demonstrated potent immunogenicity in murine models by eliciting both cellular and humoral immune responses that conferred complete protection against lethal EV-A71 challenge in neonatal murine models (Hsieh et al., 2024). According to surveillance data from the European Non-Polio Enterovirus Network (ENPEN), a study conducted between 2021 and 2022 identified 10,481 enterovirus-positive samples (6.8% positivity rate) reported by 58 institutions across 19 European countries (Simoes et al., 2024). Among these, 1,004 cases (9.6%) were confirmed as EV-D68 infections. Clinical data analysis of 969 cases revealed that 78.9% of infections occurred in children aged 0–5 years. Inactivated vaccines candidate for EV-D68 have demonstrated the capacity to elicit potent neutralizing antibodies in preclinical animal studies (Zheng et al., 2020; Senpuku et al., 2024). In summary, vaccination remains the cornerstone of enterovirus infection control, with polio immunization programs demonstrating remarkable success in disease elimination. While effective vaccines exist for poliovirus and EV-A71, the persistent circulation of EVs (including environmental poliovirus detection and emerging strains like EV-D68) underscores the need for continued vaccine development, robust surveillance systems, and sustained immunization efforts—particularly for high-risk pediatric populations. The advancement of novel vaccine platforms (e.g., nOPV2, VLPs, and live-attenuated candidates) offers promising strategies to address safety and coverage gaps in current options.

EVs have evolved sophisticated mechanisms to evade host innate immune responses, ensuring their successful replication and spread. These strategies include the inhibition of PRRs, disruption of key signaling pathways, and direct targeting of IFN signaling components. The use of viral proteases such as 2A^pro and 3C^pro to cleave host proteins is a common theme in EVs immune evasion. Additionally, EVs manipulate host factors, such as Sox4 and miRNAs, to suppress antiviral responses. Despite these evasion tactics, the development of vaccines against EVs, such as inactivated and live-attenuated formulations, offers promising strategies for controlling infections. Future research should focus on elucidating novel immune evasion mechanisms and developing targeted antiviral therapies to combat EV-induced diseases.