Enterovirus infections in children

ABSTRACT

Enteroviruses (EVs), which include numerous types, are among the most common pathogens causing infections in children. EVs can cause diverse diseases, including herpangina, hand‐foot‐and‐mouth disease, myocarditis, pneumonia, meningitis, encephalitis, and acute flaccid paralysis. Severe cases may result in pediatric mortality, posing substantial threats to child health and public health. The diversity of EV types combined with rapid viral evolution and antigenic variation presents formidable challenges for EV prevention and control, with dominant genotypes being continuously replaced by emerging variants and novel subtypes. EV invades host cells through specific receptors and hijacks cellular machinery for self‐replication, engaging in complex interactions with the host while evolving multiple mechanisms to evade host immune responses. Currently, no specific anti‐EV agents are available, and treatment remains primarily supportive and symptomatic. Effective prevention and control strategies for EV are limited, and vaccines are currently available only against selected EV serotypes. This review systematically examines EV infections from multiple perspectives, including epidemiology, virology, viral entry mechanisms, virus‐host interactions, clinical diagnosis and management, prevention and control measures, with the aim of providing insights and references for research on EV pathogenesis, precision clinical diagnosis and treatment, and the development of antiviral therapies and vaccines.

Enteroviruses (EVs) are major pathogens causing a wide spectrum of diseases in children worldwide. This review summarizes the epidemiology, virology, viral replication, and virus‐host interactions of EVs. It also highlights current advances in clinical diagnosis, antiviral therapies, and vaccine development, providing insights into future strategies for the prevention and control of EV infections.

INTRODUCTION

Enteroviruses (EVs) pose serious threats to children's health and can cause a wide spectrum of diseases including neonatal sepsis, herpangina, hand, foot, and mouth disease (HFMD), acute hemorrhagic conjunctivitis (AHC), myocarditis, pneumonia, meningitis, encephalitis, and acute flaccid myelitis (AFM). Severe cases may lead to pediatric mortality and substantial public health concerns. ¹ , ² , ³

Currently, no specific anti‐EV agents are available, and treatment of EV infections remains primarily supportive and symptomatic. Effective prevention and control strategies for EV are limited, with vaccines currently available only for selected EV serotypes. This review summarizes current knowledge on the epidemiology, etiology, viral entry mechanisms, virus‐host interactions, clinical diagnosis and management, and prevention and control of EV infections, highlighting recent advances and future directions in EV research and intervention strategies.

OVERVIEW OF EV

Biological characteristics

EVs belong to the genus Enterovirus within the family Picornaviridae and are single‐stranded positive‐sense RNA viruses. The viral particles are spherical, approximately 27–30 nm in diameter, and are non‐enveloped viruses with an icosahedral symmetry structure. The capsid comprises 60 capsid protein units and contains four structural proteins (VP1–VP4). The viral genome, a single‐stranded positive‐sense RNA of ∼7500 nucleotides, is encapsidated within the particle and flanked by 5’ and 3’ untranslated regions (UTRs), with a poly (A) tail at the 3’ end and an open reading frame in the middle that encodes a polyprotein, which is subsequently proteolytically cleaved into 11 mature proteins. Notably, some EVs possess an upstream open reading frame in the 5’ UTR, which is involved in regulating viral infection and replication (Figure 1).

FIGURE 1.

Genome structure of enterovirus. 5’UTR, 5’ untranslated region; IRES, internal ribosome entry site; VPg, viral genome‐linked protein; P1/Capsid, capsid protein region (VP1–VP4); P2/non‐structural, non‐structural region (2A–2C); P3/non‐structural, non‐structural region (3A–3D); 3’UTR, 3’ untranslated region.

The genus Enterovirus comprises both EVs and rhinoviruses. ⁴ Based on differences in genomic sequence characteristics and biological properties, EVs are classified into 15 distinct species: Enterovirus alphacoxsackie (formerly Enterovirus A), Enterovirus betacoxsackie (formerly Enterovirus B), Enterovirus coxsackiepol (formerly Enterovirus C), Enterovirus deconjuncti (formerly Enterovirus D), Enterovirus eibovi (formerly Enterovirus E), Enterovirus fitauri (formerly Enterovirus F), Enterovirus geswini (formerly Enterovirus G), Enterovirus hesimi (formerly Enterovirus H), Enterovirus idromi (formerly Enterovirus I), Enterovirus jesimi (formerly Enterovirus J), Enterovirus krodeni (formerly Enterovirus K), Enterovirus lesimi (formerly Enterovirus L), Enterovirus alpharhino (formerly Rhinovirus A), Enterovirus betarhino (formerly Rhinovirus B), Enterovirus cerhino (formerly Rhinovirus C). Within the same species, different types of EVs meet the following defining criteria: 1) Share greater than 70% amino acid identity in the structural protein P1; 2) Share greater than 70% amino acid identity in the non‐structural proteins 2C + 3CD; 3) Share a limited range of host cell receptors; 4) Share a limited natural host range; 5) Have a genome base composition (G+C), which varies by no more than 2.5%; 6) Share a significant degree of compatibility in proteolytic processing, replication, encapsidation, and genetic recombination.

The types of EVs that infect humans within the traditional classification of EVs, such as poliovirus, coxsackievirus, echovirus, and novel EV serotypes, are mainly distributed in four species: Enterovirus alphacoxsackie, Enterovirus betacoxsackie, Enterovirus coxsackiepol, and Enterovirus deconjuncti (Table 1). Rhinoviruses include three species: Enterovirus alpharhino, Enterovirus betarhino, and Enterovirus cerhino (Table 1). Rhinoviruses mainly cause respiratory infections, and there are differences in their epidemiological characteristics and prevention and control strategies compared to traditional EVs. This article will not discuss rhinovirus infection. Antigenicity and pathogenicity vary among different EV types.

TABLE 1.

Classification of human enteroviruses

Species (Former name)	Types
Enterovirus alphacoxsackie (Enterovirus A)	CVA2–8, CVA10, CVA12, CVA14, CVA16, EV‐A71, EV‐A76, EV‐A89–92, EV‐A114, EV‐A119–125
Enterovirus betacoxsackie (Enterovirus B)	CVB1–6, CVA9, E1–7, E9, E11–21, E24–27, E29–33, EV‐B69, EV‐B73–75, EV‐B77–88, EV‐B93, EV‐B97, EV‐B98, EV‐B100, EV‐B101, EV‐B106, EV‐B107, EV‐B110–114
Enterovirus coxsackiepol (Enterovirus C)	PV‐1–3, CVA1, CVA11, CVA13, CVA17, CVA19–22, CVA24, EV‐C95, EV‐C96, EV‐C99, EV‐C102, EV‐C104, EV‐C105, EV‐C109, EV‐C113, EV‐C116–118
Enterovirus deconjuncti (Enterovirus D)	EV‐D68, EV‐D70, EV‐D94, EV‐D111, EV‐D120
Enterovirus alpharhino (Rhinovirus A)	RV‑A1, RV‑A2, RV‑A7–13, RV‑A15, RV‑A16, RV‑A18–25, RV‑A28–34, RV‑A36, RV‑A38–41, RV‑A43, RV‑A45–47, RV‑A49–51, RV‑A53–68, RV‑A71, RV‑A73–78, RV‑A80–82, RV‑A85, RV‑A88–90, RV‑A94, RV‑A96, RV‑A100–109
Enterovirus betarhino (Rhinovirus B)	RV‑B3–6, RV‑B14, RV‑B17, RV‑B26, RV‑B27, RV‑B35, RV‑B37, RV‑B42, RV‑B48, RV‑B52, RV‑B69, RV‑B70, RV‑B72, RV‑B79, RV‑B83, RV‑B84, RV‑B86, RV‑B91–93, RV‑B97, RV‑B99–106
Enterovirus cerhino (Rhinovirus C)	RV‑C1–57

Abbreviations: CV, coxsackievirus; E, echovirus; EV, enterovirus; PV, poliovirus; RV, rhinovirus.

Epidemiology

EV is prevalent worldwide, and the diseases caused by EV infection are collectively referred to as enteroviral diseases. In tropical and subtropical regions, EV infection can occur throughout the year; in temperate regions, it exhibits seasonal patterns with markedly higher incidence in summer and autumn compared to winter and spring. Patients and asymptomatic carriers are the main sources of EV transmission.

EV is mainly transmitted via the fecal–oral route (e.g., through contaminated hands, food, or fomites). A small proportion of patients are infected through respiratory droplets, whereas specific serotypes such as EV‐D68 are mainly transmitted through the respiratory route. ⁵ In crowded settings such as households and schools, contact transmission can easily lead to rapid spread of EV infection, and contamination of water or food can trigger EV outbreaks.

The population is generally susceptible to EV, and only serotype‐specific immunity develops after EV infection. Children under 5 years of age are at high risk of symptomatic and severe EV infection due to their immature immune systems. Immunocompromised individuals are also at high risk of EV infection.

Genetic variation and recombination

The RNA‐dependent RNA polymerase of EV lacks proofreading capability, resulting in a high mutation rate. In addition to point mutations, genetic recombination represents another crucial mechanism driving EV evolution. ⁶ , ⁷ When different EV types or distinct EV strains of the same serotype co‐infect host cells, their genomes can undergo recombination during replication. Such exchange may occur between isogenic strains (intraserotype recombination) or across serotype boundaries (interserotype recombination). ⁸ Recombination serves as the core driver of EV evolution. Since the C4 genotype of EV‐A71 became the predominant circulating strain in China in 2004, it has generated multiple complex evolutionary branches through continuous interserotype recombination. ⁹ This “modular” genetic exchange produces genetic diversity more efficiently than point mutations, enabling EV to rapidly adapt to changes in the host environment.

MECHANISMS OF EV INVASION AND IMMUNOLOGICAL INTERACTIONS WITH HOST CELLS

Invasion and replication in host cells

The replication cycle of EV in host cells encompasses six sequential stages: attachment, entry, uncoating, genome replication and translation, viral assembly, and release (Figure 2).

FIGURE 2.

Mechanisms of enterovirus invasion into host cells. After receptor binding and endocytosis, the virion releases its (+) RNA genome into the cytoplasm. The genome is covalently linked to VPg (viral genome‐linked protein) and translated into a polyprotein that is cleaved into non‐structural proteins (2A–2C, 3A–3D) and capsid proteins (VP0, VP1, VP3). 3Dpol drives RNA replication by synthesizing a (−) RNA template for new (+) RNA on membranous replication organelles. Nascent genomes are packaged into procapsids, generating provirions that become infectious mature virions after VP0 cleavage into VP4 and VP2. Virions are released via non‐lytic extracellular vesicles or cell lysis. (Created by Figdraw).

Attachment and entry

Binding of viral particles to cellular receptors represents a critical step in EV infection, as the complexity and specificity of this interaction determine the viral host range, tissue tropism, and pathogenic mechanisms. EV receptors exhibit considerable diversity (Table 2) and can be functionally classified into attachment receptors and uncoating receptors. ¹⁰ , ¹¹ , ¹² Different EV species utilize distinct attachment receptors; for instance, EV‐D70 and coxsackievirus A (CVA) 24 primarily depend on sialic acid as the attachment receptor, whereas CVA21 employs decay‐accelerating factor (DAF) as its attachment receptor. ¹⁰ , ¹³ , ¹⁴ , ¹⁵ Certain receptors exhibit “dual functionality,” mediating both attachment and triggering or facilitating uncoating. For example, the human neonatal Fc receptor (FcRn) plays dual roles in viral entry during E18 infection, simultaneously facilitating attachment and promoting uncoating. ¹⁶

TABLE 2.

Receptors for enterovirus

Receptor	Virus
PVR (CD155)	PV
SCARB2, ANXA2, DC‐SIGN, nucleolin, vimentin, WARS, heparan sulfate, and sialic acid	EV‐A71
PSGL1	EV‐A71‐PB, CVA2/7/10/14/16
LDLR, VLDLR, and LRP	RV (minor)
ICAM1	RV (major), CVA21/24
CDHR3	RV C
KREMEN1	CVA2/3/4/5/6/10/12
SCARB2	CVA7/14/16
DAF	CVA21, CVB1/3/5, E‐3/6/7/11/12/13/19/20/21/25/29
CAR (CXADR)	CVB1/2/3/4/5/6
Integrin αVβ3	CVA9, E‐1/9
Integrin α2β1 (VLA2)	E‐1/8
Heparan sulfate	E‐5
FcRn	Echovirus
ICAM5, MFSD6, sGAGs, and sialic acid	EV‐D68
Sialic acid	EV‐D70, CVA24v

Abbreviations: ANXA2, annexin A2; DC‐SIGN, dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing non‐integrin; CAR (CXADR), coxsackievirus and adenovirus receptor (CXADR Ig‐like cell adhesion molecule); CDHR3, cadherin‐related family member 3; CV, coxsackievirus; CVA24v, variant strain of coxsackievirus 24; DAF, decay‐accelerating factor; E, echovirus; EV, enterovirus; EV‐A71‐PB, PSGL‐1‐binding EV‐A71; FcRn, neonatal Fc receptor; ICAM5, intercellular adhesion molecule 5; integrin α2β1 (VLA2), integrin alpha‐2 beta‐1 (very late antigen‐2); integrin αVβ3, integrin alpha‐V beta‐3; ICAM1, intercellular adhesion molecule 1; KREMEN1, kringle containing transmembrane protein 1; LDLR, low‐density lipoprotein receptor; LRP, low‐density lipoprotein receptor‐related protein; MFSD6, major facilitator superfamily domain‐containing 6; PSGL1, P‐selectin glycoprotein ligand‐1; PV, poliovirus; PVR (CD155), poliovirus receptor (cluster of differentiation 155); RV, rhinovirus; SCARB2, scavenger receptor class B member 2; sGAGs, sulfated glycosaminoglycans; VLDLR, very‐low‐density lipoprotein receptor; WARS, tryptophanyl‐tRNA synthetase 1.

Following high‐affinity binding between the viral capsid and its cognate receptor, the receptor‐virus complex undergoes lateral aggregation on the plasma membrane, triggering endocytic signaling that recruits adaptor protein complex 2 and clathrin to form coated pits. Subsequently, under dynamin‐mediated catalysis, the neck region undergoes scission, generating coated vesicles that complete internalization and enter the early endosomal trafficking and sorting pathway. ¹⁷ , ¹⁸ , ¹⁹ Additionally, certain serotypes can alternatively or complementarily exploit caveolae/lipid raft‐mediated endocytosis and macropinocytosis as entry routes. ²⁰ , ²¹ Collectively, receptor recognition coupled with clathrin‐mediated endocytosis constitutes the principal mechanism of EV cellular invasion, establishing the necessary spatiotemporal foundation for subsequent host‐viral interactions and lifecycle progression.

Uncoating

Following viral capsid binding to its receptor, conformational changes in capsid proteins are triggered, concurrently leading to the release of internal lipid molecules, known as “pocket factors,” that maintain viral particle stability, generating A‐particles (135S intermediates) characterized by expanded capsids and enlarged pores. ²² Subsequently, under the influence of endosomal microenvironmental factors (acidification and ionic alterations), A‐particles undergo further conformational rearrangement, during which the internal VP4 protein everts and, together with the N‐terminal region of VP1, inserts into the endosomal membrane to form a transmembrane channel. This channel facilitates the translocation of the positive‐sense RNA genome through the pore at the pentamer vertex into the cytoplasm, thereby accomplishing genome release. ²³ Concurrently, certain capsid proteins (VP4‐depleted, VP1‐everted) dissociate from the receptor, generating empty B‐particles (80S). ²³ , ²⁴ , ²⁵ Additionally, several host factors participate in EV uncoating; for instance, the adipose‐specific phospholipase A2 facilitates the translocation of the viral genome from endocytic vesicles into the cytoplasm. ²⁶

The uncoating of different viral species is triggered by their cognate receptors. For example, EV‐A71 binds its VP1 “canyon” region to specific receptors (such as scavenger receptor class B member 2 and P‐selectin glycoprotein ligand‐1) to initiate uncoating (Table 2), whereas EV‐D68 binds to the major facilitator superfamily‐domain‐containing protein 6, and echovirus interacts with the human FcRn. ²⁷ , ²⁸ , ²⁹ , ³⁰ , ³¹

Replication and translation

Replication of EV occurs within virus‐induced replication organelles (ROs), which are primarily derived from endoplasmic reticulum and Golgi membranes. ³² During infection, EV extensively remodels host intracellular membranes through the coordinated actions of viral non‐structural proteins, including 2B, 2C, and 3A, thereby promoting the formation of ROs that provide a protected platform for viral RNA synthesis. ³³ These membranous structures not only concentrate viral replication factors and RNA templates but also protect viral replication intermediates from host innate immune recognition. In addition, EV hijacks host lipid metabolism and membrane trafficking pathways to facilitate RO biogenesis and function. Specifically, viral proteins recruit phosphatidylinositol 4‐kinase IIIβ (PI4KB) to replication sites, resulting in the accumulation of phosphatidylinositol 4‐phosphate (PI4P)‐enriched membranes. These membranes promote cholesterol accumulation through oxysterol‐binding protein (OSBP)‐mediated lipid exchange. ³⁴ , ³⁵ , ³⁶ Collectively, this lipid remodeling process creates a favorable microenvironment for the assembly and activity of the viral replication complex, thereby enhancing efficient viral RNA synthesis.

Following entry of the EV genome into the cytoplasm, protein synthesis is facilitated by the internal ribosome entry site (IRES)‐mediated translation mechanism. This process is driven by IRES elements located within the 5’UTR of the viral genome, which bypass the conventional 5’ cap‐dependent translation initiation and directly recruit ribosomes to initiate translation, thereby synthesizing essential viral proteins required for genome replication. Concurrently, EV modulates the classical translation machinery of the host cell, effectively suppressing cellular protein synthesis and thereby ensuring preferential and efficient synthesis of viral proteins. ²⁶ , ³⁷

Viral replication is catalyzed by the 3D polymerase as the catalytic core enzyme, which utilizes the viral genome‐linked protein (VPg) as a primer and initiates RNA chain synthesis and elongation under the coordination of viral proteins such as 3AB. The 2C protein functions as a helicase and RNA chaperone, promoting template strand separation and maintaining the stability of the replication complex. ²⁶ Additionally, multiple host RNA‐binding proteins, including PCBP2, PABP1, and HNRNPC, participate in this intricate process. ³⁸

Recent genome‐wide functional studies, including RNA interference and CRISPR‐Cas9‐based screening approaches, have substantially advanced the understanding of host‐EV interactions. ³⁹ , ⁴⁰ These studies have identified numerous host dependency factors involved in viral replication, membrane remodeling, and intracellular trafficking, as well as antiviral restriction factors that suppress EV infection. Host factors such as acyl‐coenzyme A binding domain containing 3 (ACBD3), Golgi‐specific brefeldin A‐resistant guanine nucleotide exchange factor 1, and ADP‐ribosylation factor 1 play critical roles in RO biogenesis and viral RNA synthesis. ⁴¹ , ⁴² , ⁴³ Moreover, several interferon‐stimulated genes (ISGs) and antiviral restriction factors, including the interferon‐induced proteins with tetratricopeptide repeats family proteins and the 2’‐5’‐oligoadenylate synthetase/RNase L pathway, can inhibit EV replication through diverse antiviral mechanisms. ⁴⁴ , ⁴⁵ Collectively, these findings provide a more comprehensive understanding of the complex interplay between EV and host cellular pathways, thereby offering potential targets for antiviral therapeutic development.

Assembly and release

EV assembly initiates with the cleavage of the viral protein precursor P1 by the viral 3CD protease, assisted by heat shock protein 90, generating structural proteins VP0, VP3, and VP1. These proteins self‐assemble to form the provisional viral capsid structure. ⁴⁶ Subsequently, the viral genomic RNA directly interacts with capsid proteins through its conserved sequences and is precisely packaged into the capsid interior. The final maturation of viral particles is accompanied by the autoproteolytic cleavage of VP0 into VP2 and VP4, a process that triggers conformational rearrangement of the capsid proteins, ultimately forming infectious icosahedral virus particles. ⁴⁷

The release mechanisms of EV exhibit considerable diversity, encompassing both lytic pathways leading to host cell rupture and non‐lytic mechanisms associated with cellular autophagy and related processes. ⁴⁸ , ⁴⁹ , ⁵⁰ Further studies have revealed that following EV‐A71 infection, infected cells undergo selective extrusion from the apical surface of human intestinal organoids, thereby facilitating infection of adjacent cells. ⁵¹

Innate immune response to EV infection

Upon entering the host, EV triggers a rapid innate immune response primarily through the recognition of viral RNA by pattern recognition receptors (PRRs), principally Toll‐like receptors and retinoic acid‐inducible gene I‐like receptors. These PRRs initiate signaling cascades via adaptor proteins such as mitochondrial antiviral signaling protein (MAVS) and toll/interleukin‐1 receptor domain‐containing adaptor‐inducing interferon‐β (TRIF), leading to the activation of transcription factor interferon regulatory factor 3, interferon regulatory factor 7, and nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB). These factors translocate to the nucleus and drive the production of type I interferons as well as pro‐inflammatory cytokines. ⁵² , ⁵³ Secreted interferons subsequently induce the expression of hundreds of ISGs through the Janus kinase (JAK)‐signal transducer and activator of transcription (STAT) signaling pathway, thereby suppressing viral replication. ⁵² , ⁵³ Additionally, EV infection induces mitochondrial damage and promotes the release of mitochondrial DNA, thereby activating cyclic GMP‐AMP synthase‐stimulator of interferon genes ‐mediated antiviral immune responses. ⁵⁴ Concurrently, host innate immune cells, including macrophages and natural killer cells, contribute to viral clearance through phagocytosis and cytokine secretion. ⁵⁵ , ⁵⁶

In turn, EV has evolved highly effective countermeasures against this host defense. The viral proteases 2A and 3C serve as the primary effectors, cleaving critical host proteins involved in the innate immune response. These proteases target PRRs like melanoma differentiation‐associated gene 5, adaptor molecules such as MAVS and TRIF, and transcription factors including interferon regulatory factor 7, thereby blocking interferon production at multiple levels. ⁵⁷ , ⁵⁸ , ⁵⁹ , ⁶⁰ , ⁶¹ , ⁶² , ⁶³ , ⁶⁴ , ⁶⁵ In the downstream signaling pathways of interferon, EV suppresses JAK‐STAT signal transduction and the expression of ISGs by downregulating interferon alpha and beta receptor subunit 1 expression and cleaving interferon regulatory factor 9. ⁶⁵ , ⁶⁶ Furthermore, EV targets inflammatory pathways by degrading the transforming growth factor‐activated kinase 1 complex through its 3C protein, inhibiting the phosphorylation of IκB kinase β/ NF‐κB inhibitor αvia its 2C protein, or cleaving NOD‐like receptor protein 3, thereby suppressing NF‐κB activation and the production of inflammatory cytokines. ⁶⁷ , ⁶⁸ , ⁶⁹ These strategies collectively attenuate host immune defense, promoting viral survival and dissemination.

Adaptive immune response to EV infection

Following EV infection, cytotoxic T lymphocytes (CD8+) recognize and eliminate infected cells through dendritic cell‐mediated cross‐presentation mechanisms, with a subset of T cells differentiating into memory cells. ⁷⁰ Helper T cells (CD4+) interact with antigen‐presenting cells and differentiate into distinct subpopulations including Th1, Th2, and Th17 cells, which coordinately regulate the immune response. ⁷¹ B cell‐derived antibodies (immunoglobulin M [IgM], IgG, and IgA) not only neutralize viruses and activate the complement system, but also mediate tissue‐specific immune defense through immunoglobulin receptor‐mediated transport in diverse anatomical compartments. ⁷² , ⁷³ , ⁷⁴ EV‐specific IgM antibodies persist for 7–10 days, followed by the emergence of IgG antibodies; neutralizing antibody responses appear approximately at the second week post‐infection and reach peak titers at approximately five weeks post‐infection, and may persist lifelong. ⁷⁵ , ⁷⁶

LABORATORY DIAGNOSIS OF EV INFECTIONS

The diagnosis of EV infection primarily involves virus isolation, serological testing, and polymerase chain reaction (PCR)‐based molecular methods. Serological testing (IgM/IgG) is more relevant to epidemiologic studies than to clinical diagnosis. Driven by the need for rapid, scalable diagnostics, traditional methods such as viral culture are increasingly supplemented by molecular and serological assays. ⁷⁷ Moreover, the collection of appropriate clinical specimens is critical for the laboratory confirmation of EV infection.

Specimen collection

The selection of specimens, including throat swab, rectal swab, feces, plasma/serum, and cerebrospinal fluid (CSF), should be guided by the clinical manifestations for the diagnosis of EV infection. For example, CSF is the optimal specimen for diagnosing meningitis and encephalitis. However, if CSF is collected 2 days after symptom onset, the positivity rate becomes very low, and serological testing can provide more accurate results during the early stage. ⁷⁸

Saliva and oral fluid have recently emerged as non‐invasive alternative specimens for EV detection, particularly in pediatric populations. Compared with conventional sampling methods, saliva collection is simple, painless, and suitable for repeated sampling and large‐scale surveillance. A previous study reported sensitivities of 71% (95% confidence interval [CI], 60%–81%) for saliva samples and 48% (95% CI, 36%–60%) for mouthwash samples in the detection of rhinovirus or EV, suggesting that oral‐fluid‐based testing may serve as a useful complementary diagnostic approach. ⁷⁹

Diagnostic methods

Viral culture

Primary cell lines (e.g., human embryonic kidney cells) and continuous cell lines (e.g., rhabdomyosarcoma, Hep‐2, and HeLa cells) are widely used, with sensitivity varying by serotype. Classic cytopathic effect manifests as cell rounding, cytoplasmic vacuolization, and detachment within 3–7 days post‐inoculation. However, certain strains (e.g., EV‐D68) induce subtle cytopathic effect, necessitating prolonged observation or immunofluorescence staining for confirmation. ⁸⁰

Serological testing

IgM detection is employed in acute‐phase diagnosis, particularly during outbreaks. Commercial IgM‐capture ELISA kits targeting conserved epitopes show 90%–95% sensitivity for EV‐A71 and 65%–75% sensitivity for CVA16 but suffer from cross‐reactivity with other members of the Picornaviridae family. ⁸¹ A positive IgM result generally suggests recent or acute EV infection; however, false‐positive results may occur because of serological cross‐reactivity. Conversely, negative serological results in the early stage of infection do not rule out EV infection, as antibody production may not yet be detectable.

Molecular diagnostics

Molecular methods have become indispensable for EV diagnostics due to their rapid turnaround time, high sensitivity, and ability to address viral genetic diversity.

Reverse transcription‐PCR (RT‐PCR) targeting conserved genomic regions (e.g., 5’UTR) has become the cornerstone of molecular detection of EVs, offering >95% sensitivity and a turnaround time of <6 hours. Broad‐spectrum primers targeting 5’UTR enable pan‐EV detection, while VP1‐specific primers facilitate serotyping. Multiplex RT‐PCR panels allow simultaneous detection of the pan‐EV and different EV serotypes alongside other pathogens, thereby enhancing clinical utility. ⁸² Nevertheless, several challenges remain in the RT‐PCR detection of EVs, including primer mismatches caused by EV genetic diversity and false negatives in cases of low viremia. Positive RT‐PCR or quantitative RT‐PCR (qRT‐PCR) results usually indicate active viral replication and provide strong evidence for current EV infection. However, negative molecular results do not completely rule out infection, particularly when viral load is low, specimen collection is delayed, or inappropriate specimen types are used. Therefore, interpretation of molecular findings should be combined with clinical manifestations and epidemiological information.

qRT‐PCR enhances sensitivity and enables viral load quantification. The use of TaqMan probes labeled with distinct fluorophores (FAM, HEX, and Cy5) targeting VP4/VP2 regions improves specificity and allows co‐detection and differentiation of EV serotypes. ⁸² In addition, RNAscope in situ hybridization combined with qRT‐PCR and spatial transcriptomics has been employed to map EV RNA within infected tissues. ⁸³

Metagenomic next‐generation sequencing (mNGS) enables unbiased detection of EV and discovery of novel strains, and it can identify EVs, co‐infections with other pathogens, and novel variants in a single sequencing run. ⁸⁴ Nevertheless, detection of EV sequences by mNGS should be interpreted cautiously, as the presence of viral nucleic acids does not always indicate causative pathogenicity.

EV INFECTION AND RELATED DISEASES IN CHILDREN

EV infections exhibit broad clinical heterogeneity, ranging from asymptomatic infection or mild self‐limiting illness to severe diseases involving the central nervous system (CNS), heart, liver, respiratory tract, and other organs. The major EV‐associated diseases in children include HFMD, herpangina, encephalitis, meningitis, acute flaccid paralysis (AFP), myocarditis, hepatitis, respiratory tract infections, and AHC. The following sections summarize the epidemiological and clinical characteristics of these common EV‐related diseases in children.

EV infection in neonates

The immature immunity in neonates renders them susceptible to EV infection and at high risk for critical illness. The clinical manifestations of neonatal EV infection exhibit considerable heterogeneity; although the majority present as asymptomatic infection or fever of undetermined origin with a favorable prognosis, a minority can manifest as encephalitis/meningitis, arrhythmia, myocarditis, hepatitis accompanied by acute hepatic failure, sepsis‐like syndrome with multiple organ and system dysfunction, and coagulopathy. ⁸⁵ , ⁸⁶ , ⁸⁷ In rare instances, neonatal EV infection progresses fulminantly, leading to sudden septic shock or disseminated intravascular coagulation (DIC); once DIC occurs, the mortality rate approaches 100%. ⁸⁸ , ⁸⁹ However, large‐scale epidemiological and disease burden studies on neonatal EV infection remain limited.

A clinical study from China investigating hospitalized neonates with fever during EV outbreak seasons revealed that 39.2% (131/334) of patients tested positive for EV nucleic acid in CSF or feces. ⁹⁰ A systematic review demonstrated that among all neonates with perinatal infections, the incidence of EV infection ranged from 4.6% to 46.1%, and 38.3% of those with severe perinatal infection showed confirmed EV infection upon placental pathological examination. ⁹¹ Xu et al. conducted EV nucleic acid detection on oropharyngeal swabs collected on admission for stable neonates in a level II neonatal intensive care unit from 2020 to 2021 (over a 1‐year period), revealing a positive rate of 9.1‰ (10/1095), while the rate rose to 18.6‰ (9/483) among those who were admitted due to confirmed infectious disease. ⁹² Therefore, special attention should be paid to the diagnosis and treatment of neonatal EV infection.

Hand, foot, and mouth disease

HFMD is an acute communicable disease caused by EV, predominantly affecting children under 5 years of age. EV‐A71 and CVA16 were previously the predominant serotypes of HFMD, accounting for 60%–80% of global cases. Over the past decade, CVA6 and CVA10 have emerged as predominant serotypes, while other EV serotypes account for less than 10% of cases. ¹ , ⁹³ , ⁹⁴ , ⁹⁵ , ⁹⁶ , ⁹⁷ , ⁹⁸ , ⁹⁹ , ¹⁰⁰ , ¹⁰¹ The principal clinical features of typical HFMD cases include fever, oral vesicles or ulcers, and maculopapular rash or vesicles on the hands, feet, and buttocks, with a disease course of approximately 7–10 days; most cases have a favorable prognosis, though a minority may develop complications including encephalitis and pulmonary edema. Atypical HFMD cases are frequently accompanied by influenza‐like respiratory symptoms, with more morphologically diverse skin lesions, which may manifest as papules, vesicles, bullae, macules, or eczematiform vesicles; the distribution is also more extensive, frequently involving the trunk, limbs, and even face and neck. ¹⁰² , ¹⁰³ , ¹⁰⁴ , ¹⁰⁵ Atypical HFMD caused by CVA6, CVA4, CVA10, and other serotypes can lead to complications such as orchitis and nail changes (onycholysis or nail dystrophy). ¹⁰⁶ , ¹⁰⁷ , ¹⁰⁸ , ¹⁰⁹ , ¹¹⁰ Previous studies indicated that the proportion of severe cases associated with EV‐A71‐induced HFMD was higher than that of other EVs. China's epidemiological survey from 2008 to 2012 demonstrated that EV‐A71 accounted for 45% of severe cases, while 90% of fatal cases were associated with EV‐A71 infection. ¹¹¹ Approximately 1.1%–3.7% of survivors of severe EV‐A71 cases may develop neurological sequelae, manifesting as movement disorders (such as ataxia), weakness (paresis), or cognitive impairment; if accompanied by neurogenic pulmonary edema, survivors may experience chronic respiratory dysfunction. ¹¹² , ¹¹³

Between 1973 and 2022, more than 20 large‐scale HFMD epidemics have been reported globally, with the Asia‐Pacific region showing particularly high incidence. Singapore reported an incidence rate of 346.4/100 000 in 2009, which surged to 698.8/100 000 in 2012; ¹¹⁴ Vietnam reported more than 110 000 confirmed cases in 2011, including 170 fatal cases; ¹¹⁵ between 2008 and 2016, China reported a cumulative total of over 16.25 million cases, with an average annual incidence of approximately 1.87 million cases, including 145 000 severe cases. ¹¹⁶ The average mortality rate in China from 2008 to 2022 was 0.018/100 000; as of 2023, the cumulative number of HFMD‐related deaths in China has reached 3704 cases, with children younger than 3 years of age accounting for 83.42% of all fatal cases. ¹¹⁷

The epidemiology of HFMD is significantly influenced by season and climate. Globally, in temperate regions, epidemic peaks are concentrated in late spring and early summer (April–June); in tropical/subtropical regions such as Thailand and Vietnam, outbreaks may extend into autumn; in certain regions such as Singapore and Malaysia, sporadic cases occur year‐round. ¹¹⁸ , ¹¹⁹ , ¹²⁰ , ¹²¹ , ¹²²

Herpangina

Herpangina is an important disease caused by EV infection in children, with children under 6 years of age constituting the primary affected population, and infants aged 28 days to 1 year exhibiting the highest rate of severe disease (7.47%). ¹²³ , ¹²⁴ , ¹²⁵ The clinical manifestations of herpangina are typically characterized by fever and vesicles/ulcers in the oropharynx, with some patients experiencing convulsions triggered by abrupt elevation of body temperature; approximately 2.57%–4.29% of cases may present with neurological symptoms including lethargy, seizures, headache, or irritability. ¹²⁶ , ¹²⁷ The prognosis of herpangina is generally favorable, with approximately 90% or more of cases presenting as mild disease; the oropharyngeal vesicles/ulcers typically heal completely within 5–10 days, whereas the deep ulcers caused by rare CVA4 infections may require up to 14 days for complete healing. ¹²⁸ , ¹²⁹ Herpangina exhibits global distribution, with the Asia‐Pacific region being the primary endemic area for herpangina. Statistical data from a region in China during 2017–2022 revealed an annual incidence rate of herpangina of 4986.67 per 100 000, with the highest rate in 2018 (10 477.09 per 100 000) and the lowest in 2020 (870.88 per 100 000). ¹³⁰ Herpangina is primarily caused by the species Enterovirus alphacoxsackie viruses (including CVA2, CVA4, CVA6, CVA8, CVA10, CVA16, etc.) and EV‐A71, as well as the species Enterovirus betacoxsackie (CVA9, CVB3, E6, etc.). ¹³¹ , ¹³² , ¹³³ Herpangina occurs year‐round in tropical and subtropical regions, with the main epidemic season in temperate regions occurring in summer and autumn. ¹³³

Encephalitis and meningitis

EV is the most common etiological agent of viral encephalitis and meningitis in children, accounting for approximately 58% of meningitis or encephalitis cases in infants and children; the incidence of EV‐related encephalitis and meningitis in infants younger than 3 months is approximately 116–329 cases per 100 000. ³ , ¹³⁴ , ¹³⁵ Meningitis is the most common manifestation of EV‐related CNS involvement, most frequently occurring in infants under 1 year of age; ¹³⁶ , ¹³⁷ the majority of cases are caused by the species Enterovirus betacoxsackie infection, including coxsackievirus B (CVB) and most echoviruses. Most cases present with acute onset, featuring fever and chills; in older children, headache is typically the primary complaint, accompanied by meningeal signs, with clinical severity varying considerably. Approximately 5%–10% of patients with meningitis will manifest clinical features of encephalitis, presenting with altered level of consciousness or seizures. Multiple EV serotypes can cause encephalitis, with the most reported being CVA9, B2, and B5, as well as echovirus 6 and 9. EV encephalitis presents with variable clinical severity, with severe cases accompanied by seizures, paralysis, or coma. Certain EV serotypes, such as poliovirus, EV‐D68, and EV‐A71, specifically target the motor nuclei of the brainstem and spinal cord, causing acute cranial nerve and spinal nerve paralysis. Young children infected with EV‐A71 can develop severe and potentially fatal brainstem encephalitis accompanied by neurogenic pulmonary edema. ¹³⁸ , ¹³⁹ , ¹⁴⁰

Compared with encephalitis and meningitis caused by other viruses (such as herpes simplex virus and arboviruses), EV infection demonstrates a more favorable prognosis. ¹⁴¹ Most infants and children recover completely within 3–7 days after symptom onset. However, EV‐related CNS involvement can result in severe disease or death. Immunocompromised patients infected with EV who develop encephalitis face a higher risk of adverse outcomes and even death. ¹⁴²

Acute flaccid paralysis

AFP is a syndrome resulting from loss or dysfunction of anterior horn cells of the spinal cord due to infection or inflammation. Poliovirus was formerly the primary etiology of AFP; however, it has been nearly eliminated because of vaccination programs (as of December 2025, wild‐type poliovirus type 1 exists in only two countries—Afghanistan and Pakistan; types 2 and 3 have been eradicated); non‐poliovirus EV‐induced AFP has garnered significant attention. ¹⁴³

AFM is a subtype of AFP, with EV serving as the primary etiology, particularly EV‐D68. ¹⁴⁴ AFM presents as acute‐onset weakness in one or multiple limbs, with magnetic resonance imaging revealing spinal cord inflammation primarily located in the gray matter. Numerous EV serotypes, including poliovirus types 1, 2, and 3, and several non‐poliovirus EVs, can affect anterior horn cells, subsequently leading to acute motor weakness. ¹⁴⁴ AFM was first identified in California, the United States, in 2012 ¹⁴⁵ ; the estimated incidence of AFM in the United States is less than 1 case per 1 million population. ¹⁴⁶ Hundreds of cases have been reported in Europe, Asia, Oceania, Africa, North America, and South America, ¹⁴⁴ with a distinct seasonal pattern of outbreaks occurring every 2 years in temperate regions. The majority of AFM patients are children, with a median age of 6 years. ¹⁴⁷

In most AFM patients, upper respiratory symptoms, gastrointestinal symptoms, or fever occur within days to weeks before the onset of weakness. Once neurological symptoms appear, flaccid weakness may develop within hours to days. ¹⁴⁴ Approximately 80% of patients develop weakness in at least one upper limb, and 36% of patients experience mild tetraparesis at the onset of limb weakness. ¹⁴⁷ EV‐D68‐associated AFM most commonly causes flaccid paralysis of the upper limbs, whereas poliovirus most commonly causes that of the lower limbs. Some patients may develop cranial nerve dysfunction, bowel or bladder dysfunction, and/or sensory changes. ¹⁴⁴ , ¹⁴⁸

Myocarditis

EV is a common etiological agent of myocarditis in children and the primary cause of fulminant myocarditis in neonates, with CVB3 being a frequent pathogen; myocarditis caused by CVB3 may further progress to autoimmune myocarditis. ¹⁴⁹ , ¹⁵⁰ , ¹⁵¹

EV‐induced infectious myocarditis in children presents with diverse clinical manifestations, potentially causing varying degrees of cardiac dysfunction, including heart failure, which may be accompanied by other symptoms such as fever and respiratory symptoms. ¹⁵² , ¹⁵³ The clinical presentation of myocarditis in children may vary by age and is generally more severe in neonates. ¹⁴⁹

Definitive diagnosis requires isolation of EV or detection of viral RNA from cardiac tissue, which is generally performed only at autopsy. Consequently, the diagnosis of EV myocarditis commonly depends on detection of EV from other anatomical sites or serological evidence.

The prognosis of EV myocarditis is generally favorable; however, some cases may be fatal or require cardiac transplantation. Compared with children with dilated cardiomyopathy, children with myocarditis have lower post‐transplantation survival rates. ¹⁵⁴

Hepatitis

EV infection can cause hepatitis, hepatic necrosis, and even acute liver failure (ALF) in children, particularly in neonates and young children. EV hepatitis typically presents with acute hepatitis symptoms, such as jaundice, significantly elevated hepatic enzymes, and coagulopathy. ¹⁵⁵ Some cases can progress to ALF, characterized by hepatic encephalopathy, coagulopathy, and multi‐organ dysfunction. ¹⁵⁶ In cases accompanied by DIC, hepatic necrosis, or fulminant hepatitis, mortality can reach 27.0%. ¹⁵⁷ Common EVs causing hepatitis in children include CVB types 1–5 and echoviruses. ¹⁵⁸ , ¹⁵⁹ The severity of EV hepatitis varies depending on the viral strain and host immune status. Neonatal cases of echovirus 11 infection have an extremely poor prognosis with high mortality ¹⁶⁰ ; three cases of neonatal ALF induced by echovirus 11 infection were reported in Tokyo, Japan, from August to November 2024. All neonates developed irreversible multi‐organ failure and died. In older children, EV hepatitis cases can be managed with supportive treatment such as intravenous immunoglobulin and intensive care ¹⁶¹ ; some eventually require liver transplantation, and long‐term follow‐up data show that survivors may have residual liver dysfunction or neurological sequelae. ² , ¹⁶²

Among 427 patients with hepatitis of unknown cause reported in the European surveillance system from January 1, 2022 to June 16, 2022, 77.3% of patients were aged 5 years and under; among cases with EV testing, 15.4% (10/65) tested positive for EV, suggesting that EV may be the etiology for some of these patients. ¹⁵⁹ Some cases progressing to ALF require liver transplantation. ¹⁶¹ EV hepatitis poses a serious threat to children's health, particularly in immunocompromised or immunodeficient patients. ¹⁶³

Respiratory tract infections

EV is an important pathogen responsible for respiratory tract infections in children. A large prospective epidemiological study conducted in France demonstrated that 31% of children infected with EV had respiratory diseases, with the predominant EV serotypes being Enterovirus betacoxsackie (E3, E5–7, E11, E13, E30, CVB2, and CVB4–5), while another study revealed Enterovirus alphacoxsackie (EV‐A71 and CVA16) can also cause respiratory infections in children. ¹⁶⁴ , ¹⁶⁵ A study from China revealed that the positive detection rate of EV in acute respiratory tract infections in children ranged from 0.92% to 8.31%. EV infection was most common in children aged 2 to <6 years with peak incidence during summer and autumn. ¹⁶⁶ This study identified a total of 12 EV serotypes: CVA2, CVA4, CVA5, CVA6, CVA10, CVB3, CVB5, E5, E11, E30, PV1, and EV‐D68. Among these, the predominant serotypes detected in upper respiratory tract infections were CVA10, CVA6, and CVB3, whereas CVA2 was the predominant serotype identified in lower respiratory tract infections. In recent years, EV‐D68 infection has attracted increasing attention. In North America and Europe, EV‐D68 epidemics occur biennially. ¹⁶⁷ An EV‐D68 outbreak occurred in the United States in 2014, with 1153 confirmed cases and 14 deaths, with most cases presenting with severe pneumonia. The first case of EV‐D68 infection was reported in China in 2004, and serological studies have confirmed widespread circulation of EV‐D68 throughout the country. ⁹⁷ , ¹⁶⁸ With advances in detection technology, several novel EV serotypes have been detected in respiratory secretions of children with respiratory tract infections, such as EV‐C104, EV‐C105, EV‐C109, and EV‐C117. ¹⁶⁹ , ¹⁷⁰

Pediatric respiratory EV infections can manifest as mild upper respiratory tract symptoms, including nasal congestion and rhinorrhea, or progress to more complex conditions such as laryngitis, bronchitis, bronchiolitis, pneumonia, or asthma exacerbation. Infants, young children, and immunocompromised patients can develop severe pneumonia, potentially leading to respiratory failure and extrapulmonary complications, which may be life‐threatening. It has been reported that fatal pneumonia caused by EV infection is mainly associated with CVA4, CVA7, CVA8, CVA16, CVB1, CVB5, and EV‐D68. ¹⁷¹

Acute hemorrhagic conjunctivitis

AHC, also known as epidemic hemorrhagic conjunctivitis or colloquially as “red eye disease,” is an acute contagious eye disease primarily caused by EV infection. EV‐D70 and a variant strain of CVA24 are the principal etiological agents. ¹⁷² , ¹⁷³ , ¹⁷⁴ , ¹⁷⁵ The disease is characterized primarily by severe hyperemia and hemorrhage of the conjunctiva and subconjunctival tissue. Patients commonly present with associated symptoms including lacrimation, photophobia, foreign body sensation, and increased ocular discharge, which typically appear within 1–2 days after infection and persist for 1–2 weeks. AHC is highly contagious; the general population is universally susceptible, with predominant involvement of children and the elderly. It is generally a self‐limiting disease; however, a minority of cases may present with systemic manifestations such as fever and limb pain, and in rare instances, the disease can be fatal. ¹⁷⁶ Since its first large‐scale outbreak in 1969, AHC has triggered pandemics in multiple regions including Asia and Central/South America. AHC in China exhibits distinct seasonality and geographic characteristics, with peak incidence concentrated from June to October and predominantly affecting eastern and southern regions, including Guangxi, Guangdong, Hainan, Hubei, and Zhejiang provinces. ¹⁷⁷ , ¹⁷⁸

TREATMENTS OF EV INFECTION

The treatment of EV infection is primarily supportive and symptomatic, because no antiviral drug has yet been approved for the routine clinical application of EV infections. In recent years, multiple antiviral strategies have been actively investigated, with some progress achieved. These include direct‐acting antivirals targeting viral entry, uncoating, protease activity, and RNA replication, as well as host‐targeting agents and immunotherapeutic approaches. However, most candidate therapies remain at the experimental stage or have only been evaluated in limited clinical settings, and their translation to clinical practice continues to face substantial challenges. ¹⁷⁹ , ¹⁸⁰ , ¹⁸¹ Table 3 summarizes representative antiviral agents targeting EVs, including their targets, mechanisms of action, and developmental status.

TABLE 3.

Summary of antiviral drugs targeting enteroviruses

Antiviral strategy	Molecular target	Representative agent	Developmental status
Capsid binders	VP1 hydrophobic pocket	Pleconaril 184 , 186 , 187	Phase II clinical trials
		Pocapavir 184 , 189 , 190	Completed human poliovirus challenge studies (Phase II); development not advanced to routine clinical use due to limited efficacy and emergence of resistance
		Vapendavir 189	Phase II and Phase I clinical trials
	VP1–VP3 interprotomer pocket	Tanomastat 185	Preclinical stage (in vitro and animal studies)
2C protein inhibitors	Viral 2C protein	Jun6504 192	Preclinical stage (in vitro and animal studies)
		Fluoxetine 193 , 194	In vitro antiviral activity; limited observational clinical evidence (case report and cohort study); no confirmed clinical efficacy
3C protease inhibitors	Viral 3C protease	Rupintrivir 195	Phase II clinical trials completed; development discontinued due to lack of efficacy in natural infection studies
RdRp inhibitors	Viral 3D polymerase (RdRp)	Gemcitabine, LY2334737, Sofosbuvir 196	Preclinical stage (in vitro and animal studies)
		Ribavirin 197
Neutralizing antibodies	Viral entry/attachment	EV‐A71 and EV‐D68 mAbs 199	Preclinical stage (in vitro and animal studies)
Host‐targeting agents	Viral replication organelle	PI4KB/OSBP inhibitors 200 , 201	Preclinical stage (in vitro studies)

Abbreviations: EV, enterovirus; mABs, monoclonal antibodies; OSBP, oxysterol‐binding protein; PI4KB, phosphatidylinositol 4‐kinase IIIβ; RdRp, RNA‐dependent RNA polymerase.

Symptomatic and supportive care

Management should be tailored according to clinical phenotypes (such as neonatal viremia, meningitis/encephalitis, and myocarditis), with appropriate interventions including fluid and electrolyte management, antipyresis and analgesia, respiratory support, seizure control, and intracranial pressure management and circulatory support. Critically ill patients should be admitted to the intensive care unit. Special vigilance is warranted in neonates and infants regarding rapid disease progression and multi‐organ involvement.

Intravenous immunoglobulin

Intravenous immunoglobulin contains polyclonal antibodies capable of neutralizing viruses and modulating immune responses. It is frequently used in neonates and critically ill patients, with improved outcomes observed in some cases; however, large‐scale randomized controlled trial evidence is lacking. ¹⁸²

Interferons

Interferons induce an antiviral state through activation of the JAK‐STAT signaling pathway. A multicenter randomized double‐blind study demonstrated that topical spray of recombinant human interferon‐alpha‐2b accelerates symptom resolution in HFMD; however, evidence remains inconsistent, and additional high‐quality randomized controlled trials are needed to validate its clinical benefit. ¹⁸³

Progress on antiviral agents

Capsid binders

Capsid binders are among the most extensively studied anti‐EV agents. The EV capsid contains several druggable regions, particularly the VP1 hydrophobic pocket, the VP1‐VP3 interprotomer interface, and the 5‐fold axis involved in receptor attachment. The VP1 hydrophobic pocket is normally occupied by a lipid‐like “pocket factor” that stabilizes the virion and participates in uncoating. Classical compounds such as pleconaril and pocapavir bind to this pocket, replacing the pocket factor and locking the capsid in a rigid state. ¹⁸⁴ In addition, tanomastat has shown broad antiviral activity against EV species A–D and achieved significant protective effects in EV‐A71‐infected neonatal mice. ¹⁸⁵ However, the translation of capsid binders into routine clinical application remains challenging, mainly due to incomplete antiviral coverage, strain‐specific resistance, inconsistent efficacy, and safety concerns. ¹⁸⁶ , ¹⁸⁷ , ¹⁸⁸ , ¹⁸⁹ , ¹⁹⁰

2C protein inhibitors

The 2C protein is another promising class of antivirals involved in uncoating, RNA replication, and membrane remodeling. ¹⁹¹ Novel 2C inhibitors (Jun6504) reduce CNS viral load and prevent paralysis in EV‐D68 neonatal mouse models, but remain largely preclinical. ¹⁹² Additionally, fluoxetine, originally developed as an antidepressant, was identified as a drug repurposing candidate that inhibits EV‐B and EV‐D replication by targeting 2C. Case reports describe successful treatment of chronic enteroviral encephalitis in children with X‐linked agammaglobulinemia. ¹⁹³ However, the clinical translation of fluoxetine has been disappointing: it did not improve neurological outcomes in patients with EV‐D68‐associated AFM. ¹⁹⁴

3C protease inhibitors

The 3C protease is indispensable for EV replication and mediates the proteolytic processing of the viral polyprotein. Rupintrivir (AG7088), a 3C protease inhibitor, is effective against multiple EVs in vitro; however, due to unstable in vivo efficacy, it has not demonstrated definitive clinical benefit. ¹⁹⁵

RNA‐dependent RNA polymerase inhibitors (3Dpol inhibitors)

Nucleoside (nucleotide) analogues inhibit viral replication through chain termination or mutagenic effects. High‐throughput drug repurposing screening has identified gemcitabine as demonstrating significant antiviral activity against EV‐A71, with LY2334737 and sofosbuvir reducing viral load and improving survival in mouse models. ¹⁹⁶ Ribavirin demonstrates certain efficacy in EV‐A71 mouse models; however, clinical evidence is limited and adverse effects are substantial, precluding routine use. ¹⁹⁷ , ¹⁹⁸

Monoclonal neutralizing antibodies

Neutralizing antibodies directed against EV‐A71 and EV‐D68 demonstrate the capacity to prevent or attenuate neurological injury in vitro and in animal models, and show promise for clinical trials. ¹⁹⁹

Drugs targeting host factors

EV RNA replication occurs on virus‐induced membranous ROs. Viral 3A and 3AB proteins recruit host lipid‐regulatory factors, including ACBD3, PI4KB, and OSBP, to remodel intracellular membranes. ³³ PI4KB promotes local enrichment of PI4P, which recruits OSBP‐mediated lipid transport and cholesterol delivery to replication membranes, thereby creating a favorable environment for viral RNA replication. Agents targeting PI4KB, OSBP, or related host pathways may offer broader‐spectrum activity and a higher resistance barrier than virus‐targeting monotherapy. However, their clinical use is limited by potential cytotoxicity, interference with essential host lipid metabolism, narrow therapeutic windows, and possible viral escape through mutations. ²⁰⁰

Drugs targeting host factors such as PI4KB and OSBP can inhibit viral replication by disrupting the RO, offering potential broad‐spectrum advantages; however, they are limited by safety concerns and remain at the preclinical stage. ²⁰¹

Challenges and future directions in antiviral drug development

Several factors explain why promising antiviral candidates have not yet been translated into approved therapies. First, EVs comprise numerous species and serotypes with substantial genetic diversity; therefore, compounds active against one virus may show weak or no activity against others. Second, RNA viruses mutate rapidly, and monotherapy targeting viral proteins may readily select resistant variants, as illustrated by pocapavir‐resistant virus emerging during clinical challenge studies. ¹⁹⁰ Third, severe EV disease is relatively uncommon, unpredictable, and clinically heterogeneous, making well‐powered pediatric randomized trials difficult. Finally, CNS, myocardial, hepatic, and neonatal infections impose additional requirements for tissue penetration and safety. Future research should prioritize broad‐spectrum agents with high resistance barriers, and acceptable pediatric safety, as well as mechanism‐based combination therapy and rapid diagnostic‐guided early treatment.

PREVENTION OF EV INFECTION IN CHILDREN

Core preventive measures

Vaccination

Currently, the prevention of poliomyelitis (hereinafter referred to as “polio”) can be achieved by administering oral attenuated poliovirus vaccine and inactivated poliovirus vaccine. ²⁰² , ²⁰³ , ²⁰⁴ , ²⁰⁵ Since the widespread use of polio vaccines, the global incidence of polio has declined significantly. At present, wild poliovirus type 1 remains endemic in only Pakistan and Afghanistan, and global polio eradication has entered its final critical stage. ¹⁴³ To prevent EV‐A71 infection, inactivated EV‐A71 vaccines have been approved by the National Medical Products Administration and are currently in use in China (Table 4); however, there are no vaccines available for other human enteroviral infections.

TABLE 4.

EV‐A71 vaccines licensed in China

Vaccine name (Genotype target)	Cell line	Manufacturing enterprise	Approval date
Inactivated EV‐A71 Vaccine (C4a)	Human diploid cells	Institute of Medical Biology, Chinese Academy of Medical Sciences	December 2015
Inactivated EV‐A71 Vaccine (C4a)	Vero cells	Sinovac Biotech Co., Ltd.	December 2015
Inactivated EV‐A71 Vaccine (C4a)	Vero cells	China National Biotec Group Co., Ltd.	December 2016
Inactivated EV‐A71 Vaccine (B4)	Vero cells	Enimmune Corp.	February 2023
Inactivated EV‐A71 Vaccine (B4)	Vero cells	Medigen Vaccine Biologics Corp.	April 2023

Note: The B4‐based vaccines were independently developed in Taiwan, China, and do not represent replacements for the C4a‐based vaccines licensed in Mainland China.

Abbreviation: EV, enterovirus.

Phase III clinical trials and long‐term epidemiological surveillance data have confirmed that after infants and young children receive two doses of the EV‐A71 inactivated vaccine, neutralizing antibodies peak 1–3 months after the last dose, and protective neutralizing antibodies (≥1:16) are maintained for a median of 5–6 years. The vaccine demonstrates over 90% efficacy against severe HFMD, brainstem encephalitis, and cardiopulmonary failure caused by EV‐A71, significantly reducing EV‐A71‐related severe cases and mortality in infants in China. However, its protective efficacy against mild symptoms such as rash and fever caused by EV‐A71 is approximately 70%–80%. ²⁰⁶ , ²⁰⁷ Even if serum neutralizing antibody levels subsequently decline below the detection threshold, memory B and T lymphocytes induced by the vaccine can persist long‐term. Upon exposure to wild‐type strains, the immune recall response is rapidly activated, effectively mitigating the risk of severe disease progression. ²⁰⁸ However, since this vaccine is specifically targeted against EV‐A71 and lacks cross‐protection against other EVs such as CVA16, CVA6, and CVA10, it cannot prevent outbreaks of non‐EV‐A71 HFMD. Therefore, given the limitations of current vaccines, future research efforts will focus on the development of multivalent vaccines and broad‐spectrum EV vaccines.

Patient isolation

Early identification of EV‐infected patients (such as HFMD or herpangina patients) and timely implementation of isolation and treatment measures are essential. Patients should be kept out of crowded settings such as schools and daycare centers while symptomatic, particularly during the viral shedding period, thereby reducing the risk of virus transmission.

Interruption of transmission routes

EVs are mainly transmitted via the fecal‐oral route, respiratory droplets, and close contact. Precise protective measures should be implemented according to different transmission scenarios. Feces and respiratory secretions from infected patients, as well as contaminated objects, should be thoroughly disinfected. Good personal hygiene practices (such as frequent hand washing, avoiding raw food and untreated water) should be promoted; environmental hygiene (including regular cleaning and disinfection, vector control, proper fecal management, and prevention of food and drinking water contamination) should also be implemented.

Future optimization directions for prevention and control

Vaccine development and application

Multivalent vaccine research and development: Given the diversity of EV types, multivalent vaccines represent an important future direction. Multivalent vaccines can simultaneously contain antigens of multiple prevalent EV types, enabling the vaccine to provide protection against multiple viral infections. However, the development of multivalent vaccines faces challenges such as antigen compatibility and immune interference, difficulties in industrial production and quality control, and complex clinical evaluations. First, the immunogenicity of different EVs varies significantly, and adjuvants exhibit inconsistent adsorption capabilities across serotypes. Moreover, immune interference among components intensifies markedly with increasing valency. Second, multivalent products require individual testing of each component's antigen content, inactivation efficacy, purity, and endotoxin levels, rendering the pharmacopeial testing extremely cumbersome and leaving little room for error in quality control. Finally, clinical trials must establish separate endpoints for each valency, necessitating independent statistical analysis of confirmed protective efficacy against corresponding serotypes. This results in exponentially increasing sample sizes, follow‐up periods, and pathogen typing detection costs as valency rises.

Universal vaccine exploration: The development of universal EV vaccines holds significant strategic importance. Investigation of conserved antigenic regions of EV and application of advanced biotechnological and immunological approaches may enable development of vaccines providing protection against most enteroviral species. However, the development of broad‐spectrum vaccines also faces numerous challenges: first, the capsid‐neutralizing antigens exhibit high serotype specificity, with a severe lack of conserved surface neutralizing epitopes; second, the high‐frequency mutations facilitate immune escape, weakening broad‐spectrum immune protection; third, standardized in vitro and in vivo evaluation systems for broad‐spectrum targets remain lacking.

Optimization of vaccination strategies: Personalized vaccination strategies should be developed based on the specific characteristics of different geographic regions and populations. Additionally, vaccination schedules and immunization programs should be optimized to ensure the immunogenicity of the vaccines.

Strengthening public health prevention and control measures

Environmental hygiene management: Enhanced environmental hygiene measures are needed in critical settings such as public places, schools, kindergartens, and healthcare facilities.

Health education: Novel health education strategies should be developed to enhance relevance and efficacy, promoting public awareness of enteroviral disease prevention.

Epidemic surveillance and early warning: Surveillance systems for enteroviral diseases should be strengthened, with expanded coverage and improved sensitivity.

CONFLICT OF INTEREST

The authors declare no conflict of interest.