Retinoic acid-inducible gene I (RIG-I)-like receptors, including RIG-I and MDA5, are key cytoplasmic pattern-recognition receptors that detect viral RNA in the cytoplasm and trigger the production of interferon (IFN). Among them, MDA5, rather than RIG-I, is considered the primary sensor during picornavirus infection due to the unique features of picornaviral RNA. However, previous studies have indicated that RIG-I also possesses antiviral activity against several picornaviruses, suggesting its potential importance during viral replication. Here, we found that 3A proteins from various picornaviruses, including Senecavirus A (SVA), Enterovirus 71 (EV71), Encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), and Coxsackievirus A16 (CA16), directly target RIG-I to suppress RIG-I-like receptor-mediated IFN-β production. Mechanistically, these picornaviral 3A proteins partially share the following similar strategies to dysregulate RIG-I activation: (i) all interact with RIG-I, (ii) EMCV and FMDV 3A reduce RIG-I expression, and (iii) SVA, EV71, EMCV, and FMDV 3A impair the interaction between RIG-I and MAVS by diminishing K63-linked ubiquitination of RIG-I. These findings broaden our understanding of how picornaviruses employ nonstructural proteins to evade innate immune responses during early infection.IMPORTANCEPicornaviruses cause a broad spectrum of human and animal diseases; however, the mechanisms by which they counteract host antiviral defenses remain incompletely understood. Owing to the distinct structural features of picornaviral RNA, MDA5 is widely regarded as the primary sensor mediating antiviral responses during picornavirus infection. However, accumulating evidence suggests that RIG-I also contributes to antiviral defense. Picornaviruses have evolved various means to suppress RIG-I and MDA5 activity, thereby facilitating evasion of the innate immune response and underscoring the importance of RIG-I during picornavirus infection. This study identifies RIG-I as a conserved target of the nonstructural protein 3A from multiple picornaviruses, including Senecavirus A, Enterovirus 71, Encephalomyocarditis virus, foot-and-mouth disease virus, and Coxsackievirus A16, and uncovers both shared and virus-specific strategies that dysregulate RIG-I-mediated interferon production. Collectively, these findings expand our understanding of the antagonistic mechanisms of how picornaviruses manipulate the host innate immune system.

Despite growing health concerns about enterovirus D68 (EV-D68), its cellular entry mechanisms remain incompletely understood. In this study, we investigated the endocytic pathways exploited by the sialic acid-dependent EV-D68 TW-02795-2014 strain using pharmacological inhibitors, siRNA-mediated knockdown, dominant-negative constructs, and electron microscopic analysis. Chemical inhibition of clathrin-mediated endocytosis (CME), macropinocytosis, endosomal acidification, and other endocytic pathways reduced viral entry and protein expression, suggesting the involvement of multiple routes in viral entry. Co-localization of EV-D68 with clathrin and the effects of CME inhibitors supported CME as a major entry route. However, siRNA knockdown of CME components in RD cells inhibited the cellular uptake of transferrin, yet had no significant impact on EV-D68 infection. This was also seen in A549 cells. Notably, CME disruption by clathrin knockdown led to Rac1 upregulation and increased macropinocytosis, which facilitated EV-D68 entry. Transmission electron microscopy revealed membrane-associated EV-D68 particles organized in clusters underneath the cell surface during infection, potentially undergoing bulk internalization. In contrast, purified virions lacking membranous material strictly required CME for successful infection. These findings reveal a dual entry strategy: single (membrane-free) EV-D68 particles utilize CME, while membrane-associated virions access host cells via macropinocytosis, particularly under conditions of CME inhibition. This study highlights the complexity of EV-D68 endocytosis and underscores the importance of compensatory mechanisms in viral uptake.IMPORTANCEUnderstanding how EV-D68 enters host cells is crucial for developing antiviral strategies. This study uncovers a dual-entry mechanism used by a sialic acid-dependent EV-D68 strain: clathrin-mediated endocytosis (CME) for single virus particles, and macropinocytosis as an alternative route for membrane-associated particles or under conditions where CME is impaired. Notably, disruption of CME induces a compensatory upregulation of macropinocytosis, mediated by increased Rac1. These findings challenge prior assumptions of a singular viral entry pathway and emphasize the need to consider endocytic plasticity when designing antiviral interventions targeting EV-D68 or similar viruses.

In 2016, Panama's Surveillance Program for Influenza and Other Respiratory Viruses received 4041 samples with suspected viral respiratory infection, of which 68.78% tested positive on the respiratory viral panel, while the remaining 31.22% resulted in no etiological diagnosis. Given the need to determine which other viruses could cause these respiratory infections, a retrospective search for endemic coronaviruses and Enterovirus D68 was conducted, with the primary objective of detecting them in negative samples from that year. One hundred and twenty-seven nasopharyngeal swab samples were retrospectively selected, all of which had been previously tested using a routine respiratory viral panel for pandemic Influenza viruses, Respiratory Syncytial Virus (RSV), Human Metapneumovirus (hMPV), Parainfluenza viruses (PIV-1, PIV-2, PIV-3), Adenovirus (AdV), and Rhinovirus (RV). Of these, 70% were negative and 30% positive for at least one respiratory virus, and were subsequently classified by age group, clinical outcome and geographic location. Nucleic acids were extracted using automated equipment and amplified by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). The human RNase P gene was used as an internal control. Samples and controls were considered positive with a Ct value ≤ 35 cycles. The statistical analysis was performed using Pearson's chi-square, considering a p value < 0.05 as statistically significant. The human coronavirus 229E (HCoV-229E) (n = 3), human coranvirus OC-43 (HCoV-OC43) (n = 3) and EV-D68 (n = 17) viruses were detected, presenting viral co-detections between HCoV-229E/pandemic Influenza A/H1, HCoV-OC43/Parainfluenza type 1, Enterovirus D68/Rhinovirus, Enterovirus D68/Respiratory Syncytial Virus and Enterovirus D68/Parainfluenza type 3. Overall, 78.26% (n = 18/23) of the cases were hospitalized patients and 21.74% (n = 5/23) were outpatients. Regarding age, 52.17% (n = 12/23) of the cases corresponded to children under 5 years old, 13.04% (n = 3/23) to patients between 5 and 18 years old, and 34.78% (n = 8/23) to adults over 18 years old. The circulation of HCoV-229E, HCoV-OC43, and EV-D68 was confirmed, causing both single respiratory infections and co-detections with other viruses from the routine diagnostic panel. This is why it is crucial to expand our knowledge of the epidemiology of respiratory viruses that are not currently part of routine surveillance systems, but which could be contributing significantly to the burden of disease in the population.

Human enteric viruses frequently encounter diverse microbial communities in wastewater, where interactions with bacteria and other microorganisms can profoundly influence their environmental persistence and transmission. Increasing evidence demonstrates that bacteria can bind and stabilize enteric viruses, thereby enhancing thermal inactivation and prolonging infectivity. These interactions not only shape viral fate during wastewater treatment but also may affect the sensitivity and interpretation of wastewater-based epidemiology, an essential tool for public health surveillance. Yet significant knowledge gaps remain regarding the molecular mechanisms that govern bacterial-viral interactions and their roles in broader wastewater microbiomes. Together, advancing our understanding of transkingdom interactions in wastewater is essential for improving treatment processes, strengthening environmental surveillance, and reducing the risk of virus transmission through contaminated water systems.

Acute gastroenteritis (AGE) in children is primarily associated with pathogens such as Human Rotavirus A (HRVA), Human norovirus (HNoV), Human adenovirus (HAdV), Human astrovirus (HAstV), Human parechovirus (HPeV) and increasingly, Human enteroviruses (EVs). EVs are non-enveloped RNA viruses belonging to the Picornaviridae family, comprising over 100 subtypes classified into four species. Despite their clinical relevance, the prevalence of EVs in AGE remains underexplored in India, resulting in limited data on their epidemiology and molecular diversity. A total of 450 stool samples were collected from hospitalized children under five years of age with AGE in Pune, India, between 2017 and 2023. Molecular characterization was subsequently performed to identify and confirm the types of EVs present. EVs were detected in 11.3% (51/450) of AGE cases. HRVA remained the most frequently detected pathogen overall. Among EV-positive cases, 58.8% were viral mono-infections, while 41.2% occurred as co-infections with other enteric viruses. EVs detection was highest in children below two years of age and occurred year-round, with a marked increase during the monsoon months. Molecular characterization identified 16 distinct EVs genotypes among 28 genotyped samples, predominantly belonging to EV-B species, followed by EV-A and EV-C. Phylogenetic analysis demonstrated clustering with contemporary global strains, indicating sustained circulation and genetic diversity. Replication-competent EVs were successfully isolated from representative samples, confirming the presence of viable viruses in clinical specimens. Although rotavirus remained the predominant viral pathogen associated with pediatric AGE, EVs were consistently detected across multiple years and age groups, either as viral mono-infections or in combination with other enteric viruses. These findings suggest that non-polio EVs may contribute to the viral etiology of pediatric gastroenteritis, although their precise etiological role requires further investigation. Continued molecular surveillance and broader diagnostic approaches may help clarify their epidemiological relevance and contribution to gastroenteritis in settings with high disease burden.

Hand-foot-mouth disease (HFMD), mainly caused by Enterovirus 71 (EV71), Coxsackievirus A6 (CVA6), and Coxsackievirus A16 (CVA16), poses a serious threat to children due to its high transmissibility and potential to induce severe complications. Herein, a spatially encoded fluorescence biosensing platform that integrates a parallel entropy-driven circuit (EDC) with a combinatorial photonic crystal (cPC) array was reported for ultrasensitive, multiplex, and rapid detection of HFMD viruses. In this system, three parallel entropy-driven circuits were designed to specifically recognize the VP1 gene sequences of EV71, CVA6, and CVA16, generating distinct fluorescence signals (FAM, Cy3, and Cy5) upon target binding. Simultaneously, the cPC array, constructed from polystyrene nanospheres of three different diameters (220, 245, and 293 nm), not only acted as a spatial encoder that enabled the simultaneous discrimination of multiple signals through predefined physical zones but also served as a signal amplifier by matching its photonic band gaps to the emission wavelengths of the fluorophores. Under optimized conditions, the sensing array exhibited excellent linearity across broad dynamic ranges with the detection limits down to the femtomolar level (100.55 fM for EV71, 23.06 fM for CVA6, and 52.76 fM for CVA16). This assay also demonstrated high specificity against non-HFMD viruses and satisfactory recovery rates (94.52-102.68%) in spiked serum samples. This work reports the first application of integrating a combinatorial PC array with a parallel EDC for the detection of HFMD, offering a low-cost, user-friendly, and versatile strategy with promising potential for clinical diagnostics and public health surveillance.

Enteroviruses (EVs) are the most frequent cause of pediatric aseptic meningitis, and cause neurological complications, such as encephalitis and flaccid paralysis. Our aim was to characterize the age-specific cytokine and chemokine response profiles in the cerebrospinal fluid (CSF) and blood of neonates, infants, and children with enterovirus meningitis. We performed an ancillary study to the French multicenter BLEDI study. We selected 163 patients with confirmed EV or suspected meningitis. Demographic, clinical, and laboratory data were analyzed. Twenty-seven cytokine/chemokines were simultaneously quantified in the CSF and plasma of the patients, using a bio-plex multiplex immunoassay. Cytokine-chemokine expression increased in the CSF with the age of EV patients compared with controls: CSF IP-10, IL-6, and IL-1ra increased 13-, 11-, 5-fold, respectively, in neonates, 41-, 53-, and 20-fold in infants, and 52-, 168-, and 69-fold in children. In plasma, cytokine/chemokines were overexpressed in neonates and downregulated in children, compared with their respective controls: plasma MCP-1, IP-10, and IL-1ra increased 10-fold in neonates, and decreased threefold in children. The expression of cytokine/chemokines varied with CSF pleocytosis but not with EV types and viral load. These findings show that cytokine and chemokine responses during EV meningitis are strongly age-dependent and should be interpreted according to patient age, with neonates, infants, and children being considered separately. Our results also indicate that age stratification is essential in future studies aiming to evaluate cytokines and chemokines as biomarkers of disease severity in EV-associated neurological infections.IMPORTANCEAlthough enteroviruses (EVs) are the main cause of pediatric viral meningitis, the age-specific immune response to infection remains poorly understood. Our work is novel in combining the study of paired biological compartments (cerebrospinal fluid [CSF] and plasma), age stratification, pleocytosis status, viral load, and EV genotype, which allows a comprehensive assessment of how age shapes both local and systemic immune responses during EV meningitis. This study shows that the immunological response to EV meningitis in CSF and plasma evolves with the age of the patients. The expression of cytokines/chemokines varied with CSF pleocytosis but not with EV types and viral load. Our findings highlight that neonatal EV meningitis is associated with distinct immunopathogenic features that reflect age-dependent immune responses. These results indicate that age stratification is essential in future studies aiming to evaluate cytokines and chemokines as biomarkers of disease severity in EV-associated neurological infections.

Objective: To analyze the epidemiological characteristics of hand, foot and mouth disease (HFMD) in Hebei Province from 2008 to 2024, and quantitatively evaluate the impacts of enterovirus A71 (EV-A71) vaccination and non-pharmaceutical interventions (NPIs) during the COVID-19 pandemic period on the incidence trend of HFMD by using the interrupted time series (ITS) method. Methods: The incidence data of HFMD in Hebei from 2008 to 2024 were collected for a descriptive analysis to understand the temporal, population, and regional distributions of HFMD. ITS model was used to quantitatively evaluate the impact of EV-A71 vaccination and the NPIs during the COVID-19 pandemic period on HFMD incidence. Additionally, the children under five years old were divided into two age groups (<2 years and ≥2 years) for subgroup ITS analyses to compare the differential responses of age groups to these interventions. Results: A total of 822 740 HFMD cases were reported in Hebei during the study period, the reported cases increased firstly, then-declined, showing an obvious seasonality, with an uneven geographic distribution. The majority of cases were children aged 0-5 years, especially the scattered children, and the incidence rate was higher in boys than in girls. ITS analysis showed that following the introduction of EV-A71 vaccination in 2016, the annual decline in overall HFMD incidence accelerated from 2.00 per 100 000 (P=0.735) to 10.67 per 100 000 (P=0.186). Age-stratified analysis indicated that in children under 2 years old, HFMD incidence trend reversed from increase to decrease (P=0.014). In children aged ≥2 years, the incidence remained generally stable before and after the vaccination, with no significant immediate or long-term changes observed. In 2020, when COVID-19 control measures were taken, HFMD incidence significantly decreased in both overall population and the two age groups, with a greater decline observed in children aged ≥2 years compared with those aged <2 years. Subsequently, the incidences in both groups showed rebounds. Conclusions: From 2008 to 2024, the HFMD incidence in Hebei exhibited distinct phase-specific changes, resulting from the synergistic effects of pathogen spectrum shifts, vaccine interventions, and public health measures. Following the introduction of the EV-A71 vaccine, the incidence of HFMD showed a decreasing trend, but the intervention effect exhibited age heterogeneity, being more significant in children under 2 years old. The NPIs during the COVID-19 pandemic period significantly suppressed HFMD transmission for a short time. These findings suggest that relying solely on overall incidence is insufficient to fully evaluate type-specific vaccine effects; integrating pathogen type surveillance and age-stratified analyses can provide a more accurate evaluation of HFMD control and inform the optimization of comprehensive prevention strategies.

Enterovirus 71 (EV-A71), a neurotropic member of the Picornaviridae family, is a positive-strand RNA virus comprising of seven recognized genogroups (A-G) and has caused significant outbreaks of hand, foot, and mouth disease worldwide. Despite prior identification of D and G genogroups in India, comprehensive full-genome characterization of these indigenous lineages remains unreported. We performed next-generation sequencing (NGS) and molecular phylogenetic analyses on two Indian isolates from acute flaccid paralysis cases: R13223-IND-02 (genogroup D) and V11-2209-01 (genogroup G). The complete genomes generated through NGS were aligned with publicly available full-length and partial EV-A71 sequences using MAFFT, and phylogenetic trees were constructed employing the maximum-likelihood method. Amino acid variations were then compared across the genogroups, as well as with prototype and neurovirulent reference strains. Phylogenetic reconstruction revealed robust clustering of these strains within distinct clades (bootstrap support ≥ 99%)", divergent from globally prevalent genogroups B and C. Both D and G genogroups appear geographically restricted to India, while genogroup D persists as an endemic lineage. Comparative genomic analyses demonstrated extensive nucleotide divergence, including numerous non-synonymous substitutions across both structural (VP1-VP4) and non-structural (2A-3D) coding regions. An E-Q substitution at amino acid position 145 in the VP1 structural protein was detected in both D and G genogroups, a site functionally linked to receptor binding and mouse virulence. The findings highlight the evolutionary independence and sustained circulation of D and G genogroups in India, suggesting a unique regional evolutionary trajectory. The implications hold significant global relevance, offering valuable insights for molecular surveillance, advancing pathogenesis research, and supporting the development of genogroup-specific vaccine strategies tailored to diverse epidemiological landscapes. This study is the first to report the complete genome of EV-A71 D and G genotypes in India, and its unique mutation characteristics provide a direct target for the development of region-specific vaccines.

Acute viral gastroenteritis remains a major cause of morbidity and hospitalization among children under five years worldwide. In the Middle East, epidemiological and molecular evidence remains fragmented, particularly in the post-COVID-19 period. This systematic review and meta-analysis aimed to synthesize recent data (2020-2025) on the prevalence, genetic diversity, and co-infection patterns of enteric viruses among children aged 0-59 months in the region. A comprehensive search of PubMed/MEDLINE, Scopus, Web of Science, ScienceDirect, and the WHO Global Index Medicus identified eligible observational and molecular studies published between 1 January 2020 and 31 May 2025. Study selection, data extraction, and risk-of-bias assessment were independently conducted by two reviewers according to PRISMA 2020 guidelines. The protocol was prospectively registered in PROSPERO (CRD420251064184). Pooled prevalence estimates were calculated using a random-effects model with Freeman-Tukey double arcsine transformation. Forty-three studies, including 22,021 children tested for acute gastroenteritis (AGE) from nine Middle Eastern countries, met the inclusion criteria. Rotavirus (28 studies) was the most prevalent pathogen, with a pooled prevalence of 30.4% (95% CI: 24.3-35.8; I² = 96%, p < 0.001), followed by norovirus (12 studies) at 23.5% (95% CI: 11.4-29), adenovirus (13 studies) at 11.3% (95% CI: 8.6-17.6), and astrovirus (10 studies) at 6.0% (95% CI: 1.3-12.7). Predominant rotavirus genotypes included G1, G2, G3, and G9, commonly combined with P[8], P[4], and P[6], with G3P[8] and G1P[8] as dominant constellations. Norovirus GII.4 and recombinant GII.4[P16] strains were frequently detected. Viral co-infections were also reported, particularly involving rotavirus and other enteric viruses. Rotavirus and norovirus remain the principal viral causes of pediatric acute gastroenteritis in the Middle East and exhibit substantial genetic diversity with frequent co-infection patterns. However, marked inter-study heterogeneity and uneven geographic representation limit regional generalizability. Strengthened molecular surveillance, standardized diagnostic approaches, and continuous genotype monitoring are essential to optimize prevention strategies and vaccination policies across the region.

Genetic evolution and epidemiological dynamics of porcine epidemic diarrhea virus in Guangxi, China, from 2020 to 2023.

Depth-dependent attachment and transport of bacteriophages MS2 and ΦX174 in saturated soils: Impacts of silt and clay fraction.

Interferon epsilon (IFNε) is a unique type I IFN that, unlike other family members, is not induced by infection but is constitutively expressed in epithelial tissues. In this manuscript, we define the epithelial cell types that constitutively express IFNε in the uterus and small intestine at a single-cell resolution. We show that mice lacking IFNε lose key antiviral defenses in a tissue-dependent manner; uterine epithelial cells have diminished basal ISG expression, and key populations of cytokine-expressing enterocytes are absent from the small intestine. In the intestine, this correlates with increased susceptibility to infection with an enteric virus in mice. These findings establish IFNε as a key contributor to mucosal immunity, sustaining antiviral defenses within tissue-specific epithelial cells of both the female reproductive tract and intestine, and broaden our understanding of its role beyond traditional pathogen-induced interferon responses.

Propagation and immunological characterization of three enterovirus D68 strains using serum-free HEK293A suspension cell culture.

Enterovirus A71 (EV-A71), the primary causative agent of hand, foot, and mouth disease (HFMD), can cause severe neurological complications and even death, particularly in young children. Despite the availability of inactivated vaccines, their protective efficacy has been compromised due to frequent intra- and intertypic recombination events and ongoing mutations among circulating EV-A71 strains. To address this, we employed immunoinformatic approaches and identified conserved epitopes and constructed a multi-epitope vaccine (MEV) candidate against EV-A71. A total of 1,627 structural protein sequences from EV-A71 strains encompassing all major circulating subtypes were retrieved and aligned to generate a consensus sequence. With this consensus sequence, 11 conserved, antigenic, and non-allergenic epitopes capable of eliciting B-cell, T-cell, and interferon-gamma (IFN-γ) responses were identified. The constructed MEV demonstrated superior immunological potential with a high antigenicity score of 0.94 and was predicted to be non-allergenic and non-toxic. Structural characterization via AlphaFold 3 and 300ns molecular dynamics (MD) simulations confirmed the formation of a stable β-strand framework. Molecular docking followed by trajectory-stabilized interaction analysis revealed that the MEV maintains a high-affinity and stable binding profile with Toll-like receptor 3 (TLR-3). To ensure optimal translational efficiency, the vaccine gene was codon-optimized with a GC content of 52.8%, and the protein was successfully expressed in a bacterial system. Collectively, this study provides a high-performance MEV candidate with robust structural stability and potent immunogenicity, offering a promising and cost-effective strategy for broad-spectrum protection against EV-A71.

The oral in fecal-oral: Exploring the overlooked relationship between saliva and enteric viruses.

Neutral sphingomyelinase 2 knockdown attenuates disease severity and modulates immune responses in enterovirus A71-infected mice.

Enteric viruses leverage bacteria to boost infectivity and disinfection resistance.

Enterovirus D68 (EV-D68) has been implicated in clusters of acute flaccid myelitis (AFM) and severe respiratory illness; however, the magnitude and consistency of association across settings and over time remain uncertain. We quantified the association between EV-D68 detection and acute neurologic outcomes (particularly AFM) and explored design- and time-related heterogeneity. Following PRISMA 2020 and MOOSE guidance, we systematically identified observational studies reporting associations between EV-D68 detection and neurologic outcomes. Random effects meta-analysis was performed using REML with Knapp-Hartung adjustment; heterogeneity was summarised with τ2 and I2. Prespecified moderators were examined with meta-regression. Small-study effects were evaluated using contour-enhanced funnel plots, Egger and Peters tests, and PET-PEESE bias-adjusted models. PROSPERO registration: 1152300. Across 98 studies, the pooled odds ratio (OR) was 1.39 (95% CI 1.14-1.69), with high heterogeneity (I2=98.9%; prediction interval 0.24-8.15). By design, respiratory surveillance studies showed stronger association (OR 1.59, 95% CI 1.35-1.86, k=78) whereas AFM case-control studies did not (OR 0.86, 95% CI 0.40-1.86, k=20). In multivariable meta-regression, study design and calendar year explained about 47% of inter-study variance. Predicted ORs declined from 2014 to 2022 across regions. Funnel asymmetry and small-study effects were suggested; PET-PEESE bias-adjusted estimates remained above the null. The EV-D68 outcome association is context-dependent and time-varying. Surveillance datasets enriched during outbreak waves drive the pooled signal, while AFM case-control designs yield attenuated estimates after adjustment. Standardising diagnostics and integrating design-specific surveillance will improve risk estimation and AFM preparedness.

Comparative immunogenic and structural characterization of virus-like particle and inactivated whole-virion vaccines against enterovirus D68