Enterovirus 71 (EV71) infection poses a global public health challenge, especially in infants and young children, with severe cases leading to fatal consequences. EV71 infection modulates various biological processes of the host and evades host immunity through multiple mechanisms. The balance of mitochondrial dynamics is important for cellular homeostasis. However, the mechanisms underlying EV71-induced cellular damage via mitophagy remain unclear. In the current study, we showed that EV71 infection significantly reduced the total and mitochondrial ATP contents in cells, as well as the expression of mitochondrial proteins TOM20 and TIM23. Then, EV71 infection increased the protein levels of PINK1, Parkin, and LC3B, suggesting that EV71 infection triggers the mitophagy. Silencing PINK1 caused a significant reduction in viral replication, while overexpressing Parkin promoted the replication of EV71. Moreover, CsA treatment, as a mitophagy inhibitor, alleviated pathological damage and suppressed the replication of EV71 in vivo. Mechanistic study showed that silencing PINK1 inhibited the cleavage of MAVS by EV71, while overexpressing Parkin enhanced the cleavage of MAVS by EV71, suggesting that PINK1-mediated mitophagy was involved in regulating innate immunity. Furthermore, we found that EV71 infection promoted the release of mitochondria carrying EV71 virions into the extracellular environment, which mediated infection of other cells, thus facilitating virus spreading. In addition, we also demonstrated that the extracellular mitochondria induced the degradation of MAVS and mitophagy promoted the release of mitochondria in EV71-infected HeLa cells. In conclusion, these findings suggest that EV71 infection induces PINK1-mediated mitophagy, which inhibits innate immunity and facilitates virus replication.

Enterovirus A71 (EV-A71) is the major causative pathogen for severe hand-foot-mouth disease (HFMD), a predominantly childhood-associated communicable disease. The mechanisms that children manifest severe disease progression while adults typically exhibit milder or asymptomatic infections remain incompletely characterized, which hinders the development of effective therapy against this disease. Herein, using the newborn mouse model of EV-A71 infection, we uncovered that the underdevelopment of T cells closely associated with the severity of EV-A71 infection, and EV-A71 infection dramatically impaired T-cell immune response. Moreover, the dysfunction of T-cell immunity contributes to the pathogenesis of EV-A71 infection, as the loss of T cells made neonatal mice highly vulnerable to EV-A71 infection. To further assess the relationship between T-cell immunity and HFMD, we enrolled a cohort of 145 pediatric patients with laboratory-confirmed EV-A71 infection and found that the compromised T-cell immune response is associated with the severity of EV-A71-caused HFMD in these children. Furthermore, we found that the treatment of newborn mice with Astragaloside A, a saponin from the medicinal herb Astragalus membranaceus, showed potent in vivo therapeutic efficacy against EV-A71 infection in a T-cell-dependent manner. In conclusion, these findings uncover the interaction between EV-A71 infection and T-cell immunity, provide novel insights onto the physiological impacts of T cells on the pathogenesis of EV-A71 infection and HFMD, and find a promising immunotherapeutic strategy to treat this viral disease.

Enterovirus A71 (EV-A71) is a major cause of severe hand, foot, and mouth disease (HFMD) among children in the Asia-Pacific region. Here we review randomised clinical trial data and post-licensure effectiveness studies of inactivated EV-A71 vaccines following their development and licensure in China since late 2015. We searched PubMed, Web of Science Core Collection, Elsevier ScienceDirect, China National Knowledge Infrastructure (CNKI), and Wanfang Data on 20 May 2024 with no date restriction in English and Mandarin, using key terms including "EV-A71", "enterovirus 71", "hand, foot, and mouth disease", "HFMD", "vaccine", "efficacy", "effectiveness", "protection" and "impact". Phase III randomized controlled trials (RCTs) reporting vaccine efficacy and observational studies on effectiveness were eligible for inclusion. We excluded studies that evaluated non-laboratory confirmed HFMD associated outcomes, abstracts, reviews, comments, animal studies, cross-sectional studies, and modelling studies. Summary measures of vaccine efficacy and effectiveness were based on random-effects models. After screening, 14 articles were eligible for inclusion, including 6 reporting 4 different phase III RCTs. Estimated efficacy of 2 doses of EV-A71 vaccine against EV-A71 associated HFMD 1 year after vaccination ranged from 90.0% to 97.9%, with an overall estimate for all products of 95.6% (95% Confidence Interval: 92.1, 97.5). Efficacy remained high at 26 months post-vaccination, ranging from 94.7% to 94.8%. The estimated overall effectiveness of 2-dose vaccination against any EV-A71-HFMD across five test-negative case-control studies was 84.3% (95% CI: 75.2, 90.0) among children aged 0-12 years. Effectiveness was higher in older children compared to younger children (85.3% (72.9, 92.1) vs. 79.8% (61.2, 89.5)) and higher against severe compared with non-severe EV-A71-HFMD (90.0% (80.0, 95.0) vs. 76.5% (50.0, 89.0)). The effectiveness declined in more recent studies with longer follow-up. Inactivated EV-A71 vaccines offer a high level of protection against EV-A71 HFMD. Longer term studies are needed to evaluate the persistence of protection beyond 2 years post-vaccination.

Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease, which can lead to further infections and spreads via the oral cavity; however, the mechanism of how EV-A71 infects the human oral cavity remains unclear. Screening and validation using small-interfering RNAs and chemical inhibitors showed that EV-A71 entry into human oral epidermoid carcinoma KB cells was independent of clathrin-, caveolin-, endophilin-, dynamin-, and macropinocytosis-mediated pathways. However, the clathrin-independent carriers/GPI-anchored-protein-enriched endosomal compartment (CLIC/GEEC) pathway is crucial for EV-A71 entry into KB cells and normal human oral epithelial cells (NHOEC), which requires the entire actin cytoskeleton and membrane cholesterol. Inhibition of GBF1, ARF1, CDC42, PICK1, GRAF1, and IRSp53, the key molecules of the CLIC/GEEC pathway, significantly suppressed EV-A71 entry and infection. Confocal microscopy showed that internalized EV-A71 colocalized with CDC42 and GRAF1. Knockdown of CDC42 or GRAF1 reduced the number of internalized viral particles, which were predominantly localized at the plasma membrane. By using the sucrose density gradient centrifugation, EV-A71 and glycosylphosphatidylinositol-anchored GFP (GPI-GFP) were observed within the same low-density components. Furthermore, endocytosed EV-A71 was colocalized with GRAF1 and GPI-GFP and transported to the late endosomes. Mouse experiments demonstrated that the GBF1 inhibitor, Golgicide A, significantly reduced EV-A71 infection-induced mortality. Immunohistochemical staining and histopathological section analysis revealed that Golgicide A markedly decreased the viral load in brain tissue and oral epithelium, and alleviated the pathological damage induced by the virus in brain tissue. Our findings reveal a novel pathway for EV-A71 entry into KB cells and provide a new target for drug development.

Prodiginins are a large family of at least 34 pyrrolic compounds, including the well-studied red pigment prodigiosin. Prodiginins are produced by several microorganisms displaying broad biological activities, including antimicrobial, antiviral, antiparasitic, antiproliferative, and immunosuppressive activities. The present review aims to compile and analyze the main physicochemical and biological properties and mechanisms of action of prodiginins for microbial disease treatment, particularly SARS-CoV-2 disease and opportunistic infections related to COVID-19. The interaction of prodigiosin, as a model molecule, with cellular membranes, potential drug delivery devices, and toxicological studies, and in silico studies using molecular dynamics showed that the prodigiosin motif, which interacts with lipids, opens a new door for the potential therapeutic use of prodiginins.

Enterovirus D68 (EV-D68) is an emerging pathogen causing severe respiratory infections, and the immune evasion mediated by EV-D68 structural protein has been under discussion for several years. Our early research has identified that EV-D68 structural protein VP3 targets specifically the interferon regulatory factor 7 to inhibit type I interferon signaling, but not interferon regulatory factor 3, which is indispensable for mitochondrial antiviral signaling protein (MAVS)-activated type I interferon signaling. Interestingly, in this study, we found that VP3 co-localizes and interacts with MAVS. Furthermore, VP3 acts as a negative regulator of MAVS/Sendai virus-activated NF-κB signaling pathway. Overexpression of VP3 can promote EV-D68 replication and reverse MAVS-mediated inhibition of virus replication. The mechanism of the interaction between VP3 and MAVS may be that VP3 not only disrupts the mitochondrial membrane potential but also leads to the release of MAVS from mitochondria. Moreover, VP3 binds to the transmembrane domain of MAVS with mitochondrial membrane localization function, which provides support for the mechanism of action. Finally, in our study, we found that VP3 interaction with MAVS to inhibit NF-κB activation is a mechanism that is prevalent in enteroviruses. Overall, our data demonstrate that the interaction between VP3 and MAVS can be used by enteroviruses to evade host innate immunity as a broad-spectrum strategy.IMPORTANCEEnterovirus D68 (EV-D68), as an emerging pathogen, has resulted in a rising number of pediatric infections worldwide since its initial outbreak in the United States in 2014. This virus can cause severe respiratory illnesses and is linked to acute flaccid myelitis. In this article, we report that the structural protein VP3 of EV-D68 inhibits the activation of the NF-κB signaling pathway by targeting mitochondrial antiviral signaling protein (MAVS). Further studies demonstrate that VP3 can induce mitochondrial damage, resulting in the loss of MAVS localization in mitochondria. These findings suggest that the interaction between VP3 and MAVS may represent a mechanism by which EV-D68 suppresses the activation of the NF-κB signaling pathway, facilitating immune evasion and promoting viral replication. Our study suggests potential therapeutic strategies for enterovirus-related viral diseases and the development of novel antiviral drugs.

The Enterovirus genus contains two major subgroups: rhinovirus (RV) species A-C and enterovirus (EV) ones A-D. While RV only infects the respiratory system, the EV can cause a wide variety of diseases, ranging from non-specific febrile illness to severe neurologic complications. To date, no curative treatments are commercially available. Our research team had recently developed EV-A71 inhibitors. To improve their activity and broaden their spectrum, we performed optimization of the structure following an iterative cycle of chemical modulations. As a result, we obtained two broad-spectrum inhibitors with micromolar activity against these 3 types of viruses (OM1260: EC50 (MRC-5, EV-A71) = 1.15 μM; EC50 (RD, EV-A71) = 4.38 μM; EC50 (MRC-5, E30) = 0.41 μM; EC50 (MRC-5, CVA24) = 1.15 μM; HR-568: EC50 (MRC-5, EV-A71) = 3.25 μM; EC50 (RD, EV-A71) = 1.53 μM; EC50 (MRC-5, E30) = 0.40 μM; EC50 (MRC-5, CVA24) = 1.22 μM). Docking studies shed light on structure-activity relationships, while time-of-drug addition assays confirmed their intervention during the early step of viral replication. Eventually, some pharmacokinetic modelling has been carried out to evaluate their druggability. All these results showed that OM1260 and HR-568 are promising candidates for further development.

Enterovirus 71 (EV71) is a major cause of hand, foot, and mouth disease, particularly affecting pediatric populations worldwide. The role of ZYG11B, a CUL2-complex-associated E3 ubiquitin ligase from the Zyg-11 family, in antiviral defense against EV71 remains unclear. To our knowledge, this study is the first to reveal that ZYG11B targets EV71 VP1 for proteasomal degradation via the ubiquitin-proteasome pathway, with CRL2ZYG11B complex activity specifically driving K33-linked ubiquitination. Mass spectrometry and immunoprecipitation analyses confirmed the interaction between ZYG11B and VP1 and identified key domains required for binding both VP1 and CUL2. Comparative analyses showed that VP1 ubiquitination sites are highly conserved across related enteroviruses, including CA6, CA16, and EVD68. Functional assays further demonstrated that ZYG11B restricts these viruses, highlighting its potential as a broad-spectrum antiviral target. These findings establish ZYG11B as a critical effector in host antiviral responses and support its therapeutic potential for managing enterovirus infections.

E3 ubiquitin ligases and deubiquitinases have become important topics of competition between viruses and hosts. Here, we identified CRL2ZYG11B as an E3 ubiquitin ligase complex capable of degrading structural protein VP1 of enteroviruses, making ZYG11B a broad-spectrum antiviral factor. We first proposed the inhibitory effect of ZYG11B on viruses and identified the structural domains of ZYG11B connecting substrates and CUL2, providing new targets for the design of antiviral drugs.

Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD), leading to a serious health threat to young children. Probiotics are effective at treating or preventing gastrointestinal infections, especially viral infections. Probiotics against EV71 are mainly traditional lactic acid-producing bacteria, and most of them have been proven to be effective only in vitro. Here, we report that the marine bacterium Paraliobacillus zengyii X-1125 (P. zengyii) has promising anti-EV71 activity. The antiviral effect of P. zengyii against EV71 was assessed in different cell lines, and the viral RNA levels and titers were obviously reduced after treatment with P. zengyii. Furthermore, we established an EV71-infected mouse model to evaluate its antiviral efficacy in vivo. The oral administration of P. zengyii significantly decreased the viral loads in the hindlimb muscles, spleens, and ileums. Further research revealed that P. zengyii enhances the expression of type I interferon (IFN-I) in EV71-infected cells. Similarly, transcriptome analysis indicated that the expression of interferon-stimulated genes (ISGs) in EV71-infected mice significantly increased after P. zengyii treatment. Taken together, the results of this study indicated that P. zengyii markedly reduces EV71 infection by regulating the IFN response both in vivo and in vitro, providing a potential means to work against EV71 infection.

Emerging infectious diseases such as COVID-19 and Disease X, which was detected in the Democratic Republic of the Congo in early December 2024, underscore the importance of developing new virucidal, antiviral, and antimicrobial compounds. The virucidal activity of natural products, including tiliacorinine (1), dioscorine (2), racemosol (3), and terrein (4), against influenza A virus (H1N1), human coronavirus 229E (HCoV-229E), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and enterovirus 71 (EV71) were evaluated using the American Society for Testing and Materials E1053-20 method. Racemosol (3) from Bauhinia malabarica had the most potent virucidal activity against the H1N1, HCoV-229E, and SARS-CoV-2 viruses, followed by terrein (4), a metabolite of the fungus Aspergillus terreus. Racemosol (3) exhibited virucidal activity with a log reduction of 4 (99.99 % viral reduction) against H1N1, HCoV-229E, and SARS-CoV-2 at a concentration of 0.1250 mg/mL. The alkaloids tiliacorinine (1) from Tiliacora triandra and dioscorine (2) from Dioscorea hispida exhibited weaker virucidal activity than racemosol (3) and terrein (4). Compounds 1, 2, and 4 showed weak virucidal activity against the EV71 virus, while racemosol (3) displayed moderate activity with a log reduction of 3.813 at the concentration of 0.1250 mg/mL. This work underscores the importance of natural products as sources of virucidal agents, which may be useful for the future threats of emerging and re-emerging viral diseases.

To analyze the epidemiological characteristics of Hand, Foot and Mouth Disease (HFMD) reinfection and its influencing factors in Jiulongpo District from 2009 to 2023 to provide targeted prevention and control recommendations for key factors.

HFMD cases in Jiulongpo District of Chongqing were derived from the China Information System for Disease Control and Prevention from 2009 to 2023. Descriptive analysis was used to analyze the epidemiological characteristics of HFMD reinfection, spatial autocorrelation to analyze the regional clustering, and binary logistic regression to analyze the influencing factors.

From 2009 to 2023, 4,764 HFMD reinfection cases involving 2,436 individuals were reported in Jiulongpo District, with a reinfection rate of 5.48%. The interval between the two infections ranged from 26 to 3,863 days, and 71.51% of patients were reinfected within 2 years. There was a bimodal distribution in time (April-July and October-November). In the population, the reinfection rate was 5.87% in males and 4.93% in females, 3.97% in scattered children and 7.89% in kindergarten children, 8.61% in children >3 years old, and 4.68% in children ≤3 years old. There was a spatial positive correlation of HFMD reinfection in Jiulongpo District, with hot spots concentrated in the rural area and cold spots in the urban area. The multifactorial logistic regression analysis showed that reinfection risk was higher in non-epidemic years, male, rural areas, >3 years old, and kindergarten children (p < 0.05).

Post-epidemic prevention and control measures should prioritize interventions to target reinfection, focusing on children in rural areas and kindergartens. Improve rural infrastructure and sanitation, raise disease awareness in kindergartens, train healthcare workers, and promote hygiene to reduce HFMD reinfection.

Glioma, the deadly primary intracranial tumor, poses challenges in clinical treatment due to its infiltrative growth and resistance to radiation. Oncolytic virus therapy holds potential for the treatment of malignant gliomas, but its application is impeded by the requirement for intracranial injections due to the presence of blood-brain barrier (BBB). In this study, to overcome this limitation, the study develops a nanocapsule encapsulating the recombinant oncolytic virus EV-A71-miR124T, enabling the treatment of glioma through intravenous administration. It is demonstrated that the nanocapsule can cross the BBB and selectively release oncolytic virus at the tumor site, resulting in targeted and specific killing of glioma cells. In mice with implanted intracranial orthotopic gliomas, intravenous administration of the nanocapsule suppresses tumor growth and significantly extends survival time. Consequently, the study establishes an effective treatment method for malignant gliomas using an oncolytic virus nanocapsule through intravenous administration. These findings provide a new strategy for oncolytic virus therapy in glioma treatment and offer perspectives for targeted therapies of other brain tumors and diseases.

Enterovirus 71 (EV-71) is a major pathogenic factor that causes hand, foot, and mouth disease in young children and infants. Given the limited treatments for EV-71 infection, discovering new host factors and understanding the mechanisms involved will aid in combating this viral infection. Neutral sphingomyelinase-2 (nSMase-2, encoded by SMPD3) is a crucial cellular cofactor in viral infection. We found that EV-71 infection increased nSMase-2 expression in African green monkey kidney cells (Vero cells). Knockdown of nSMase-2 by small interfering RNA obviously decreased the viral replication and infectivity. Furthermore, the knockdown of nSMase-2 reduced autophagy-associated proteins expression. Collectively, our findings uncovered a potential mechanism of nSMase-2 supporting EV-71 infection.

Enterovirus is a well-known cause of infection in all age groups, with particular importance for neonates with both vertically and horizontally transmission. Neonatal clinical manifestations are highly variable and mostly is asymptomatic, but severe infections are described such as myocarditis, meningitis, encephalitis, hepatitis, coagulopathy, pneumonia and viral sepsis. Since 2022, The World Health Organization has recently reported an increasing number of severe neonatal infection associated with a new variant of Echovirus-11 (E-11). Many of the infants described with E-11 infections in a case reports series were preterm, male and twins. Despite the criticality of neonatal disease, the clinical management remains primarily supportive and therapeutic options are unfortunately few.

We present the case of male dichorionic diamniotic late-preterm twins, born in December 2023 at Filippo del Ponte Hospital in Varese (Northen Italy) with early Echovirus-11 infection. They had two dramatically different clinical scenarios and one of them developed a severe and fatal hemorrhage-hepatitis syndrome.

Our experience has the purpose to emphasize public health attention to Echovirus-11 neonatal infections and their rare dramatically neonatal clinical presentations. Prematurity, host genetic predisposition and vertical transmission seem to be strong risk factors for severe infections, but it still need to be elucidated. According to previous Italian report, a surveillance protocols in all cases with unexpected clinical presentations and sequencing complete genome in order to better understand typing and molecular characterization of emerging and re-emerging pathogenic variants and new strains are strongly recommend in Italy.

Gastroparesis, a chronic digestive disorder characterized by delayed gastric emptying, often results from diabetes, post-surgical complications, autoimmune diseases, and neurological disorders. In approximately 50% of cases, the cause is idiopathic gastroparesis (IGD). Recent studies suggest a link between chronic enteroviral infection and persistent gastrointestinal symptoms, including delayed gastric emptying. This study investigates the effects of a silydianin-rich extract from Silybum marianum seeds on enteroviral infections in vitro and the mitigation of delayed gastric emptying in mice. Silydianin, a key bioactive compound known for its liver-protective and antioxidant properties, has not been extensively studied for its impact on enteroviral infections and gastroparesis.

NMR spectroscopy (1H, 13C, DEPT 135 and 2D, and HSQC) and HRMS identified silydianin as the primary compound, with minor flavonolignans. This study assessed the cytotoxicity and antiviral activity of the extract at various stages of the viral life cycle, including virucidal activity, cell protection, and post-infection effects, using neutral red assays in RD cells, with results confirmed by real-time PCR. The viruses studied included coxsackievirus B2, coxsackievirus A10, poliovirus SL-1, and enterovirus EV71. The impact on delayed gastric emptying was evaluated in a mouse model using doses of 100 and 200 mg/kg compared to a control group receiving physiological saline.

The silydianin-rich extract showed consistent antiviral activity, with the highest selectivity index (SI) for EV71 (4.08) during virucidal activity. It provided moderate cell protection, with EC50 values ranging from 120.88 to 186.10 µg/mL and SI values from 2.20 to 3.39. Post-infection treatment showed varying efficacy, with coxsackie A10 demonstrating the highest SI (3.90). In vivo, the extract at 200 mg/kg significantly improved gastric emptying to 96.47% and slightly increased gastrointestinal transit from 50.33% to 61.46%.

These results suggest that silydianin may be effective for treating enteroviral infections and enhancing intestinal function, making it a promising candidate for gastroparesis treatment and warranting further research.

Human Enterovirus A71 (EV-A71) is the primary pathogen responsible for severe hand, foot, and mouth disease (HFMD). Vaccination plays a crucial role in controlling its spread. Although inactivated vaccines have been approved, there is growing interest in developing new candidates using advanced platforms. mRNA vaccines, widely used for enveloped viruses, are less studied for non-enveloped viruses like EV-A71. This study investigates the potential of an mRNA vaccine targeting the EV-A71 VP1 protein.

A nucleoside-modified mRNA vaccine encoding the VP1 protein of EV-A71, encapsulated in lipid nanoparticles (LNPs), was developed. Immunogenicity and protective efficacy were evaluated in BALB/c and neonatal A129 mice, respectively. Immune responses were assessed by ELISA, micro-neutralization assays, ELISpot, and intracellular cytokine staining (ICS). Passive protection was tested by transferring immune sera to neonatal mice challenged with EV-A71.

The VP1 mRNA-LNP vaccine elicited robust humoral and cellular immunity, including high levels of VP1-specific IgG, neutralizing antibodies, and a Th1-biased T-cell response. Notably, the mRNA vaccine outperformed the inactivated vaccine in eliciting cellular immunity. Immune sera provided complete protection against lethal EV-A71 challenge, significantly reducing viral load and pathology.

This study demonstrates that the mRNA vaccine exhibits significant potential for combating non-enveloped viruses. These findings highlight the promising role of mRNA platforms in advancing vaccine development against non-enveloped viral pathogens, offering new avenues for future research and clinical applications.

Enterovirus A71 (EV-A71) is a common small RNA virus that is highly neuroinvasive. Emerging evidence indicates that the complement fragment C5a and its receptor C5aR1 are important drivers of neuroinflammation. However, the potential role of the C5a-C5aR1 axis in EV-A71 encephalitis remains largely elusive. Our previous studies revealed that EV-A71 can infect astrocytes and result in complement activation in vivo. Here, we investigated how complement factors interact with astrocytes to promote a severe inflammatory response upon EV-A71 infection. Our data revealed that EV-A71 infected mainly astrocytes and caused astrocyte activation in the mouse brain, which was further verified in patients with EV-A71 infection and U87-MG cells. Notably, EV-A71 infection led to activation of the C5a-C5aR1 axis in U87-MG cells, and knockdown (siC5aR1) or blockade (PMX53) of C5aR1 significantly suppressed EV-A71-induced astrocyte activation and proinflammatory cytokine (e.g., CXCL1) production. Next, the activation of the C5a-C5aR1 axis in mouse astrocytes was confirmed. Compared with C5aR1 knockout mice, wild-type mice presented more severe symptoms and lower survival rates after EV-A71 infection. C5aR1 deficiency or blockade significantly reduced EV-A71-induced pathological damage and proinflammatory cytokine production in the mouse brain. Importantly, an increased level of soluble C5a was strongly correlated with the severity of symptoms in patients with EV-A71 infection. By using confocal microscopy, primary astrocytes, and human specimens, we observed that the increase in CXCL1 levels resulted mainly from astrocytes. Neutralizing CXCL1 significantly alleviated the neuropathological changes caused by EV-A71 infection, and the production of CXCL1 in astrocytes was regulated by p38 MAPK signaling. Taken together, our findings indicate that the activation of the C5a-C5aR1 axis in astrocytes facilitates the neuropathological changes resulting from EV-A71 infection, emphasizing the potential role of p38 MAPK-mediated CXCL1 production in these alterations.

Enterovirus A71 (EV-A71) is a common small RNA virus with highly neuroinvasive tendencies. Our previous studies took the view that EV-A71 could infect astrocytes and result in complement activation in vivo. We investigated how complement interacts with astrocytes to promote a severe inflammatory response upon EV-A71 infection in the study. As expected, our data demonstrate that EV-A71 triggers robust activation of the C5a-C5aR1 axis in astrocytes and that knockout or blockade of C5aR1 in animals exposed to lethal doses of EV-A71 significantly enhances survival by diminishing the production of the chemokines CXCL1 and IL-6. In addition, neutralizing CXCL1 significantly alleviates the neuropathogenesis caused by EV-A71 infection. Thus, inhibiting the C5a-C5aR1 axis has emerged as a potential therapeutic strategy to mitigate neural damage caused by EV-A71 infection.

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease (HFMD), and can cause severe cerebral complications and even fatality in children younger than 5 years old. However, there is no specific medication for EV71 infection in clinical practice. Our previous studies had identified the 6-thioguanine (6-TG), an FDA-approved anticancer drug, as a potential antiviral agent, but its anti-EV71 activity is largely unknown, therefore, we aim to explore the antiviral effect of 6-TG on EV71.

6-TG significantly suppressed EV71 mRNA level, VP1 protein expression, and viral progeny production in HT-29 cells. In EV71-infected HT-29 cells, the 50% cytotoxicity concentration of 6-TG (CC50) was > 2000 µM and the 50% inhibitory concentration of 6-TG against EV71 (IC50) was 0.9302 µM. Interestingly, the selectivity index (SI) value of 6-TG against EV71 was > 2150.1, which was higher than the SI value (> 66.7) of ribavirin. Mechanistically, 6-TG treatment reduced the expression of baculoviral IAP repeat containing 3 (BIRC3), and further inhibited EV71 replication by attenuating BIRC3-mediated the complete autophagy.

6-TG exerted a significant inhibitory effect on EV71 infection in vitro and prevented EV71-induced the complete autophagy by decreasing BIRC3 expression. Our work provided a basis for the further development of 6-TG as a therapy for EV71-associated HFMD.

Enterovirus 71 (EV71) is a common pathogen responsible for hand, foot, and mouth disease (HFMD), leading to severe neurological complications and even death. However, the mechanisms underlying severe EV71-induced disease remain unclear, and no effective specific treatments are available. In this study, we successfully infected mice of different ages using a mouse-adapted EV71 strain, resulting in disease and mortality. We compared immune system responses between infected and uninfected mice of different ages to identify key pathogenic targets during EV71 infection. Our findings revealed that the level of Group 3 Innate Lymphoid Cells (ILC3s) in mice negatively correlated with the severity of disease induced by EV71 infection. We conducted anti-ILC3 cytokine injections and cytokine neutralizing antibody experiments on 14-day-old EV71-infected mice. The results showed that the cytokine IL-17 secreted by ILC3 cells had a mild protective effect, while IL-22 promoted inflammatory responses. Our research demonstrates that ILC3 cells play a dual role in EV71 infection. These findings not only clarify key immune factors in the progression of EV71-induced disease but also provide a promising approach for the early diagnosis and treatment of severe EV71 infections.

Human enterovirus 71 (EV-A71), a member of the Picornaviridae family, is predominantly associated with hand, foot, and mouth disease in infants and young children. Additionally, EV-A71 can cause severe neurological complications, including aseptic meningitis, brainstem encephalitis, and fatalities. The molecular mechanisms underlying these symptoms are complex and involve the viral tissue tropism, evasion from the host immune responses, induction of the programmed cell death, and cytokine storms. This review article delves into the EV-A71 life cycle, with a particular emphasis on recent advancements in understanding the virion structure, tissue tropism, and the interplay between the virus and host regulatory networks during replication. The comprehensive review is expected to contribute to our understanding of EV-A71 pathogenesis and inform the development of antiviral therapies and vaccines.

Exosomes, small extracellular vesicles secreted by various cells, play crucial roles in the pathogenesis and treatment of oral diseases. Recent studies have highlighted their involvement in orthodontics, periodontitis, oral squamous cell carcinoma (OSCC), and hand, foot, and mouth disease (HFMD). Exosomes have a positive effect on the inflammatory environment of the oral cavity, remodeling and regeneration of oral tissues, and offer promising therapeutic options for bone and periodontal tissue restoration. In OSCC tumor-derived exosomes promote cancer progression through cell proliferation, migration, invasion, and angiogenesis, and serve as potential biomarkers for early diagnosis and prognosis. Additionally, engineered exosomes constructed specifically based on exosome properties hold great promise for targeted drug delivery and regenerative therapies such as bone regeneration in orthodontics and periodontal healing. With continued research, exosomes hold great potential for improving diagnosis and treatment in oral diseases, advancing personalized and regenerative therapies.

Enterovirus 71 (EV71) is a major pathogen that causes hand, foot, and mouth disease (HFMD) in infants and children. Notably, no clinically approved drugs specifically target EV71. The EV71 2A protease (2Apro), a cysteine protease produced by the virus, is essential for the virus' replication and has a significant impact on the functioning of host cells. Thus, it presents a valuable target for the discovery of antiviral medications. In this study, based on the monomers and their derivatives in the Library of Traditional Chinese Medicine (TCM), we performed virtual screening and biological experiments. We identified a derivative of a traditional herbal monomer, Etoposide, commonly isolated from the roots and rhizomes of Podophyllum spp. Etoposide inhibited replication of EV71 A, B, C, and CVA16 viruses in a concentration-dependent manner in a variety of cell lines with minimal cytotoxicity. Furthermore, both molecular dynamics simulations and site-directed mutagenesis assays revealed that Etoposide inhibited the activity of the EV71 2A protease by mainly binding to two residues, Y89 and P107. The findings indicate that Etoposide serves as a promising inhibitor of the EV71 2Apro, demonstrating strong antiviral properties and positioning itself as a formidable candidate for clinical trials against EV71.IMPORTANCEWe first used a drug screening approach focused on monomeric compounds and their derivatives from traditional Chinese medicine to identify an EV71 2Apro inhibitor-Etoposide. We then performed biological experiments to validate that Etoposide suppresses the replication of the EV71 virus in a concentration-dependent manner with minimal cytotoxicity to various cell lines. Remarkably, it shows inhibitory activity against EV71 A, B, C, and CVA16, suggesting that Etoposide may be a potential broad-spectrum inhibitor. We revealed a novel mechanism that Etoposide inhibits EV71 proliferation by targeting 2Apro, and the interactions with Y89 and P107 are of great importance. The findings suggest that Etoposide serves as a promising inhibitor of EV71 2Apro, demonstrating significant antiviral properties. It stands out as a strong candidate for broad-spectrum applications in clinical research.

Inflammatory cells infiltration in the cerebrospinal fluid is a hallmark of severe enterovirus 71 (EV71) infection, but which type of immune cells are critical for severe EV71 infection remains unclear. Here, we observe that both neutrophils and macrophages are increased in the brains of patients and mice with severe EV71 infection, and the depletion of neutrophils but not macrophages results in a marked enhancement of survival of EV71-infected mice. Furthermore, CCR1/3 may play an important role in CCL3 facilitating the accumulation of neutrophils in the brains of patients. Inhibition of CCL3 by anti-CCL3 antibodies or selected miRNAs significantly reduces the neutrophils infiltration in brains and the mortality of EV71-infected mice. Collectively, CCL3-mediated neutrophils recruitment into the brain contributes to the severe immunopathology of EV71 infection, which provides a potential diagnostic and therapeutic target for EV71 infection.

Field-effect transistor (FET) sensors are attractive for the label-free detection of target biomolecules, offering ultrahigh sensitivity and a rapid response. However, conventional methods for modifying biomolecular probes on sensors often involve intricate and time-consuming procedures that require specialized training. Herein, we propose a simple and versatile approach to functionalize floating-gate (FG) FET sensors by exploiting the strong binding ability of polyvalent interactions and the three-dimensional structure of densely functionalized spherical nucleic acids (SNAs). Crucially, the SNAs can be easily deposited onto a dielectric layer under mild conditions, ensuring stable immobilization of the probes. Further, the SNAs show efficient and robust immobilization on various dielectric layers including Y2O3, Ta2O5, and HfO2, forming conjugates that resist denaturation by various agents. By modifying the DNA sequence within the SNAs, we achieved highly sensitive FG-FET biosensors for DNA, adenosine triphosphate, and viral nucleic acids at the attomolar level. For clinical samples detection, unamplified enterovirus 71 RNA at levels as low as 0.13 copies μL-1 was detected within 100 s. Moreover, the sensor attained 100% accuracy for analyte detection in both positive and negative samples. Our findings provide a general and simple method for fabricating FET-based biochemical sensors and demonstrate that the SNA-modified FG-FET biosensor is a versatile and reliable integrated platform for ultrasensitive biomarker detection.

Enterovirus-A71 (EV-A71) is the second most common causative agent after coxsackievirus A16 of hand, foot, and mouth disease. The capsids of EV-A71 consist of 60 copies of each of the four viral structural proteins (VP1-VP4). VP1 is highly exposed and surface accessible, playing a central role in virus particle assembly, attachment, and entry. To gain insight into the role of highly conserved residues at positions 75, 78, and 88 in the capsid protein VP1 in these processes, an alanine-scanning analysis was performed using an infectious cDNA clone of EV-A71. Our study revealed that the substitutions of VP1-T75A, VP1-T78A, and VP1-G88A could affect the assembly of the virus capsid proteins, resulting in the production of abnormal virions with reduced infectivity. Specifically, the substitution of VP1-T75A affected the maturation cleavage of the VP0 precursor, leading to deficiencies in binding to receptor scavenger receptor class B2 (SCARB2), viral attachment, internalization, and even uncoating. For the mutants of T78A and G88A, a significant reduction in virion-associated genomic RNA was observed, suggesting that more noninfectious empty particles were produced during viral assembly. Interestingly, the VP1-T75A variant showed weak replication in cell cultures but demonstrated increased virulence in BALB/c neonatal mice, which might be due to the difference in viral receptors among mammalian species. Taken together, our data revealed the important role of the highly conserved residues T75, T78, and G88 in VP1 protein in the infectivity of EV-A71. Characterizing these novel determinants of EV-A71 virulence would contribute to rationally developing effective treatments and broadly protective vaccine candidates.

EV-A71 causes hand, foot, and mouth disease in children. In this study, we discovered three highly conserved residues at positions 75, 78, and 88 of the capsid protein VP1 as the potential virulence determinants of EV-A71, which can influence viral replication by regulating the assembly of EV-A71. Mechanistic studies revealed that VP1-T75A could affect the maturation cleavage of the VP0 precursor, resulting in deficiencies in binding to the receptor SCARB2, viral attachment, internalization, and even uncoating. For the mutants of T78A and G88A, more noninfectious empty particles were produced during viral assembly. The discovery of these novel determinants of EV-A71 virulence will promote the study of the pathogenesis of enteroviruses.

Enteroviruses and rhinoviruses are highly diverse, with over 300 identified types. Reverse transcription-polymerase chain reaction (RT-PCR) assays targeting their VP1, VP4, and partial VP2 (VP4-pVP2) genomic regions are used for detection and identification. The VP4-pVP2 region is particularly sensitive to RT-PCR detection, making it efficient for clinical specimen analysis. However, a standard type identification method using this region is lacking. This study aimed to establish such a method by examining the divergence of VP4-pVP2 amino acid sequences between enterovirus and rhinovirus prototypes. Pairwise analysis of 249 types indicated a 95% threshold for enterovirus intra-species identification but not for rhinovirus prototypes. Protein BLAST search analyses of representative enterovirus prototypes, including EV-A71, EV-D68, CVA6, CVA10, CVA16, and polioviruses (PVs), validated the 95% threshold for typing, with a few exceptions such as PV1-PV2 and CVA6-CVA10, as well as some EV-C types. This study proposes a criterion for typing based on VP4-pVP2 amino acids, which can aid in rapid enterovirus diagnosis during routine clinical or environmental surveillance and emergency outbreaks. Our research confirms the reliability of the suggested VP4-pVP2-based threshold for typing and its potential application in laboratory settings.

Tauroursodeoxycholic acid (TUDCA) is a naturally occurring hydrophilic bile acid that alleviates endoplasmic reticulum (ER) stress and inhibits apoptosis, thereby protecting cells. Previous studies have shown that enterovirus 71 (EV71) infection modulates ER stress and induces autophagy to assist viral replication. This study observed the effects of TUDCA pretreatment on HeLa and Vero cells infected with EV71, finding that TUDCA inhibits EV71 replication in TUDCA pretreated HeLa and Vero cells in a dose-dependent manner. We found that TUDCA pretreatment inhibited EV71 replication by regulating three branches of UPR, that is up-regulated ATF6, down-regulated both PERK and IRE1. The results also indicated that autophagy which is downstream of UPR, was inhibited either. The results indicate that TUDCA inhibits EV71 replication by regulating UPR sensor proteins and autophagy following ER stress.

In the context of the near-global eradication of wild poliovirus, the significance of non-polio enteroviruses (NPEVs) in causing acute flaccid paralysis (AFP) and their impact on public health has gained increased attention. This research, conducted from 2001 to 2021, examined stool samples from 1597 children under 15 years in São Paulo, Brazil, through the AFP/Poliomyelitis Surveillance Program, detecting NPEVs in 6.9% of cases. Among the 100 NPEV-positive strains analyzed, 90 were genotyped through genomic sequencing of the partial VP1 region, revealing a predominance of EV-B species (58.9%), followed by EV-A (27.8%) and EV-C (13.3%). This study identified 31 unique NPEV types, including EV-A71, CVB2, and E11, as the most prevalent, along with the first documented occurrence of CVA19 in Brazil. These findings emphasize the importance of NPEV genotyping in distinguishing AFP from poliomyelitis, enhancing understanding of these viruses' epidemiology. Moreover, it ensures that AFP cases are correctly classified, contributing to the effective surveillance and eradication efforts for poliomyelitis.

Enterovirus A71 (EV-A71) is the main pathogen causing hand, foot and mouth disease (HFMD) in children and occasionally associated with neurological diseases such as aseptic meningitis, brainstem encephalitis (BE) and acute flaccid paralysis. We report here that cellular pseudokinase tribbles 3 (TRIB3) facilitates the infection of EV-A71 via dual mechanisms. In one hand, TRIB3 maintains the metabolic stability of scavenger receptor class B member 2 (SCARB2), the bona fide receptor of EV-A71, to enhance the infectious entry and spreading of the virus. On the other hand, TRIB3 facilitates the replication of EV-A71 RNA in a SCARB2-independent manner. The critical role of TRIB3 in EV-A71 infection and pathogenesis was further demonstrated in vivo in mice. In comparison to wild-type C57BL/6 mice, EV-A71 infection in TRIB3 knockdown mice (Trib3+/-) resulted in significantly lower viral loads in muscular tissues and reduced lethality and severity of clinical scores and tissue pathology. In addition, TRIB3 also promoted the replication of coxsackievirus B3 (CVB3) and coxsackievirus A16 (CVA16) in vitro. In conclusion, our results suggest that TRIB3 is one of key host cellular proteins required for the infection and pathogenesis of EV-A71 and some other human enteroviruses and may thus be a potential therapeutic target for combating the infection of those viruses.

Enteroviruses (EV) are common and can cause severe diseases, particularly in young children. However, the information of EV infection in infants in China is limited due to the vast population size and extensive geographical area of the country. Here, we conducted a retrospective multicenter analysis of available EV data to assess the current epidemiological situation in the infant population in southern China.

The study enrolled infants with suspected EV infection from 34 hospitals across 12 cities in southern China between 2019 to 2022, and the confirmation of EV was done using RT-PCR and VP1 gene sequencing.

Out of 1221 infants enrolled, 330 (27.03%) were confirmed as EV-infected. Of these, 260 (78.79%) were newborns aged 0-28 days. The EV belonged to three species: EV-B (80.61%), EV-A (11.82%), and human rhinovirus (7.58%). Newborns were more susceptible to EV-B than older infants (p < 0.001). Within EV-B, we identified 15 types, with coxsackievirus (CV) B3 (20.91%), echovirus (E) 11 (19.70%), and E18 (16.97%) being the most common. The predominant EV types changed across different years. EV infection in infants followed a seasonal pattern, with a higher incidence from May to August. Furthermore, perinatal mother-to-child EV transmission in 12 mother-newborn pairs were observed.

Our study is the first to demonstrate the emergence and widespread circulation of EV-B species, mainly CVB3, E11, and E18, in southern China, primarily affecting young infants. This research provides valuable insights for future epidemic assessment, prediction, as well as the elimination of mother-to-child transmission.

The Picornaviridae are a large group of positive-sense, single-stranded RNA viruses, and most research has focused on the Enterovirus genus, given they present a severe health risk to humans. Other picornaviruses, such as foot-and-mouth disease virus (FMDV) and senecavirus A (SVA), affect agricultural production with high animal mortality to cause huge economic losses. The 3Dpol protein of picornaviruses is widely known to be used for genome replication; however, a growing number of studies have demonstrated its non-polymerase roles, including modulation of host cell biological processes, viral replication complex assembly and localization, autophagy, and innate immune responses. Currently, there is no effective vaccine to control picornavirus diseases widely, and clinical therapeutic strategies have limited efficiency in combating infections. Many efforts have been made to develop different types of drugs to prohibit virus survival; the most important target for drug development is the virus polymerase, a necessary element for virus replication. For picornaviruses, there are also active efforts in targeted 3Dpol drug development. This paper reviews the interaction of 3Dpol proteins with the host and the progress of drug development targeting 3Dpol.

The public health and social measures (PHSMs) for mitigation/control of COVID-19 pandemic influenced the transmission dynamics of many other infectious diseases, including respiratory syncytial virus (RSV) infection, and hand, foot and mouth disease (HFMD) and their disease-burden. This study aimed to infer the transmission dynamics of these respiratory viruses and assess the impact of COVID-19 PHSMs on their community activity. We developed a compartmental framework to infer the transmission dynamics of RSV and HFMD in Hong Kong and South Korea from January 2014 to May 2024. We assessed the impact of PHSMs by comparing the change in virus transmissibility, reproduction number and population susceptibility before, during, and after the COVID-19 pandemic period. A significant reduction in RSV and HFMD activity was observed starting in January 2020, with a resurgence since late 2021. Transmissibility of both diseases decreased by 46 % - 95 % during the lull, while population susceptibility was estimated to increase by maximum of 19 %. On relaxation of the PHSMs, the transmissibility were recovered up to 70 % in Hong Kong and nearly 100 % in South Korea in 2023 with significant epidemics for these viruses. Strict implementation of COVID-19 PHSMs led to low RSV and HFMD activity, but the absence of community infection resulted in reductions in population immunity, and slightly larger epidemics when these diseases re-emerged following the COVID-19 pandemic.

RNA modification is a key posttranscriptional process playing various biological roles, and one which has been reported to exist extensively in cellular RNAs. Interestingly, recent studies have shown that viral RNAs also contain a variety of RNA modifications, which are regulated dynamically by host modification machinery and play critical roles in different stages of the viral life cycle. In this review, we summarize the reports of four typical modifications reported on viral RNAs, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), N4-acetylcytosine (ac4C), and N1-methyladenosine (m1A), describe the molecular mechanisms of these modification processes, and illustrate their impacts on viral replication, pathogenicity, and innate immune responses. Notably, we find that RNA modifications in different viruses share some common features and mechanisms in their generation, regulation, and function, highlighting the potential for viral RNA modifications and the related host machinery to serve as the targets or bases for the development of antiviral therapeutics and vaccines.

Enterovirus A71 (EV-A71) is a major cause of hand-foot-and-mouth disease (HFMD), particularly in cases that involve complications affecting the nervous system or cardiopulmonary function. In South America, EV-A71 has primarily been identified through studies of acute flaccid paralysis (AFP) and other neurological disorders. In September 2022, two children from a small city in Uruguay were hospitalized with presumptive rhombencephalitis, exhibiting symptoms of HFMD. EV-A71 was identified through RT-PCR and next-generation sequencing of stool and skin lesion samples. A maximum-likelihood phylogenetic analysis of the P1 coding region classified the Uruguayan strains as part of an emerging lineage, primarily reported in Europe over the past decade, known as the C1-like lineage. The findings presented here represent the first detection of the EV-A71 C1-like lineage in cases of HFMD and encephalitis reported from South America, underscoring the urgent need to enhance surveillance for HFMD, aseptic meningitis, encephalitis, and AFP, in countries facing challenges in establishing effective surveillance programs related to enteroviruses and associated diseases.

Coxsackievirus (CV) A6 has emerged as an important causative agent in global outbreaks of hand, foot, and mouth disease (HFMD), which typically presents as a mild illness with a large generalized rash, herpes. However, some patients can develop encephalitis, pneumonia, myocarditis and liver injury. Our previous study took the view that CVA6 could replicate in mouse liver, leading to acute liver injury; however, the precise underlying mechanism remains elusive.

10-day-old wild-type (WT, C57BL/6J) and NLRP3 knock-out (KO) mice were intraperitoneal (i.p.) inoculated with a lethal dose of the CVA6 strain. The muscle homogenate supernatant from normal mice was used to inoculate mock-infected mice. At 5 days post infection (dpi), the mouse liver was taken out for histopathological analyses and molecular biology experiments.

Our in vivo experiments demonstrated that CVA6 caused severe liver injury in mice, as evidenced by pathological changes in liver slices, elevated liver injury markers (e.g., AST, ALT, LDH) and pro-inflammatory cytokines (e.g., IL-6, MCP-1, TNF-α, IL-1β). Further results revealed the activation of NLRP3 inflammasome characterized by the increase in the expression of NLRP3, Cleaved-Casp-1 (p20), mature IL-1β and IL-18. Importantly, upon CVA6 infection, NLRP3 KO mice exhibited attenuated pathological damage and reduced levels of pro-inflammatory cytokines production (e.g., TNF-α and IL-1β) compared with WT mice. Finally, increased levels of blood ALT, AST, LDH were strongly correlated with the severity of CVA6 patients.

Collectively, our findings suggest that the activation of NLRP3 inflammasome is involved in CVA6 infection-induced acute liver injury, providing novel insights into CVA6 infection associated adverse clinical outcomes.