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Enterovirus A71 (EV-A71) and coxsackievirus A6 (CVA6) primarily cause hand, foot and mouth disease and have emerged to cause potential fatal neurological and systemic manifestations. However, limited surveillance data collected through passive surveillance systems hampers characterization of their epidemiological dynamics. We fit a series of catalytic models to age-stratified seroprevalence data for EV-A71 and CVA6 collected in England at three time points (2006, 2011 and 2017) to estimate the force of infection (FOI) over time and assess possible changes in transmission. For both serotypes, model comparison does not support the occurrence of important changes in transmission over the study period, and we find that a declining risk of infection with age and / or seroreversion are needed to explain the seroprevalence data. Furthermore, we provide evidence that the increased number of reports of CVA6 during 2006-2017 is unlikely to be explained by changes in surveillance. Therefore, we hypothesize that the increased number of CVA6 cases observed since 2011 must be explained by increased virus pathogenicity. Further studies of seroprevalence data from other countries would allow to confirm this. Our results underscore the value of seroprevalence data to unravel changes in the circulation dynamics of pathogens with weak surveillance systems and large number of asymptomatic infections., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kamau et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In this study, we analyzed the dynamic molecular epidemiology of herpangina based on pharyngeal swabs and demographic data collected from children with herpangina monitored in Tongzhou district in China from January 2021 to December 2022. A total of 1022 herpangina cases were diagnosed. Out of 225 samples collected, 56.4% (127/225) were positive for non-polio enterovirus, with seven genotypes identified: coxsackievirus A4 (CV-A4), CV-A6, CV-A10, CV-A2, CV-A16, CV-B3, and CV-A8. The predominant genotypes associated with herpangina changed during and after the COVID-19 pandemic, with the predominant genotypes being CV-A4 and CV-A6 in 2021 and CV-A10 and CV-A6 in 2022., Competing Interests: Declarations Conflict of interest The authors declare there are no competing interests. Ethical approval All of the participants provided informed consent. The Peking University Institutional Review Board Office granted ethical approval to carry out the study within its facilities (IRB00001052-19005)., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Neural stem cells have intact innate immune responses that protect them from virus infection and cell death. Yet, viruses can antagonize such responses to establish neuropathogenesis. Using a forebrain organoid model system at two developmental time points, we identified that neural stem cells, in particular radial glia, are basally primed to respond to virus infection by upregulating several antiviral interferon-stimulated genes. Infection of these organoids with a neuropathogenic Enterovirus-D68 strain, demonstrated the ability of this virus to impede immune activation by blocking interferon responses. Together, our data highlight immune gene signatures present in different types of neural stem cells and differential viral capacity to block neural-specific immune induction., (© 2024. The Author(s).)

Enterovirus A71 (EVA71) is a leading causative agent of hand, foot, and mouth disease, posing a significant threat to the health of young children, particularly in the Asia-Pacific region. Currently, there is no specific antiviral drug for EVA71 infection; therefore, early and rapid diagnosis is critical for disease prevention and control. Here, we report the development of a simple, rapid, and sensitive detection method for EVA71 infection using reverse transcription-multiple cross displacement amplification (RT-MCDA) combined with nanoparticle-based lateral flow biosensors (LFB). In the RT-MCDA system, a set of 10 primers was designed to target the highly conserved region of the VP1 gene of EVA71 and amplify the genes in an isothermal amplification device. The RT-MCDA amplification reaction products could then be identified by visual detection reagent (VDR) and LFB without the need for specialized equipment. The results demonstrated that the optimal reaction condition for the EVA71-RT-MCDA assay was 65℃ for 40 min. The EVA71-RT-MCDA assay could detect as low as 40 copies of plasmid and 50 copies of pseudotyped virus in a reaction. No cross-reaction was found between EVA71 strains and non-EVA71 strains. For 125 clinical anal swab samples, with EVA71-RT-MCDA assay, 30 samples were positive, which was in consistent with the the conventional real-time quantitative reverse transcription polymerase chain reaction assays. The entire procedure, including a 15-min specimen processing step, a 40-min MCDA reaction, and result reporting within 2 min, was completed in less than 60 min. In conclusion, the EVA71-RT-MCDA-LFB assay targeting the VP1 gene is a rapid, highly sensitive, simple, and specific test that could be widely applied in point-of-care settings and basic medical facilities in rural areas., (© 2024 Wiley Periodicals LLC.)

Enteroviruses (EV) initiate replication by binding to their cellular receptors, leading to the uncoating and release of the viral genome into the cytosol of the host cell. Neutralising antibodies (NAbs) binding to epitopes on enteroviral capsid proteins can inhibit this infectious process through several mechanisms of neutralisation in vitro. Fc-mediated antibody effector functions such as antibody-dependent cell-mediated cytotoxicity and antibody-dependent cellular phagocytosis have also been described for some EV. However, antibody binding to virions does not always result in viral neutralisation. Non-neutralising antibodies, or sub-neutralising concentrations of antibodies, can enhance infection of viruses, leading to more severe pathologies. This phenomenon, known as antibody-dependent enhancement (ADE) of infection, has been described in vitro and/or in vivo for EV including poliovirus, coxsackievirus B and EV-A71. It has been shown that ADE of EV infection is mediated by FcγRs expressed by monocytes, macrophages, B lymphocytes and granulocytes. Antibodies play a crucial role in the diagnosis and monitoring of infections. They are valuable markers that have been used to establish a link between enteroviral infection and chronic diseases such as type 1 diabetes. Monoclonal and polyclonal antibodies targeting enteroviral proteins have been developed and shown to be effective to prevent or combat EV infections in vitro and in vivo. In addition, vaccines are under development, and clinical trials of vaccines are underway or have been completed, providing hope for the prevention of diseases due to EV. However, the ADE of the infection should be considered in the development of anti-EV antibodies or safe vaccines., (© 2024 The Author(s). Reviews in Medical Virology published by John Wiley & Sons Ltd.)

Congregate homeless shelters are disproportionately affected by infectious disease outbreaks. We describe enterovirus epidemiology across 23 adult and family shelters in King County, Washington, USA, during October 2019-May 2021, by using repeated cross-sectional respiratory illness and environmental surveillance and viral genome sequencing. Among 3,281 participants >3 months of age, we identified coxsackievirus A21 (CVA21) in 39 adult residents (3.0% [95% CI 1.9%-4.8%] detection) across 7 shelters during October 2019-February 2020. We identified enterovirus D68 (EV-D68) in 5 adult residents in 2 shelters during October-November 2019. Of 812 environmental samples, 1 was EV-D68-positive and 5 were CVA21-positive. Other enteroviruses detected among residents, but not in environmental samples, included coxsackievirus A6/A4 in 3 children. No enteroviruses were detected during April 2020-May 2021. Phylogenetically clustered CVA21 and EV-D68 cases occurred in some shelters. Some shelters also hosted multiple CVA21 lineages.

Enteroviruses contain multiple serotypes and can cause severe neurological complications. The intricate life cycle of enteroviruses involving dynamic virus-receptor interaction hampers the development of broad therapeutics and vaccines. Here, using function-based screening, we identify a broadly therapeutic antibody h1A6.2 that potently protects mice in lethal models of infection with both enterovirus A71 and coxsackievirus A16 through multiple mechanisms, including inhibition of the virion-SCARB2 interactions and monocyte/macrophage-dependent Fc effector functions. h1A6.2 mitigates inflammation and improves intramuscular mechanics, which are associated with diminished innate immune signalling and preserved tissue repair. Moreover, cryogenic electron microscopy structures delineate an adaptive binding of h1A6.2 to the flexible and dynamic nature of the VP2 EF loop with a binding angle mimicking the SCARB2 receptor. The coordinated binding mode results in efficient binding of h1A6.2 to all viral particle types and facilitates broad neutralization of enterovirus, therefore informing a promising target for the structure-guided design of pan-enterovirus vaccine., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Enteroviruses, which infect via the gut, have been implicated in type 1 diabetes (T1D) development. Prolonged faecal shedding of enterovirus has been associated with islet autoimmunity. Additionally, enteroviral proteins and viral RNA have been detected in the pancreatic islets of individuals with recent-onset T1D, implicating their possible role in beta cell destruction. Despite this, no approved antiviral drugs currently exist that specifically target enterovirus infections for utilisation in disease interventions. Drug repurposing allows for the discovery of new clinical uses for existing drugs and can expedite drug discovery. Previously, the cancer drug Vemurafenib demonstrated unprecedented antiviral activity against several enteroviruses. In the present study, we assessed the efficacy of Vemurafenib and an analogue thereof in preventing infection or reducing the replication of enteroviruses associated with T1D. We tested Vemurafenib in intestinal epithelial cells (IECs) and insulin-producing beta cells. Additionally, we established a protocol for infecting human stem cell-derived islets (SC-islets) and used Vemurafenib and its analogue in this model. Our studies revealed that Vemurafenib exhibited strong antiviral properties in IECs and a beta cell line. The antiviral effect was also seen with the Vemurafenib analogue. SC-islets expressed the viral receptors CAR and DAF, with their highest expression in insulin- and glucagon-positive cells, respectively. SC-islets were successfully infected by CVBs and the antiviral activity of Vemurafenib and its analogue was confirmed in most SC-islet batches. In summary, our observations suggest that Vemurafenib and its analogue warrant further exploration as potential antiviral agents for the treatment of enterovirus-induced diseases, including T1D., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Type 1 diabetes (T1D), a severe disease requiring intensive insulin treatment, carries an increased risk for complications and reduced lifespan. Certain viruses have been implicated in T1D's etiology, with 'live', replicating enteroviruses (EVs) recently found in the pancreas at diagnosis. This discovery prompted a trial to slow down disease progression using antiviral drugs. A 6-month treatment combining pleconaril and ribavirin in new-onset T1D patients preserved residual insulin production after 1 year, unlike placebo. The results support the theory that viruses may cause T1D in genetically susceptible individuals. A low-grade, persistent viral infection may initiate a cascade of pathogenic mechanisms initially involving the innate immune system, inducing β-cell stress and neoantigen release, leading to autoimmunity, and eventually the destruction of insulin-producing β-cells., Competing Interests: Declaration of interests The author has no conflicts of interest to declare., (Copyright © 2024 The Author. Published by Elsevier Ltd.. All rights reserved.)

Enterovirus D68 (EV-D68), a respiratory RNA virus in the family Picornaviridae, is implicated as a potential etiological agent for acute flaccid myelitis in preteen adolescents. The absence of a specific therapeutic intervention necessitates the development of an effective animal model for EV-D68. The AG129 mouse strain, characterized by the double knockout of IFN-α/β and IFN-γ receptors on the 129 genetic background, has been proposed as a suitable model for EV-D68. The goals of this study were to assess the effect of a nonmouse-adapted EV-D68 strain (US/MO/14-18947, NR-49129) in AG129 (IFN-α/β and IFN-γ receptors null), A129 (IFN-α/β receptor null), G129 (IFN-γ receptor null), and the 129 background strain (129S2/SvPasCrl) when infected intraperitoneally at 10 d of age. Both AG129 and A129 strains demonstrated similar clinical signs (paralysis, paresis, lethargy, dyspnea [characterized by prominent abdominal respiration], and morbidity requiring euthanasia) induced by EV-D68. While G129 and 129S2 strains also exhibited susceptibility to EV-D68, the severity of clinical signs was less than in AG129 and A129 strains, and many survived to the experimental endpoint. Histopathological and immunohistochemical data confirmed EV-D68 tropism for the skeletal muscle and spinal cord and suggest that the dyspnea observed in infected mice could be attributed, in part, to lesions in the diaphragmatic skeletal muscles. These findings contribute valuable insights into the pathogenesis of EV-D68 infection in this mouse model and provide investigators with key information on virus dose and mouse strain selection when using this mouse model to evaluate candidate EV-D68 therapeutics.

Background: Hirschsprung's disease (HSCR) is a congenital enteric nervous system (ENS) disorder. Genetics cannot explain most sporadic cases. To explore the relationship between pathogen infection, autoantibodies, innate immune, and HSCR. Methods: Pathogen microarray was conducted in the serum of the prospective neonatal abdominal distension (NAD) cohort, consisting of 56 children followed for at least 6 months until the final diagnosis of HSCR was determined or excluded. We conducted an autoantibody microarray in an HSCR cohort, which is comprised of diagnosed HSCR patients (HSCR) and healthy control subjects (HC). RNA-seq of colon tissues from aganglionic and ganglionic segments of HSCR patients was performed. Results: Experimental results show that the serum lgM and lgG of enterovirus 71 (EV71) were significantly higher in HSCR than in the gastrointestinal dysfunction (GI) group, with a prediagnose value reaching area under the curve (AUC) over 0.76. We discovered that a group of autoantibodies were significantly higher in HSCR including neuronal pentraxin 1 (NPTX1), amyloid, neuron lysate, and myelin-associated oligodendrocytic basic protein (MOBP) than that in the HC group. These four autoantibodies could distinguish HSCR from the HC group, with a combined AUC of over 0.90 using both serum IgG and IgM. Further analysis showed that wide activation of innate immune pathways, including toll-like receptor (TLR) signaling pathway, neutrophil-to-lymphocyte ratio (NLR) signaling pathway, red cell distribution width to lymphocyte ratio (RLR) signaling pathway, and cyclic adenosine monophosphate (cAMP) signaling pathway in aganglionic compared to ganglionic segments of HSCR. Conclusion: This study suggested that virus-triggered autoimmunity may contribute to HSCR through activation of innate immunity, which facilitates the diagnosis and prevention of HSCR., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Weiyong Zhong et al.)

Because host kinases are key regulators of multiple signaling pathways in response to viral infections, we previously screened a kinase inhibitor library using rhabdomyosarcoma cells and human intestinal organoids in parallel to identify potent inhibitors against EV-A71 infection. We found that Rho-associated coiled-coil-containing protein kinase (Rock) inhibitor efficiently suppressed the EV-A71 replication and further revealed Rock1 as a novel EV-A71 host factor. In this study, subsequent analysis found that a variety of vascular endothelial growth factor receptor (VEGFR) inhibitors also had potent antiviral effects. Among the hits, Pazopanib, with a selectivity index as high as 254, which was even higher than that of Pirodavir, a potent broad-spectrum picornavirus inhibitor targeting viral capsid protein VP1, was selected for further analysis. We demonstrated that Pazopanib not only efficiently suppressed the replication of EV-A71 in a dose-dependent manner, but also exhibited broad-spectrum anti-enterovirus activity. Mechanistically, Pazopanib probably induces alterations in host cells, thereby impeding viral genome replication and transcription. Notably, VEGFR2 knockdown and overexpression suppressed and facilitated EV-A71 replication, respectively, indicating that VEGFR2 is a novel host dependency factor for EV-A71 replication. Transcriptome analysis further proved that VEGFR2 potentially plays a crucial role in combating EV-A71 infection through the TSAd-Src-PI3K-Akt pathway. These findings expand the range of potential antiviral candidates of anti-enterovirus therapeutics and suggest that VEGFR2 may be a key host factor involved in EV-A71 replication, making it a potential target for the development of anti-enterovirus therapeutics., Importance: As the first clinical case was identified in the United States, EV-A71, a significant neurotropic enterovirus, has been a common cause of hand, foot, and mouth disease (HFMD) in infants and young children. Developing an effective antiviral agent for EV-A71 and other human enteroviruses is crucial, as these viral pathogens consistently cause outbreaks in humans. In this study, we demonstrated that multiple inhibitors against VEGFRs effectively reduced EV-A71 replication, with Pazopanib emerging as the top candidate. Furthermore, Pazopanib also attenuated the replication of other enteroviruses, including CVA10, CVB1, EV-D70, and HRV-A, displaying broad-spectrum anti-enterovirus activity. Given that Pazopanib targets various VEGFRs, we narrowed the focus to VEGFR2 using knockdown and overexpression experiments. Transcriptomic analysis suggests that Pazopanib's potential downstream targets involve the TSAd-Src-PI3K-Akt pathway. Our work may contribute to identifying targets for antiviral inhibitors and advancing treatments for human enterovirus infections., Competing Interests: The authors declare no conflict of interest.

Enterovirus 71 (EV71) belongs to the family of Picornaviridae ; it could cause a variety of illnesses and pose a great threat to public health worldwide. Currently, there is no specific drug treatment for this virus, and a better understanding of virus-host interaction is crucial for novel antiviral development. Here, we find that the class III phosphatidylinositol 3-kinase, VPS34, is an essential host factor for EV71 infection. VPS34 inhibition with either shRNA or specific chemical inhibitor significantly reduces EV71 infection. Meanwhile, EV71 infection upregulates phosphatidylinositol 3-phosphate (PI3P) production in viral replication organelles (ROs), while the depletion of PI3P by phosphatase overexpression inhibits EV71 infection. In addition, the PI3P-binding protein, double FYVE-containing protein 1 (DFCP1), is also required for an efficient replication of EV71. DFCP1 could interact with viral 2C protein and facilitate viral association with lipid droplets (LDs), which are important lipid sources for viral RO biogenesis. Taken together, these results indicate that EV71 virus exploits the VPS34-PI3P-DFCP1-LDs pathway to promote viral RO formation and viral infection, and they also illuminate novel targets for antiviral development.IMPORTANCEEnterovirus 71 (EV71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD) and other serious complications, which are big threats to children under 5 years old. Unravelling the interactions between virus and the host cells will open new avenues in antiviral research. Here, we found the class III phosphatidylinositol 3-kinase, VPS34, and its effector, double FYVE-containing protein 1 (DFCP1), were essential for EV71 infection, both of which could support EV71 viral replication by enhancing the biogenesis of viral replication organelles (ROs). As DFCP1 localizes to lipid droplets, hijacking of these host factors will enable viral utilization of lipids from LDs for the generation of membrane structures during RO biogenesis. In addition, the VPS34 kinase inhibitor was found to be potent against EV71 infection; therefore, this study also brings up a novel target for future anti-EV71 drug development., Competing Interests: The authors declare no conflict of interest.

Ubiquitin modification of viral proteins to degrade or regulate their function is one of the strategies of the host to resist viral infection. Here, we report that ubiquitin protein ligase E3C (UBE3C), an E3 ubiquitin ligase, displayed inhibitory effects on EV-A71 replication. UBE3C knockdown resulted in increased viral protein levels and virus titers, whereas overexpression of UBE3C reduced EV-A71 replication. To explore the mechanism by which UBE3C affected EV-A71 infection, we found that the C-terminal of UBE3C bound to 2C protein and facilitated K33/K48-linked ubiquitination degradation of 2C K268. Moreover, UBE3C lost its ability to degrade 2C K268R and had a diminished inhibitory impact against the replication of recombinant EV-A71-FY-2C K268R. In addition, UBE3C also promoted ubiquitination degradation of the 2C protein of CVB3 and CVA16 and inhibited viral replication. Thus, our findings reveal a novel mechanism that UBE3C acts as an enterovirus host restriction factor, including EV-A71, by targeting the 2C protein., Importance: The highly conserved 2C protein of EV-A71 is a multifunctional protein and plays a key role in the replication cycle. In this study, we demonstrated for the first time that UBE3C promoted the degradation of 2C K268 via K33/K48-linked ubiquitination, thereby inhibiting viral proliferation. Our findings advance the knowledge related to the roles of 2C in EV-A71 virulence and the ubiquitination pathway in the host restriction of EV-A71 infection., Competing Interests: The authors declare no conflict of interest.

Enteroviruses have been a historical concern since the identification of polioviruses in humans. Wild polioviruses have almost been eliminated, while multiple species of non-polio enteroviruses and their variants co-circulate annually. To date, at least 116 types have been found in humans and are grouped into the species Enterovirus A-D and Rhinovirus A-C. However, there are few available antiviral drugs, especially with a universal pharmaceutical effect. Here, we demonstrate that peptide P25 from EV-D68 has broad antiviral activity against EV A-D enteroviruses in vitro. P25, derived from the HI loop and β-I sheet of VP1, operates through a conserved hydrophilic motif -R---K-K--K- and the hydrophobic F near the N-terminus. It could prevent viral infection of EV-A71 by competing for the heparan sulfate (HS) receptor, binding and stabilizing virions by suppressing the release of the viral genome. P25 also inhibited the generation of infectious viral particles by reducing viral protein synthesis. The molecular docking revealed that P25 might bind to the pocket opening area, a potential target for broad-spectrum antivirals. Our findings implicate the multiple antiviral effects of peptide P25, including blocking viral binding to the HS receptor, impeding viral genome release, and reducing progeny particles, which could be a novel universal anti-enterovirus drug candidate.

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 infections and its clinical impact during the fall-winter season of 2021-2022. From 19 European countries, 58 institutes reported 10 481 (6.8%) EV-positive samples of which 1004 (9.6%) were identified as EV-D68 (including 852 respiratory samples). Clinical data were reported for 969 cases; 78.9% of infections were reported in children (0-5 years); and 37.9% of cases were hospitalized. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases including 6 diagnosed with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of 2 novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale European EV-D68 upsurge with severe clinical impact and the emergence of B3-derived lineages., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The intricate connection between the gut and the brain involves multiple routes. Several viral families begin their infection cycle in the intestinal tract. However, amongst the long list of viral intestinal pathogens, picornaviruses, and astroviruses stand out for their ability to transition from the intestinal epithelia to central or peripheral nervous system cells. In immunocompromised, neonates and young children, these viral infections can manifest as severe diseases, such as encephalitis, meningitis, and acute flaccid paralysis. What confers this remarkable plasticity and makes them efficient in infecting cells of the gut and the brain axes? Here, we review the current understanding of the virus infection along the gut-brain axis for some enteric viruses and discuss the molecular mechanisms of their attenuation., (© 2024. The Author(s).)

We report a considerable increase in enterovirus D68 (EV-D68) cases since July 2024, culminating in an ongoing outbreak of acute respiratory infections in northern Italy, accounting for nearly 90% of all enterovirus infections. The outbreak was identified by community- and hospital-based surveillance systems, detecting EV-D68 in individuals with mild-to-severe respiratory infections. These strains belonged to B3 and a divergent A2 lineage. An increase in adult cases was observed. Enhanced surveillance and molecular characterisation of EV-D68 across Europe are needed.

To clarify the epidemiology of enterovirus D68 (EV-D68), an enterovirus rarely identified in the 20th century, we performed seroepidemiological analysis against EV-D68 using sera collected in 1976, 1985, 1990, 1999, 2009, and 2019, as well as Yamagata isolate (EVD68/Yamagata.JPN/2023-89), in Yamagata, Japan. The neutralizing antibody (Ab)-positive rates for those under 20 years old were 61.0%, 82.5%, 84.3%, 46.7%, 50.5%, and 67.9%, in each year, whereas the rates for those above 20 years old were between 93.4% and 99.1%. Generally, geometric mean titers (GMTs）increased with age among children and the total GMT in each year was 25.4, 49.2, 37.2, 30.8, 29.5, and 33.9, from 1976 to 2019, respectively. The findings in this Yamagata-based study showed that the seroprevalence of EV-D68 over the last four decades has increased with age among children, as a susceptible group, and then reaches a plateau of over approximately 80% among adults. This study clearly revealed that EV-D68 was stably transmitted among children in the 20th century, when EV-D68 detection was quite rare., (© 2024 Wiley Periodicals LLC.)

Human Enterovirus 71 (EV71) has emerged as one of the predominant causative agents of hand, foot and mouth disease (HFMD) with global impact. Despite the inactivated vaccine being licensed, other vaccine candidates based on advanced technology platforms are under development. In this report, we rationally designed and constructed two DNA-launched live attenuated vaccine candidates (pDL-EV71) under the control of specific promoters. In vitro and in vivo transfection with pDL-EV71 driven by the CMV promoter successfully yielded fully infectious EV71. More importantly, the administration of pDL-EV71 did not cause clinical symptoms following intracranial or intramuscular inoculation in neonatal and IFNα/βR -/- mice, demonstrating its safety profile. Moreover, a single-dose or two-dose immunization with pDL-EV71 elicited robust neutralizing antibodies against EV71 as well as an antigen-specific cellular response in mice. A single-dose immunization with 10 ​μg of pDL-EV71 conferred complete protection against lethal EV71 infection in neonates born to immunized maternal mice. Overall, our present results demonstrate that pDL-EV71 is a safe and effective vaccine candidate against EV71 for further development., Competing Interests: Conflict of interest Prof. Cheng-Feng Qin is an editorial board member for Virologica Sinica and was not involved in the editorial review or the decision to publish this article. All authors declare that there are no competing interests., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Innate immune cells have been acknowledged as trainable in recent years. While intestinal tuft cells are recognized for their crucial roles in the host defense against intestinal pathogens, there remains uncertainty regarding their trainability. Enterovirus 71 (EV71), a prevalent enterovirus that primarily infects children but rarely infects adults. At present, there is a significant expansion of intestinal tuft cells in the EV71-infected mouse model, which is associated with EV71-induced interleukin-25 (IL-25) production. Further, we found that IL-25 pre-treatment at 2 weeks old mouse enabled tuft cells to acquire immune memory. This was evidenced by the rapid expansion and stronger response of IL-25-trained tuft cells in response to EV71 infection at 6 weeks old, surpassing the reactivity of naïve tuft cells in mice without IL-25-trained progress. Interestingly, IL-25-trained intestinal tuft cells exhibit anti-enteroviral effect via producing a higher level of IL-25. Mechanically, IL-25 treatment upregulates spermidine/spermine acetyl-transferase enzyme (SAT1) expression, mediates intracellular polyamine deficiency, further inhibits enterovirus replication. In summary, tuft cells can be trained by IL-25, which supports faster and higher level IL-25 production in response to EV71 infection and further exhibits anti-enteroviral effect via SAT1-mediated intracellular polyamine deficiency. Given that IL-25 can be induced by multiple gut microbes during human growth and development, including shifts in gut flora abundance, which may partially explain the different susceptibility to enteroviral infections between adults and children., (© 2024. The Author(s).)

Persistent enterovirus B infection has been proposed as an important contributor to the etiology of type 1 diabetes. We leveraged extensive bulk RNA-sequencing (RNA-seq) data from α-, β-, and exocrine cells, as well as islet single-cell RNA-seq data from the Human Pancreas Analysis Program (HPAP), to evaluate the presence of enterovirus B sequences in the pancreas of patients with type 1 diabetes and prediabetes (no diabetes but positive for autoantibodies). We examined all available HPAP data for either assay type, including donors without diabetes and with type 1 and type 2 diabetes. To assess the presence of viral reads, we analyzed all reads not mapping to the human genome with the taxonomic classification system Kraken2 and its full viral database augmented to encompass representatives for all 28 enterovirus B serotypes for which a complete genome is available. As a secondary approach, we input the same sequence reads into the STAR aligner using these 28 enterovirus B genomes as the reference. No enterovirus B sequences were detected by either approach in any of the 243 bulk RNA libraries or in any of the 79 single-cell RNA libraries. While we cannot rule out the possibility of a very-low-grade persistent enterovirus B infection in the donors analyzed, our data do not support the notion of chronic viral infection by these viruses as a major driver of type 1 diabetes., (© 2024 by the American Diabetes Association.)

Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0. We have demonstrated an essential role of VP0 E096 in VP0 cleavage independent of RNA encapsidation and generated a new model of capsid maturation, supported by bioinformatic analysis. This provides a molecular basis for RNA-dependence, where RNA induces conformational changes required for VP0 maturation, but that RNA packaging itself is not sufficient to induce maturation. These data have implications for understanding production of infectious virions and potential relevance for future vaccine and antiviral drug design., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kingston et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Enterovirus A71 (EV-A71), as a neurotropic virus, mainly affects infants and young children under the age of 5. EV-A71 infection causes hand-foot-mouth disease and herpetic angina, and even life-threatening neurological complications. However, the molecular mechanism by which EV-A71 induces nervous system damage remains elusive. The viral protease 3C plays an important role during EV-A71 infection and is also a key intersection of virus-host interactions. Previously, we used yeast two-hybrid to screen out the host protein Double-stranded RNA-binding protein Staufen homolog 2 (Stau2), an important member involved in neuronal mRNA transport, potentially interacts with 3C., Methods: We used coimmunoprecipitation (Co-IP) and immunofluorescence assay (IFA) to confirm that EV-A71 3C interacts with Stau2. By constructing the mutant of Stau2, we found the specific site where the 3C protease cleaves Stau2. Detection of VP1 protein using Western blotting characterized EV-A71 viral replication, and overexpression or knockdown of Stau2 exhibited effects on EV-A71 replication. The effect of different cleavage products on EV-A71 replication was demonstrated by constructing Stau2 truncates., Results: In this study, we found that EV-A71 3C interacts with Stau2. Stau2 is cleaved by 3C at the Q507-G508 site. Overexpression of Stau2 promotes EV-A71 VP1 protein expression, whereas depletion of Stau2 by small interfering RNA inhibits EV-A71 replication. Stau2 is essential for EV-A71 replication, and the product of Stau2 cleavage by 3C, 508-570 aa, has activity that promotes EV-A71 replication. In addition, we found that mouse Stau2 is also cleaved by EV-A71 3C at the same site., Conclusions: Our research provides an example for EV-A71-host interaction, enriching key targets of host factors that contribute to viral replication., (© 2024. The Author(s).)

Enterovirus-D68 (EV68) has emerged as a global health concern over the last decade with severe symptomatic infections resulting in long-lasting neurological deficits and death. Unfortunately, there are currently no FDA-approved antiviral drugs for EV68 or any other non-polio enterovirus. One particularly attractive class of potential drugs are small molecules inhibitors, which can target the conserved active site of EV68-3C protease. For other viral proteases, we have demonstrated that the emergence of drug resistance can be minimized by designing inhibitors that leverage the evolutionary constraints of substrate specificity. However, the structural characterization of EV68-3C protease bound to its substrates has been lacking. Here, we have determined the substrate specificity of EV68-3C protease through molecular modeling, molecular dynamics (MD) simulations, and co-crystal structures. Molecular models enabled us to successfully characterize the conserved hydrogen-bond networks between EV68-3C protease and the peptides corresponding to the viral cleavage sites. In addition, co-crystal structures we determined have revealed substrate-induced conformational changes of the protease which involved new interactions, primarily surrounding the S1 pocket. We calculated the substrate envelope, the three-dimensional consensus volume occupied by the substrates within the active site. With the elucidation of the EV68-3C protease substrate envelope, we evaluated how 3C protease inhibitors, AG7088 and SG-85, fit within the active site to predict potential resistance mutations.

To gather national level data on Israeli neonatal HSV (NHSV) infection and to evaluate the distinct clinical characteristics of NHSV and neonatal enteroviral meningitis (NEM). Israeli NHSV patients, hospitalized between January 2015 and April 2022 in 22 medical centers were assessed, together with NEM patients, hospitalized at Sheba Medical Center during the same period. NHSV demographic and clinical characteristics were documented and compared to those of NEM. Eighty-five NHSV (73% males) and 130 NEM (62% males) patients were included. The incidence of NHSV was 5.9/100 000 live births, the common phenotype and HSV type were SEM (53%) and HSV1 (91%), respectively. Horizontal transmission was suspected in 50% cases (of which 67% underwent a Jewish ritual circumcision with direct wound sucking, 33% had relatives with highly suspicious herpetic lesions). Compared with NEM, NHSV tends to present with rash (14% vs. 60%, p-value < 0.01) and seizures (0% vs. 6%, p-value 0.02), while fever, irritability and poor feeding appear more frequently in NEM (94% vs. 18%, p-value < 0.01; 37% vs. 1%, p-value < 0.01; 25% vs. 1%, p-value < 0.01 respectively). Of NEM patients, 28% were treated with acyclovir. Our results mark a decrease in the incidence rate of NHSV in Israel and a prominent mode of horizontal infection acquisition. We underscore the unique localized phenotype of NHSV, in contrast to enterovirus, which tends to cause a systemic disease with constitutional symptoms. These findings should be considered when evaluating the need for comprehensive empirical treatment for HSV in the context of neonatal fever, or according to a certain clinical presentation., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Non-polio enteroviruses (NPEV) cause significant disease worldwide. Population-based sero-surveillance, by measuring antibodies against specific NPEV types, provides additional information on past circulation and the prediction for future upsurges. Virus neutralisation assays (VNA), the current method of choice for measuring NPEV type specific antibodies, are not entirely standardised. Via the European Non-Polio Enterovirus Network, we organised a VNA quality assessment in which twelve laboratories participated. We provided five echovirus (E) types (E1, E18, E30 G2, E30 G6 and E6) and intravenous immunoglobulins (IVIG) as a sample for the NPEV VNA quality assessment. Differences in VNA protocols and neutralising Ab (nAb) titres were found between the participating laboratories with geometric coefficients of variation ranging from 10.3-62.9 %. Mixed-effects regression analysis indicated a small but significant effect of type of cell line used. Harmonisation of cell line passage number, however, did not improve variation between laboratories. Calibration by making use of a reference sample, reduced variation between laboratories but differences in nAb titres remained higher than two log2 dilution steps. In conclusion, sero-surveillance data from different laboratories should be compared with caution and standardised protocols are needed.

We performed a comparative, retrospective analysis (March 2019-April 2023) of children diagnosed with non-polio enterovirus (NPEV) central nervous system (CNS) infections (n = 47 vs. 129 contemporaneous controls without NPEV, all <18 years old), requiring cerebrospinal fluid (CSF) testing upon presentation to hospital. We found that showed that admissions decreased during pandemic restrictions (13% vs. controls 33%, p = 0.003). The median age of children with NPEV was 41 days (IQR: 18-72), most were male (n = 76, 59%) and were less likely to present with symptoms of irritability (11% vs. controls 26%, p = 0.04), but more likely to be febrile (93% vs. controls 73%, p = 0.007), have higher respiratory rates (mean 44 bpm, SD 11, vs. controls 36 bpm, SD 14, p = 0.001), higher heart rates (mean 171 bpm, SD 27 vs. controls 141 bpm, SD 36, p < 0.001), higher CSF protein (median 0.66 g/L, interquartile range [IQR] 0.46-1.01, vs. controls 0.53 mg/mL, IQR 0.28-0.89, p = 0.04), higher CSF white cell count (WCC) (median WCC 9.5×10 6 /L, IQR 1-16 vs. controls 3.15×10 6 /L, IQR 2.7-3.6, p < 0.001), but lower CSF glucose (median 2.8 mmol/L, IQR 2.4-3.1 vs. controls 3.1 mmol/L, IQR 2.7-3.6, p < 0.001). Phylogenetic analysis showed that these NPEVs originated from Europe (EV A71, CV B4, E21, E6, CV B3, CV B5, E7, E11, E18), North America (CV B4, E18), South America (E6), Middle East (CV B5), Africa (CV B5, E18), South Asia (E15), East/Southeast Asia (E25, CV A9, E7, E11, E18), and Australia (CV B5)., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Background: Global cyclical outbreaks of human enterovirus infections has positioned human enterovirus A71 (EV-A71) as a neurotropic virus of clinical importance. However, there remains a scarcity of internationally approved antivirals and vaccines., Methods: In pursuit of repurposing drugs for combating human enteroviruses, we employed a comprehensive pharmacophore- and molecular docking-based virtual screen targeting EV-A71 capsid protein VP1-4, 3C protease, and 3D polymerase proteins. Among 15 shortlisted ligand candidates, we dissected the inhibitory mechanism of Tanomastat in cell-based studies and evaluated its in vivo efficacy in an EV-A71-infected murine model., Findings: We demonstrated that Tanomastat exerts dose-dependent inhibition on EV-A71 replication, with comparable efficacy profiles in enterovirus species A, B, C, and D in vitro. Time-course studies suggested that Tanomastat predominantly disrupts early process(es) of the EV-A71 replication cycle. Mechanistically, live virus particle tracking and docking predictions revealed that Tanomastat specifically impedes viral capsid dissociation, potentially via VP1 hydrophobic pocket binding. Bypassing its inhibition on entry stages, we utilized EV-A71 replication-competent, 3D pol replication-defective, and bicistronic IRES reporter replicons to show that Tanomastat also inhibits viral RNA replication, but not viral IRES translation. We further showed that orally administered Tanomastat achieved 85% protective therapeutic effect and alleviated clinical symptoms in EV-A71-infected neonatal mice., Interpretation: Our study establishes Tanomastat as a broad-spectrum anti-enterovirus candidate with promising pre-clinical efficacy, warranting further testing for potential therapeutic application., Funding: MOE Tier 2 grants (MOE-T2EP30221-0005, R571-000-068-592, R571-000-076-515, R571-000-074-733) and A∗STARBiomedical Research Council (BMRC)., Competing Interests: Declaration of interests HF received grants from Synthetic Biology Translational Research Program, School of Medicine, National University of Singapore., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

This paper presents a unique case of double meningitis with enterovirus and reactivated varicella-zoster virus without shingles in an immunocompetent male teenager, a case that offers many important medical lessons, all "gravitating" around physiopathological reasoning of any clinical case in general., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: Enteroviruses (EV) comprises many different types and are the most common cause of aseptic meningitis. How the virus affects the brain including potential differences between types are largely unknown. Measuring biomarkers in CSF is a tool to estimate brain damage caused by CNS infections., Methods: A retrospective study was performed in samples from 38 patients with acute neurological manifestations and positive CSF-EV RNA ( n  = 37) or serum-IgM ( n  = 1). The EV in 17 samples were typed by sequencing. The biomarkers neurofilament light (NFL), glial fibrillary acidic protein (GFAP), S-100B protein, amyloid-β (Aβ) 40 and Aβ42, total-tau (T-tau) and phosphorylated tau (P-tau) were measured and compared with data derived from a control group ( n  = 19)., Results: There were no increased levels of GFAP ( p  ≤ 0.1) nor NFL ( p  ≤ 0.1) in the CSF of patients with EV meningitis ( n  = 38) compared with controls. However, we found decreased levels of Aβ42 ( p  < 0.001), Aβ40 ( p  < 0.001), T-tau ( p  ≥ 0.01), P-tau ( p  ≤ 0.001) and S-100B ( p  ≤ 0.001). E30 ( n  = 9) and CVB5 ( n  = 6) were the most frequent EV-types identified, but no differences in biomarker levels or other clinical parameters were found between the infecting virus type. Seven patients who were followed for longer than one month reported remaining cognitive impairment, although no correlations with biomarker concentrations were observed., Conclusion: There are no indication of neuronal or astrocyte damage in patients with EV meningitis. Yet, decreased concentrations of Aβ40, Aβ42, P-tau and T-tau were shown, a finding of unknown importance. Cognitive impairment after acute disease occurs, but with only a limited number of patients analysed, no conclusion can be drawn concerning any association with biomarker levels or EV types.

Objectives: A new variant of echovirus 11 (E11) infection is a major health concern in neonates. Here, we describe the clinical and virological characteristics of enterovirus (EV) infections in children hospitalized with acute respiratory infection in Southern Italy., Methods: Between July 2022 and August 2023, 173 EV infections were identified. Demographic and clinical characteristics, comorbidities, and coinfections were analyzed. Genotypes were identified by sequencing of VP1. Whole-genome sequencing of five E11 strains was performed., Results: Case numbers peaked in July 2022, November-December 2022, and June-July 2023. Coxsackievirus A2 was identified in 36.7%, coxsackievirus B5 in 13.8%, echovirus E11 in 9.2%, and EV-D68 in 6.4% of cases. No child had critical symptoms or a severe infection. The only neonate infected by E11 recovered fully after 5 days in hospital. Phylogenetic analysis revealed that four E11 strains were closely related to divergent lineage I E11 strains identified in France and Italy., Conclusions: The new variant of E11 was identified in children in Southern Italy. Although the cases were mild, the data suggest that transmission routes and host factors are likely to be main drivers for the development of potentially severe diseases. Systematic epidemiological/molecular surveillance will help us better understand the clinical impact of EV infections and develop preventive strategies., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In Iran, which is at high risk of the Wild Poliovirus (WPV) and Vaccine-Derived Poliovirus (VDPV) importation due to its neighborhood with two polio endemic countries, Pakistan and Afghanistan, Environmental Surveillance (ES) was established in November 2017. Sistan-Balouchestan province was chosen for the ES due to its vicinity with Pakistan and Afghanistan. Five sewage collection sites in 4 cities (Zahedan, Zabol, Chabahar and Konarak) were selected in the high-risk areas. Since the establishment of ES in November 2017 till the end of 2023, 364 sewage specimens were collected and analyzed. The ES detected polioviruses which have the highest significance for polio eradication program, that is, Wild Poliovirus type 1 (WPV1) and Poliovirus type 2 (PV2). In April and May 2019, three of 364 (0.8%) sewage specimens from Konarak were positive for imported WPV1. According to phylogenetic analysis, they were highly related to WPV1 circulating in Karachi (Sindh province) in Pakistan. PV2 was also detected in 5.7% (21/364) of the sewage specimens, most of which proved to be imported from the neighboring countries. Of 21 isolated PV2s, 7 were VDPV2, of which 5 proved to be imported from the neighboring countries as there was VDPV2 circulating in Pakistan at the time of sampling, and 2 were ambiguous VDPVs (aVDPV) with unknown source. According to the findings of this study, as long as WPV1 and VDPV2 outbreaks are detected in Iran's neighboring countries, there is a definite need for continuation and expansion of the environmental surveillance., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bovine enterovirus (BEV) infection manifests as a spectrum of clinical signs affecting the respiratory, gastrointestinal, and reproductive systems in cattle. Outbreaks of this disease results in large economic losses to the bovine industry worldwide. Currently there are no efficacious vaccines and medicines to prevent BEV infection. In the present study, reverse transcription-polymerase chain reaction was used to amplify the VP1 and VP2 genes of BEV, enabling the synthesis of a chimeric recombinant protein which contained partial sequences from both proteins. Subsequently, the emulsified purified proteins with Freund's adjuvant were used for triple-fold immunization of 4-week-old Institute of Cancer Research (ICR) mice. The mice were subsequently subjected to a challenge assay which elicited an immune response that was characterized by elevated titers of BEV-specific neutralizing antibodies. Notably, the results showed that the purification of pET32a-VP1 and pET32a-VP2 proteins markedly curtailed virus excretion and mitigated the histopathological damage usually associated with BEV infections. These were observed in the small intestines, kidneys and brain in infected animals. It also alleviated clinical symptoms such as hypothermia and weight loss. In summary, this study offers a theoretical and practical basis for BEV vaccine development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A method that has rapidly evolved for detection of viral pathogens are loop-mediated isothermal amplification (LAMP) assays. The available LAMP assays usually target the most common viral strains, including enteroviruses, but for the atypical enterovirus D68 strain VR-1197 this method has not yet been developed. Enterovirus D68 are known for severe respiratory distress in children, and atypical strains are less likely to be detected by traditional methods. This study targets the atypical EVD68 strain VR-1197 and have developed a rapid detection method saving time when differentiating enterovirus strains. This study present method development and review the sensitivity and specificity compared to traditional RT-qPCR, and wet lab cross reactivity with other airway pathogens. The EVD68 VR-1197 assay can be a rapid POC (Point of care) test for atypical EVD68 VR-1197 and have the potential as reliable detection method with minimal technological requirements., Competing Interests: Declaration of Competing Interest Authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

To characterize enteroviruses (EVs) circulating in farm animals in Central African Republic (CAR), we screened 192 stools of animals under 12 months belonging to family farms located in or near Bangui. To assess whether EV exchanges exist between these animals and humans, we also screened 195 stools of children who lived in contact with farm animals, as well as control stools of 358 children with no contact with farm animals. EVs were typed based on their capsid sequences.In children, all EVs belonged to species A, B and C, with EV-Cs accounting for 60%. Some EV-Cs shared recent common ancestors with lineages of vaccine-derived poliovirus that emerged in the country in 2019-2020. In animals, we identified EV-Gs that belonged to 10 different types, including a previously unknown one that we named EV-G28, while no EV-E or EV-F were observed. The CAR EV-Gs were genetically closely related to specimens sampled in other continents and some of them harboured the torovirus-derived insertion already reported in some EV-Gs. The worldwide circulation of EV-Gs is likely due the massive international trade of live animals. Besides, two human EV-Cs (coxsackievirus A17 and coxsackievirus A24) were detected in pigs, suggesting that these viruses could cross the species barrier. Our work provides original data on the epidemiology and ecology of EVs circulating among herd animals in Africa.

A positive-sense (+) single-stranded RNA (ssRNA) virus (e.g. enterovirus A71, EV-A71) depends on viral polypeptide translation for initiation of virus replication after entry. We reported that EV-A71 hijacks Hsp27 to induce hnRNP A1 cytosol redistribution to initiate viral protein translation, but the underlying mechanism is still elusive. Here, we show that phosphorylation-deficient Hsp27-3A (Hsp27 S15/78/82A ) and Hsp27 S78A fail to translocate into the nucleus and induce hnRNP A1 cytosol redistribution, while Hsp27 S15A and Hsp27 S82A display similar effects to the wild type Hsp27. Furthermore, we demonstrate that the viral 2A protease (2A pro ) activity is a key factor in regulating Hsp27/hnRNP A1 relocalization. Hsp27 S78A dramatically decreases the IRES activity and viral replication, which are partially reduced by Hsp27 S82A . However, Hsp27 S15A displays the same activity as the wild-type Hsp27. Peptide S78 potently suppresses EV-A71 protein translation and reproduction through blockage of EV-A71-induced Hsp27 phosphorylation and Hsp27/hnRNP A1 relocalization. A point mutation (S78A) on S78 impairs its inhibitory functions on Hsp27/hnRNP A1 relocalization and viral replication. Taken together, we demonstrate the importance of Ser78 phosphorylation of Hsp27 regulated by virus infection in nuclear translocation, hnRNP A1 cytosol relocation, and viral replication, suggesting a new path (such as peptide S78) for target-based antiviral strategy.

Enterovirus A71 (EV-A71) causes Hand, Foot, and Mouth Disease and has been clinically associated with neurological complications. However, there is a lack of relevant models to elucidate the neuropathology of EV-A71 and its mechanism, as the current models mainly utilize animal models or immortalized cell lines. In this study, we established a human motor neuron model for EV-A71 infection. Single cell transcriptomics of a mixed neuronal population reveal higher viral RNA load in motor neurons, suggesting higher infectivity and replication of EV-A71 in motor neurons. The elevated RNA load in motor neurons correlates with the downregulation of ferritin-encoding genes. Subsequent analysis confirms that neurons infected with EV-A71 undergo ferroptosis, as evidenced by increased levels of labile Fe 2+ and peroxidated lipids. Notably, the Fe 2+ chelator Deferoxamine improves mitochondrial function and promotes survival of motor neurons by 40% after EV-A71 infection. These findings deepen understanding of the molecular pathogenesis of EV-A71 infection, providing insights which suggest that improving mitochondrial respiration and inhibition of ferroptosis can mitigate the impact of EV-A71 infection in the central nervous system.

Human enteroviruses are highly prevalent world-wide. Up to more than 100 subtypes of enteroviruses can cause several diseases, including encephalitis, meningitis, myocarditis, hand-foot-mouth disease, conjunctivitis, respiratory diseases, and gastrointestinal diseases, thus posing a great threat to human health. This study aimed to investigate the epidemiological characteristics of enterovirus in children in Hangzhou, China before and after the COVID-19 outbreak. Systematic monitoring of enterovirus infections was performed by collecting samples from the children admitted to the inpatient wards and outpatient departments in the Children's Hospital, Zhejiang University School of Medicine, between January 2019 and May 2023. A commercial real-time RT PCR kit was utilized to detect enteroviruses. Among the 34,152 samples collected, 1162 samples, accounting for 3.4% of the samples, were tested positive for enteroviruses. The annual positive rates of the enteroviruses were 5.46%, 1.15%, 4.43%, 1.62%, and 1.96% in 2019, 2020, 2021, 2022, and May 2023, respectively. The positivity rate of the enteroviruses was highest among children aged 3-5 years and 5-7 years. Moreover, the monthly positivity rate of enterovirus infection ranged from 0.32% to 10.38%, with a peak in June and July. Serotypes, especially EV71 and CA16, causing severe symptoms such as HFMD, were decreasing, while the proportion of unidentified serotypes was on the rise. The incidence of enteroviruses in Hangzhou was higher in children aged 1-3 years and 7-18 years., (© 2024. The Author(s).)

The recent emergence and rapid response to severe acute respiratory syndrome coronavirus 2 was enabled by prototype pathogen and vaccine platform approaches, driven by the preemptive application of RNA vaccine technology to the related Middle East respiratory syndrome coronavirus. Recently, the National Institutes of Allergy and Infectious Diseases identified nine virus families of concern, eight enveloped virus families and one nonenveloped virus family, for which vaccine generation is a priority. Although RNA vaccines have been described for a variety of enveloped viruses, a roadmap for their use against nonenveloped viruses is lacking. Enterovirus D68 was recently designated a prototype pathogen within the family Picornaviridae of nonenveloped viruses because of its rapid evolution and respiratory route of transmission, coupled with a lack of diverse anti-enterovirus vaccine approaches in development. Here, we describe a proof-of-concept approach using a clinical stage RNA vaccine platform that induced robust enterovirus D68-neutralizing antibody responses in mice and nonhuman primates and prevented upper and lower respiratory tract infections and neurological disease in mice. In addition, we used our platform to rapidly characterize the antigenic diversity within the six genotypes of enterovirus D68, providing the necessary data to inform multivalent vaccine compositions that can elicit optimal breadth of neutralizing responses. These results demonstrate that RNA vaccines can be used as tools in our pandemic-preparedness toolbox for nonenveloped viruses.

Acute gastroenteritis (AGE) is a common pediatric infection that remains a significant cause of childhood morbidity and mortality worldwide, especially in low-income regions. Thus, the objective of this study was to detect human adenovirus (HAdV) and non-polio enterovirus (NPEV) in fecal samples from the Gastroenteritis Surveillance Network, and to identify circulating strains by nucleotide sequencing. A total of 801 fecal samples were tested using qPCR/RT-qPCR, and 657 (82.0%) were inoculated into HEp-2C and RD cell lines. The HAdV and NPEV positivity rates obtained using qPCR/RT-qPCR were 31.7% (254/801) and 10.5% (84/801), respectively, with 5.4% (43/801) co-detection. Cytopathic effect was observed in 9.6% (63/657) of patients, 2.7% (18/657) associated with HAdV, and 6.2% (41/657) associated with NPEV after testing by ICC-PCR. A comparison of the two methodologies demonstrated an agreement of 93.5% for EVNP and 64.4% for HAdV. These two viruses were detected throughout the study period, with HAdV positivity rates ranging from 41% in Amapá to 18% in Pará. The NEPV varied from 18% in Pará/Rondônia to 3% in Acre. The most affected age group was over 60 months for both HAdV and NPEV. Samples previously positive for rotavirus and norovirus, which did not show a major difference in the presence or absence of diarrhea, fever, and vomiting, were excluded from the clinical analyses of these two viruses. These viruses circulated over five years, with a few months of absence, mainly during the months corresponding to the waves of SARS-CoV-2 infection in Brazil. Five HAdV species were identified (A, B, C, D, and F), with a greater predominance of HAdV-F41 (56.5%) followed by HAdV-C (15.2%). Three NPEV species (A, B, and C) were detected, with serotypes E14 (19.3%) and CVA-24 (16.1%) being the most prevalent. The present study revealed a high diversity of NPEV and HAdV types circulating in children with AGE symptoms in the northern region of Brazil., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Alves et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Enterovirus (EV) infections have various symptoms and severe complications, including death. To determine EV prevalence and EV types in Slovenia, data on over 25 000 EV RNA tests for diagnostics and surveillance from 2014 to 2023 were analyzed. Altogether, 3733 cerebrospinal fluid (CSF) and 21 297 respiratory (sentinel and clinical) samples were tested for EV RNA. EV typing was performed on all residual EV-positive CSF samples and on subset of respiratory specimens. Altogether, 1238 samples tested positive for EV RNA: 238 (6.4%) CSF and 1000 (4.7%) respiratory samples. EV-positive patients were predominantly male (p < 0.001). Many EV-positive CSF samples were from infants under 3 months (33.1%), whereas most EV-positive respiratory samples were from children 1 to 2 years old (49.2%). Echovirus 30 (E-30) was most frequent in CSF (33.0%), followed by CV-B5 (13.8%) and E-6 (13.8%). CV-A6 was most frequent in respiratory samples (16.0%), followed by EV-D68 (7.6%) and CV-A5 (7.4%). EV types in CSF and respiratory samples show diverse dynamics, with some outbreaks indicated. A significant difference was found in the EV detection rate between CSF and respiratory samples by age. Various EV types were characterized, showing that some EV types are more neurotropic or cause more severe infections., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Enteroviruses are important human pathogens with diverse serotypes, posing a major challenge to develop vaccines for individual serotypes, the success of polio vaccines in controlling and eradicating polio, along with the recent emergence and high prevalence of enterovirus-caused infectious diseases, highlights the importance of enterovirus vaccine development. Given our previous report on enteroviruses weakened by the 2 A S/T125A mutation, we assessed the potential of the EV-A71 2A-125A mutant as a vaccine candidate to address this challenge. We found that the 2A-125A mutant caused transient mild symptoms, low viral loads, and no significant pathological changes mild pathological changes in hSCARB2-KI mice, producing long-lasting cross-neutralizing antibodies against two EV-A71 wild strains. Pre-exposure to the 2A-125A mutant substantially protected against the EV-A71 Isehara wild-type strain, causing minor pathologies, significantly reducing muscle and lung inflammation, and preventing neurological damage, with reduced viral loads in vivo. Pre-exposure also distinctly suppressed the expression of pro-inflammatory cytokines, correlating to the severity of clinical symptoms. Collectively, the EV-A71 2A-125A mutant was attenuated and could generate a robust and protective immune response, suggesting its potential as a vaccine candidate and global solution for specific enterovirus vaccine development., (© 2024 Wiley Periodicals LLC.)

In December 2023, we observed through hospital-based surveillance a severe outbreak of enterovirus D68 infection in pediatric inpatients in Dakar, Senegal. Molecular characterization revealed that subclade B3, the dominant lineage in outbreaks worldwide, was responsible for the outbreak. Enhanced surveillance in inpatient settings, including among patients with neurologic illnesses, is needed.

Human enteroviruses (HEV) can cause a range of diseases from mild to potentially life-threatening. Identification and genotyping of HEV are crucial for disease management. Existing typing methods, however, have inherent limitations. Developing alternative methods to detect HEV with more virus types, high accuracy, and sensitivity in an accessible manner presents a technological and analytical challenge. Here, a sequence-specific nanoparticle barcode (SSNB) method is presented for simultaneous detection of 10 HEV types. This method significantly increases sensitivity, enhancing detection by 10-10 6 times over the traditional multiplex hybrid genotyping (MHG) method, by resolving cross-interference between the multiple primer sets. Furthermore, the SSNB method demonstrates a 100% specificity in accurately distinguishing between 10 different HEV types and other prevalent clinical viruses. In an analysis of 70 clinical throat swab samples, the SSNB method shows slightly higher detection rate for positive samples (50%) compared to the RT-PCR method (48.6%). Additionally, further assessment of the typing accuracy for samples identified as positive by SSNB using sequencing method reveals a concordance rate of 100%. The combined high sensitivity and specificity level of the methodology, together with the capability for multiple type analysis and compatibility with clinical workflow, make this approach a promising tool for clinical settings., (© 2024. The Author(s).)

Hand, foot and mouth disease (HFMD), mainly caused by enterovirus 71 (EV71), has frequently occurred in the Asia-Pacific region, posing a significant threat to the health of infants and young children. Therefore, research on the infection mechanism and pathogenicity of enteroviruses is increasingly becoming important. The 3D polymerase, as the most critical RNA-dependent RNA polymerase (RdRp) for EV71 replication, is widely targeted to inhibit EV71 infection. In this study, we identified a novel host protein, AIMP2, capable of binding to 3D polymerase and inhibiting EV71 infection. Subsequent investigations revealed that AIMP2 recruits the E3 ligase SMURF2, which mediates the polyubiquitination and degradation of 3D polymerase. Furthermore, the antiviral effect of AIMP2 extended to the CVA16 and CVB1 serotypes. Our research has uncovered the dynamic regulatory function of AIMP2 during EV71 infection, revealing a novel antiviral mechanism and providing new insights for the development of antienteroviral therapeutic strategies., Competing Interests: Conflict of interest Prof. Ke Lan is an editorial board member for Virologica Sinica and was not involved in the editorial review or the decision to publish this article. The authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Background: Non-polio enteroviruses (EV) and human parechoviruses (HPeV) are known etiological agents of meningoencephalitis in neonates. However, reports of neuroradiological findings and neurodevelopmental outcomes in this population are scarce., Objectives: to describe clinical characteristics, neuroradiological findings and, in a subset of patients, neurodevelopmental outcomes in a cohort of infants with EV or HPeV meningoencephalitis within 60 days of life., Study Design: clinical/laboratory data, neuroradiological findings (cranial ultrasound, cUS, brain magnetic resonance imaging, MRI), and neurodevelopmental outcomes assessed by Ages and Stages Questionnaires - third edition were prospectively collected., Results: overall, 32 infants with EV (21, 67.8 %) or HPeV (11, 28.2 %) meningoencephalitis were enrolled. Infants with HPeV (73 %: type 3 HPeV) presented more frequently with seizures (18.2 % vs. 0, p value=0.03), lymphopenia (1120 vs. 2170 cells/mm 3 , p = 0.02), focal anomalies at electroencephalography (EEG) (63.6 vs. 23.8 %, p = 0.03), and pathological findings at MRI (72.7 % vs. 15.8 %, p value=0.004) compared to those affected by EV. cUS was not significantly altered in any of the enrolled infants. All infants with EV meningoencephalitis evaluated at 12-24 months and at 30-48 months were normal. Two out of the 7 infants with HPeV meningoencephalitis showed some concerns in gross motor (1/7, 14.3 %) or in problem solving (1/7, 14.3 %) function at 30-48 months of age., Conclusions: In our cohort, neonates infected by HPeV had more severe clinical manifestations, more alterations at brain MRI, and some signs of long-term neurodevelopmental delay. Our data highlight the heterogeneity of manifestations in infants with EV or HPeV meningoencephalitis, and the need for long-term follow-up of those infected by HPeV in the neonatal period., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Increasing evidences suggest that the methyltransferase NSUN2 catalyzes 5-methylcytosine (m5C) modifications on viral RNAs, which are essential for the replication of various viruses. Despite the function of m5C deposition is well characterized, other potential roles of NSUN2 in regulating viral replication remain largely unknown. In this study, the m5C modified residues catalyzed by NSUN2 on enterovirus 71 (EV71) RNAs were mapped. NSUN2, along with m5C modifications, played multiple roles during the EV71 life cycle. Functional m5C modified nucleotides increased the translational efficiency and stability of EV71 RNAs. Additionally, NSUN2 was found to target the viral protein VP1 for binding and promote its stability by inhibiting the ubiquitination. Furthermore, both viral replication and pathogenicity in mice were largely attenuated when functional m5C residues were mutated. Taken together, this study characterizes distinct pathways mediated by NSUN2 in regulating EV71 replication, and highlights the importance of its catalyzed m5C modifications on EV71 RNAs for the viral replication and pathogenicity., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Pathogens of the enterovirus genus, including poliovirus and coxsackieviruses, typically circulate in the summer months suggesting a possible positive association between warmer weather and transmission. Here we evaluate the environmental and demographic drivers of enterovirus transmission, as well as the implications of climate change for future enterovirus circulation. We leverage pre-vaccination era data on polio in the US as well as data on two enterovirus A serotypes in China and Japan that are known to cause hand, foot, and mouth disease. Using mechanistic modeling and statistical approaches, we find that enterovirus transmission appears positively correlated with temperature although demographic factors, particularly the timing of school semesters, remain important. We use temperature projections from Coupled Model Intercomparison Project Phase 6 (CMIP6) to simulate future outbreaks under late 21st-century climate change for Chinese provinces. We find that outbreak size increases with climate change on average, though results differ across climate models depending on the degree of wintertime warming. In the worst-case scenario, we project peak outbreaks in some locations could increase by up to 40%., (© 2024. The Author(s).)

Background: Coronary heart disease (CHD) is a common cardiovascular disease that is associated with altered gut microbiota. Enteroviruses, an essential component of the gut microbiome, may play an important role in disease progression. However, the relationship between enteroviruses and CHD remains unclear. The development of high-throughput sequencing technologies has facilitated research on the interconnections between viruses and disease-related metabolites., Methods and Results: Mice were fed a high-fat diet (CHD group) or chow diet (Sham group) for 12 weeks, and ligation of the left anterior descending coronary artery was performed at the end of week 8. After 4 weeks, all animals were euthanised. Subsequently, the animals were evaluated for basic haemato-biochemical parameters and cardiac function, and aorta staining was performed. Based on enteroviral metagenomics and serum UPLC-MS/MS metabolomics analyses, we evaluated the association between enteroviral groups and serum metabolites of CHD mouse model. A high-fat diet and coronary ligation enabled the establishment of the CHD mouse model. Notably, the enterovirus spectrum of the sham group was significantly different from that of the CHD group, with 24 viral communities of different family and species classification, such as Tsarbombavirus, Mingyongvirus, Claudivirus, and Firehammervirus, exhibiting significant differences. In addition, 731 Differential metabolites were detected in the serum of both groups of mice. Correlation network analysis revealed a close relationship between various metabolites related to lipid metabolism and different viruses, including Tsarbombavirus, Mingyongvirus, Claudivirus, and Firehammervirus., Conclusions: An animal model of CHD, characterised by lipid disturbance and myocardial ischaemia, was established using a high-fat diet and ligation of the left anterior descending branch of the coronary artery. Tsarbombavirus, Firehammervirus, Mingyongvirus, and Claudivirus were associated with metabolites in the lipid metabolism pathway. The results indicate that Tsarbombavirus may be the main genus interacting with CHD-related metabolites in mice. Conclusively, the findings of our study provide novel insights into the potential relationship enterovirus groups and metabolites associated with CHD., (© 2024. The Author(s).)