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Background: 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., Case Presentation: 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., Conclusions: 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., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Written informed consent was obtained from the patients’ parents for the publication of this case report and accompanying images. Competing interests: The authors declare that they have no competing interests., (© 2025. The Author(s).)

Human enteric viruses can remain infective in surface waters for extended periods of time, posing a public health risk. Microbial activity contributes to the inactivation of waterborne enteric viruses, but while individual bacteria-virus interactions have been characterized, the importance of microbial diversity remains unknown. Here, we experimentally manipulated the diversity of bacterial communities from Lake Geneva across three seasons using a dilution-to-extinction approach and monitored the inactivation and genome decay of echovirus 11, a member of the Enterovirus genus. Long-read sequencing of the 16S rRNA gene revealed diversity gradients ranging between 373 and 2,722 bacterial species. Compared to sterile controls, echovirus 11 inactivation was enhanced by the presence of active bacteria and depended both on season and sample dilution. Throughout all seasons, the highest inactivation (between 3.0 and 7.9 log 10 fold reduction in infectivity over 96 h) was observed in the least diluted incubations (i.e., the highest bacterial richness). Genome decay exhibited a 24-h lag and was less pronounced than the corresponding infectivity loss (ranging between 2.3 and 3.8 log 10 fold over 96 h), indicating that microbial inactivation primarily targets the echovirus 11 capsid. We found a positive-saturating relationship between bacterial species richness and viral inactivation, suggesting functional redundancy and pointing toward the importance of rare species for viral inactivation. Biomarker analysis revealed several clades of bacteria, particularly members of Chitinophagaceae , to be significantly associated with echovirus 11 inactivation. Overall, these findings suggest that high microbial diversity enhances the capacity of surface waters to rid themselves of contamination by enteric viruses and hence protects public health.IMPORTANCEHuman enteric viruses in natural waterbodies pose a public health risk. Microorganisms, particularly bacteria, contribute to the inactivation of enteroviruses, thereby mitigating this risk. We use experimental manipulations of lake water bacterial diversity to unravel the importance of diversity for the inactivation of echovirus 11, a model human pathogen. Our findings suggest that bacterial diversity is important for echovirus 11 inactivation and that specific, but numerically rare, bacteria present in the surface water of Lake Geneva across different seasons contribute to viral inactivation. These findings contribute to our understanding of the inactivation of human enteric viruses in natural waterbodies-a hitherto understudied ecosystem service., Competing Interests: The authors declare no conflict of interest.

Fulminant myocarditis (FM) is a severe inflammatory condition of the myocardium that often results in sudden death, particularly in young individuals. In this study, we employed single-nucleus and spatial transcriptomics to perform a comprehensive analysis of coxsackievirus B3 (CVB3)-induced FM in A/J mice, spanning seven distinct time points pre- and post-treatment. Our findings reveal that mesothelial cells play a critical role in the early stage of myocarditis by acting as primary targets for CVB3 infection. This triggers the activation of macrophages, initiating a cascade of inflammation. Subsequently, pro-inflammatory Inflammatory_Mac and T cells infiltrate the myocardium, driving tissue damage. We also identified Cd8 + effector T cells as key mediators of cardiomyocyte injury. These cells release cytotoxic molecules, particularly IFN-γ, which modulates the expression of Spi1, a factor implicated in exacerbating cardiomyocyte death and amplifying disease progression. Therapeutic interventions targeting the IFN-γ/Spi1 axis demonstrated significant efficacy in FM models. Notably, intravenous immunoglobulin (IVIG) treatment reduced mortality, suppressed viral proliferation, and mitigated the hyperinflammatory state of FM. IVIG therapy also downregulated IFN-γ and Spi1 expression, underscoring its immunomodulatory and therapeutic potential. This comprehensive spatiotemporal transcriptomic analysis provides profound insights into the pathogenesis of FM and highlights actionable therapeutic targets, paving the way for more effective management strategies for this life-threatening condition., Competing Interests: Competing interests: Daowen Wang is the editorial board member of Signal Transduction and Targeted Therapy, but he has not been involved in the process of manuscript handling. Other authors declare no conflict of interest. Ethics: Animal experiments in this study were approved by the Institutional Animal Research Committee of Tongji Medical College (permit No. TJH-202311007) and the Ethics Committee of BGI (permit No. BGI-IRB A22019). All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Studies involving human participants were reviewed and approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (permit No. TJ-IRB20221265). Written informed consent was obtained from the legal representatives of the deceased individuals prior to sample collection. All procedures adhered to ethical standards and regulations for the use of human biological materials., (© 2025. The Author(s).)

Viral aseptic meningitis is a neuroinflammatory condition that occurs when viruses gain access to the central nervous system (CNS) and induce inflammation. The blood-brain barrier (BBB) is comprised of brain endothelial cells (BECs) that stringently regulate the passage of molecules, toxins, and pathogens from the circulation into the CNS. Through their unique properties, such as complex tight junctions, reduced rates of endocytosis, expression of efflux transporters, and restricted expression of leukocyte adhesion molecules, the BBB is often able to limit pathogen entry into the brain; however, certain neurotropic pathogens, such as coxsackievirus B3 (CVB3) are able to infect the CNS. We have previously demonstrated that CVB3 can infect and disrupt induced pluripotent stem cell-derived brain-like endothelial cells (iBECs), but the host response to this infection remains unknown. Here, we investigate global host transcriptional changes during CVB3 infection of iBECs using RNA sequencing. We validated our data set by exploring pathways altered by CVB3 using quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay of upregulated cytokines and interferon signaling molecules., Importance: Coxsackievirus B3 (CVB3) is a leading cause of viral aseptic meningitis that can produce severe disease in susceptible individuals. To gain access to the central nervous system, CVB3 must cross central nervous system barriers, such as the blood-brain barrier. Previously, we have shown that CVB3 infects a human stem cell-derived brain-like endothelial cell model. Here, we report the global transcriptome of stem cell-derived brain-like endothelial cells to CVB3 infection and provide proof-of-concept validation of the dataset using molecular biology techniques. These data could inform novel mechanisms of CVB3-mediated blood-brain barrier dysfunction., Competing Interests: The authors declare no conflict of interest.

Three throat swab samples from infants and young children in Hubei Province were identified as positive for enteroviruses. All subjects were one month old, and the onset of symptoms occurred in June; these cases were sporadic and exhibited no clear epidemiological associations. The results of VP1 gene amplification analysis indicated that all three infant cases were attributed to Echovirus 30 (ECHO30). The ECHO30 strain isolated from the three samples was classified as Cluster Ⅲ within the H gene subtype. Whole genome amplification analysis revealed that the ECHO30 strain from Hubei exhibited the highest homology with ECHO30/MW586892/Ashburton/17WQ2027G/USA/2007-08-20 from GenBank, demonstrating nucleotide and amino acid homologies of 97.6% and 99.3%, respectively. Recombination analysis indicated the presence of recombination events in the non-structural protein region of the ECHO30 Hubei strain, with recombinant fragments potentially originating from ECHO30/KY888274/14-397/Germany/2013 and ECHO18 strains Echo18/MN815811/BJ2018-S6363/Beijing/2018. This study provides a comprehensive analysis of the genetic evolution of the ECHO30 strain prevalent in Hubei Province, contributing essential data for the prevention of ECHO30 infections.

In 2022-23, several European countries reported paediatric acute liver failure (ALF) with enterovirus infection. In August-November 2024, three neonatal cases of ALF with echovirus 11 (E11) were reported in Tokyo, Japan. All neonates developed irreversible multiple-organ failure and died. The E11 strain belonged to the new lineage 1, which was the same as strains isolated from neonatal ALF cases in Europe in 2022-23.

Recently, we demonstrated that the oncolytic Coxsackievirus B3 (CVB3) strain PD-H can be efficiently adapted to resistant colorectal cancer cells through dose-dependent passaging in colorectal cancer cells. However, the method is time-consuming, which limits its clinical applicability. Here, we investigated whether the manufacturing time of the adapted virus can be reduced by replacing the dose-based passaging with volume-based passaging. For this purpose, the murine colorectal carcinoma cell line MC38, resistant to PD-H-induced lysis, was initially infected with PD-H at 0.1 multiplicity of infection (MOI). For subsequent passages, 15-30 µL of a 1:10 dilution of the cell culture supernatant was transferred to fresh MC38 cells early after virus-induced cell lysis became visible. By virus passage 10, complete cell lysis of MC38 cells was achieved. Sequencing of the passage 10 virus (P-10) revealed two nucleotide substitutions in the 5' UTR and six amino acid changes in the viral polyprotein compared to the PD-H founder. P-10, however, consisted of a heterogeneous virus population. Therefore, the detected mutations were introduced into the cDNA of PD-H, from which the recombinant virus PD-MC38 was generated. PD-MC38 exhibited significantly enhanced replication and lytic activity in MC38 cells compared to PD-H, whereas its oncolytic activity in other colorectal cancer cell lines was comparable to or even lower than that of PD-H. These findings demonstrate that volume-based passaging is suitable to generate tumor cell-specific adapted PD-H. Moreover, compared to the dose-dependent passaging, volume-based passaging significantly reduced the time required to generate the adapted virus.

The development of pancreatic cancer requires both, acquisition of an oncogenic mutation in KRAS as well as an inflammatory insult. However, the physiological causes for pancreatic inflammation are less defined. We show here that oncogenic KRas-expressing pre-neoplastic lesion cells upregulate coxsackievirus (CVB) and adenovirus receptor (CAR). This facilitates infections from enteroviruses such as CVB3, which can be detected in approximately 50% of pancreatic cancer patients. Moreover, using an animal model we show that a one-time pancreatic infection with CVB3 in control mice is transient, but in the presence of oncogenic KRas drives chronic inflammation and rapid development of pancreatic cancer. We further demonstrate that a knockout of CAR in pancreatic lesion cells blocks these CVB3-induced effects. Our data demonstrate that KRas-caused lesions promote the development of pancreatic cancer by enabling certain viral infections., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Purpose: Recently, cases of serious illness in newborns infected with Echovirus 11 have been reported in Europe, including Italy. Here, we report the case of a newborn diagnosed with disseminated Echovirus 11 infection, which occurred in October 2023 in the Province of Bolzano, Italy., Methods: A molecular screening, by Real-Time RT-PCR, was employed to analyse the cerebrospinal fluid, blood and stool samples, and nasal swabs. The entire viral genome was sequenced using both Illumina and Nanopore technologies., Results: The patient was admitted to hospital due to fever. Molecular testing revealed the presence of enterovirus RNA. Typing confirmed the presence of Echovirus 11. The patient was initially treated with antibiotic therapy and, following the diagnosis of enterovirus infection, also with human immunoglobulins. Over the following days, the patient remained afebrile, with decreasing inflammation indices and in excellent general condition. Genomic and phylogenetic characterization suggested that the strain was similar to strains from severe cases reported in Europe., Conclusions: Despite the low overall risk for the neonatal population in Europe, recent cases of Echovirus 11 have highlighted the importance of surveillance and complete genome sequencing is fundamental to understanding the phylogenetic relationships of Echovirus 11 variants., Competing Interests: Declarations. Consent to participate and consent to publish: Both parents of the newborn consented to participate in this study and to have the related data published. Both parents signed a written informed consent. Conflict of interest: The authors have no conflict of interest to declare., (© 2024. The Author(s).)

Echovirus 11 (E11) has gained attention owing to its association with severe neonatal infections. From 2018 to 2023, a surge in severe neonatal cases and fatalities linked to a novel variant of genotype D5 was documented in China, France, and Italy. However, the prevention and control of E11 variants have been hampered by limited background data on the virus circulation and genetic variance. Therefore, the present study investigated the circulating dynamics of E11 and the genetic variation and molecular evolution of genotype D5 through the collection of strains from the national acute flaccid paralysis (AFP) and hand, foot, and mouth disease (HFMD) surveillance system in China during 2000-2022 and genetic sequences published in the GenBank database. The results of this study revealed a prevalent dynamic of E11 circulation, with D5 being the predominant genotype worldwide. Further phylogenetic analysis of genotype D5 indicated that it could be subdivided into three important geographic clusters (D5-CHN1: 2014-2019, D5-CHN2: 2016-2022, and D5-EUR: 2022-2023). Additionally, variant-specific (144) amino acid mutation sites and positive-selection pressure sites (132, 262) were identified in the VP1 region. Cluster-specific recombination patterns were also identified, with CVB5, E6, and CVB4 as the major recombinant viruses. These findings provide a preliminary landscape of E11 circulation worldwide and basic scientific data for further study of the pathogenicity of E11 variants.

Coxsackievirus B5 (CVB5) is a major pathogen responsible for hand-foot-mouth disease, herpangina, and even severe death. The mechanisms underlying CVB5-induced diseases are not fully elucidated, and no specific antiviral treatments are currently available. Circular RNAs (circRNAs), a closed-loop molecular structure, have been reported to be involved in virus infectious diseases. However, their roles and mechanisms in CVB5 infection remain largely unknown. In this study, we identify that CircPTPN11 is significantly upregulated following CVB5 infection in RD cells. Characteristic analysis reveals that the expression of CircPTPN11 is both time- and dose-dependent upon CVB5 infection and is specific to intestinal tissue. Moreover, CircPTPN11 inhibits CVB5 replication by activating IRF3 in the type-I interferon (IFN-I) pathway. Further underneath mechanism shows that CircPTPN11 indirectly regulates CVB5 replication by sponging miR-152-3p, and miR-152-3p influences CVB5 replication by interacting with the gene coding for signal regulatory protein alpha (SIRPA). In conclusion, this study suggests that CircPTPN11 targets SIRPA by sponging miR-152-3p, thereby inhibiting the replication and proliferation of CVB5. These findings provide a molecular target for the diagnosis and treatment of CVB5 infection., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Background: 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., Methods: 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., Results: 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., Conclusion: 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.

Echovirus 11 has emerged as a major public health concern, causing sepsis in neonates in many European countries in recent years. In Africa, especially West Africa, where resources and diagnostic capacities are limited, only sporadic cases have been reported. To better understand the recent molecular epidemiology of E11 in West Africa, we characterized twenty-three echovirus 11 strains isolated through the acute flaccid paralysis and environmental surveillance systems for polio from 2013 to 2023, using high-throughput sequencing. Our data are noteworthy due to identifying for the first time a recombinant strain from an acute flaccid paralysis case and represent the first focus to date on molecular characterization of echovirus 11 in West Africa. Moreover, our data show that echovirus 11 diverged from 1970 (95% HPD range, 1961-1979) and evolved into four distinct clades, with the virus spread from West Africa to Europe, exhibiting two introductions in France around 2017, from Senegal and Guinea. Furthermore, the in silico analysis reveals four non-conservative amino acid substitutions in the VP1 sequences of the European strains associated with neonatal sepsis in newborns and a conserved amino acid motif in the VP1 protein toward enterovirus genotypes. Our data provide new insights into the epidemiology of echovirus 11 and point to the crucial need to implement specific surveillance programs targeting non-polio enteroviruses for the rapid identification of emerging or re-emerging enterovirus species, particularly in Africa.

Echovirus type 18 (E18) is a member of the genus Enterovirus of the family Picornaviridae. In this study, we investigated the characteristics of E18 infections in hospitalized adults with meningoencephalitis that occurred during an unusual epidemic in south-western Hungary in mid-2023. Five (6.1%) out of 82 cerebrospinal fluid specimens that were tested were positive for an enterovirus, four of which were E18 (OR372160 and PP861087-PP861090). Headache (100%), fever (75%), retrobulbar pain (50%), nausea (50%), joint/limb pain (50%), exanthema, photophobia, and vomiting were the most common symptoms. Sequence analysis showed that these viruses were related to unpublished emerging E18 strains from France (2022/2023) and China (2019/2020). Further study is necessary to monitor the circulation of epidemic/pandemic E18 variants over time., (© 2024. The Author(s).)

Coxsackievirus B2 (CVB2) is a member of the enterovirus group known to induce a spectrum of illnesses, from mild to severe. In the summer of 2022, an unusual outbreak of enteroviral central nervous system (CNS) infections occurred that was attributed to CVB2. Cerebrospinal fluid (CSF) samples collected from patients in 2015-2022 were tested for enterovirus via RT-PCR, followed by Sanger sequencing for positive cases. CVB2 samples were further sequenced in the P1 region using NGS. A total of 30 CSF samples were identified as CVB2, with 60% of these cases occurring between June and August 2022. The 2022 CVB2 variants were associated with severe clinical symptoms, including encephalitis (50%) and ataxia (27.8%). Samples from 2015 to 2020 were also included due to the absence of these symptoms. Phylogenetic analysis revealed that CVB2 strains from 2019 to 2020 were also distinct from those obtained in 2022. Amino acid analysis of the capsid proteins VP1, VP2, and VP3 identified three unique substitutions with potential antigenic significance in the 2022 variant: S67A in VP2, and T93A and E274D in VP1. These substitutions were not present in earlier strains or reported in the literature, suggesting they may influence the virus's virulence. The clinical observations from this study highlight new patterns of CVB2 infection in the CNS that had not been previously observed. Additionally, it identifies unique genetic changes in the 2022 CVB2 variant that may account for the increased virulence seen in the 2022 outbreak. However, further analysis is required to validate this assumption., (© 2024 Wiley Periodicals LLC.)

Enteroviruses contain conserved RNA structures at the extreme 5' end of their genomes that recruit essential proteins 3CD and PCBP2 to promote genome replication. However, the high-resolution structures and mechanisms of these replication-linked RNAs (REPLRs) are limited. Here, we determined the crystal structures of the coxsackievirus B3 and rhinoviruses B14 and C15 REPLRs at 1.54, 2.2 and 2.54 Å resolution, revealing a highly conserved H-type four-way junction fold with co-axially stacked sA-sD and sB-sC helices that are stabilized by a long-range A•C•U base-triple. Such conserved features observed in the crystal structures also allowed us to predict the models of several other enteroviral REPLRs using homology modeling, which generated models almost identical to the experimentally determined structures. Moreover, our structure-guided binding studies with recombinantly purified full-length human PCBP2 showed that two previously proposed binding sites, the sB-loop and 3' spacer, reside proximally and bind a single PCBP2. Additionally, the DNA oligos complementary to the 3' spacer, the high-affinity PCBP2 binding site, abrogated its interactions with enteroviral REPLRs, suggesting the critical roles of this single-stranded region in recruiting PCBP2 for enteroviral genome replication and illuminating the promising prospects of developing therapeutics against enteroviral infections targeting this replication platform., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

BackgroundIn 2023, a European alert was issued regarding an increase in severe enterovirus (EV) neonatal infections associated with echovirus 11 (E11) new lineage 1.AimTo analyse E11-positive cases between 2019 and 2023 to investigate whether the new lineage 1 circulated in Spain causing severe neonatal infections.MethodsEV-positive samples from hospitalised cases are sent for typing to the National Reference Enterovirus Laboratory. Available samples from 2022-23 were subjected to metagenomic next-generation sequencing.ResultsOf 1,288 samples genotyped, 103 were E11-positive (98 patients: 6 adults, 33 neonates, 89 children under 6 years; male to female ratio 1.9). E11 detection rate was similar before and after detection of the new lineage 1 in Spain in June 2022 (9.7% in 2019 vs 10.6% in 2023). The proportion of E11-infected ICU-admitted neonates in 2019-2022 (2/7) vs 2022-2023 (5/12) did not significantly differ (p = 0.65). In severe neonatal infections, 4/7 E11 strains were not linked to the new lineage 1. The three novel E11 recombinant genomes were associated with severe (n = 2) and non-severe (n = 1) cases from 2022-2023 and clustered outside the new lineage 1. Coinfecting pathogenic viruses were present in four of 10 E11-positive samples.ConclusionThe emergence of the new lineage 1 is not linked with an increase in incidence or severity of neonatal E11 infections in Spain. The detection of two novel E11 recombinants associated with severe disease warrants enhancing genomic and clinical surveillance.

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.)

Enteroviruses such as coxsackievirus B3 are identified as a common cause of viral myocarditis, but the potential mechanism of its replication and pathogenesis are largely unknown. The genomes of a variety of viruses contain N6-methyladenosine (m 6 A), which plays important roles in virus replication. Here, by using the online bioinformatics tools SRAMP and indirect immunofluorescence assay (IFA), we predict that the CVB3 genome contains m 6 A sites and found that CVB3 infection could alter the expression and cellular localization of m 6 A-related proteins. Moreover, we found that 3-deazaadenosine (3-DAA), an m 6 A modification inhibitor, significantly decreased CVB3 replication. We also observed that the m 6 A methyltransferases methyltransferase-like protein 3 (METTL3) and METTL14 play positive roles in CVB3 replication, whereas m 6 A demethylases fat mass and obesity-associated protein (FTO) or AlkB homolog 5 (ALKBH5) have opposite effects. Knockdown of the m 6 A binding proteins YTH domain family protein 1 (YTHDF1), YTHDF2 and YTHDF3 strikingly decreased CVB3 replication. Finally, the m 6 A site mutation in the CVB3 genome decreased the replication of CVB3 compared with that in the CVB3 wild-type (WT) strain. Taken together, our results demonstrated that CVB3 could exploit m 6 A modification to promote viral replication, which provides new insights into the mechanism of the interaction between CVB3 and the host.

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.)

Enteroviruses cause a wide range of disorders with varying presentations and severities, and some enteroviruses have emerged as serious public health concerns. These include Coxsackievirus B3 (CVB3), an active causative agent of viral myocarditis, and Coxsackievirus B4 (CVB4), which may accelerate the progression of type 1 diabetes. The 3C proteases from CVB3 and CVB4 play important roles in the propagation of these viruses. In this study, the 3C proteases from CVB3 and CVB4 were expressed in Escherichia coli and purified by affinity chromatography and gel-filtration chromatography. The crystals of the CVB3 and CVB4 3C proteases diffracted to 2.10 and 2.01 Å resolution, respectively. The crystal structures were solved by the molecular-replacement method and contained a typical chymotrypsin-like fold and a conserved His40-Glu71-Cys147 catalytic triad. Comparison with the structures of 3C proteases from other enteroviruses revealed high similarity with minor differences, which will guide the design of 3C-targeting inhibitors with broad-spectrum properties.

Self-amplifying RNAs (saRNAs) are versatile vaccine platforms that take advantage of a viral RNA-dependent RNA polymerase (RdRp) to amplify the messenger RNA (mRNA) of an antigen of interest encoded within the backbone of the viral genome once inside the target cell. In recent years, more saRNA vaccines have been clinically tested with the hope of reducing the vaccination dose compared to the conventional mRNA approach. The use of N1-methyl-pseudouridine (1mΨ), which enhances RNA stability and reduces the innate immune response triggered by RNAs, is among the improvements included in the current mRNA vaccines. In the present study, we evaluated the effects of this modified nucleoside on various saRNA platforms based on different viruses. The results showed that different stages of the replication process were affected depending on the backbone virus. For TNCL, an insect virus of the Alphanodavirus genus, replication was impaired by poor recognition of viral RNA by RdRp. In contrast, the translation step was severely abrogated in coxsackievirus B3 (CVB3), a member of the Picornaviridae family. Finally, the effects of 1mΨ on Semliki forest virus (SFV), were not detrimental in in vitro studies, but no advantages were observed when immunogenicity was tested in vivo., (© 2024. The Author(s).)

RNA viruses have notoriously high mutation rates due to error-prone replication by their RNA polymerase. However, natural selection concentrates variability in a few key viral proteins. To test whether this stems from different mutation tolerance profiles among viral proteins, we measured the effect of >40,000 non-synonymous mutations across the full proteome of coxsackievirus B3 as well as >97% of all possible codon deletions in the nonstructural proteins. We find significant variation in mutational tolerance within and between individual viral proteins, which correlated with both general and protein-specific structural and functional attributes. Furthermore, mutational fitness effects remained stable across cell lines, suggesting selection pressures are mostly conserved across environments. In addition to providing a rich dataset for understanding virus biology and evolution, our results illustrate that incorporation of mutational tolerance data into druggable pocket discovery can aid in selecting targets with high barriers to drug resistance., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Álvarez-Rodríguez 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.)

After the first phase of the COVID-19 pandemic in Europe, a new highly pathogenic variant of echovirus 11 (E11) was detected. The aim of this study was to analyze the genetic diversity of Polish E11 environmental and clinical strains circulating between 2017 and 2023 as well as compare them with E11 strains isolated from severe neonatal sepsis cases reported in Europe between 2022 and 2023. Additionally, the study explores the effectiveness of environmental monitoring in tracking the spread of new variants. For this purpose, the complete sequences of the VP1 capsid protein gene were determined for 266 E11 strains isolated in Poland from 2017 to 2023, and phylogenetic analysis was performed. In the years 2017-2023, a significant increase in the detection of E11 strains was observed in both environmental and clinical samples in Poland. The Polish E11 strains represented three different genotypes, C3, D5 and E, and were characterized by a high diversity. In Poland, the intensive circulation of the new variant E11, responsible for severe neonatal infections with a high mortality in Europe, was detected in the years 2022-2023. This investigation demonstrates the important role of environmental surveillance in the tracking of enteroviruses circulation, especially in settings with limited clinical surveillance.

Echovirus 11 (E11) has gained attention owing to its association with severe neonatal infections. Due to the limited data available, the World Health Organization (WHO) considers public health risk to the general population to be low. The present study investigated the genetic variation and molecular evolution of E11 genomes collected from May to December 2023. Whole genome sequencing (WGS) was performed for 16 E11 strains. Phylogenetic analysis on WG showed how all Italian strains belonged to genogroup D5, similarly to other E11 strains recently reported in France and Germany all together aggregated into separate clusters. A cluster-specific recombination pattern was also identified using phylogenetic analysis of different genome regions. Echovirus 6 was identified as the major recombinant virus in 3C pro and 3D pol regions. The molecular clock analysis revealed that the recombination event probably occurred in June 2018 (95% HPD interval: Jan 2016-Jan 2020). Shannon entropy analyses, within P1 region, showed how 11 amino acids exhibited relatively high entropy. Five of them were exposed on the canyon region which is responsible for receptor binding with the neonatal Fc receptor. The present study showed the recombinant origin of a new lineage of E11 associated with severe neonatal infections., (© 2024 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Objectives: Following the alert of echovirus 11 (E-11) infection in neonates in EU/EEA Member States, we conducted an investigation of E-11 circulation by gathering data from community and hospital surveillance of enterovirus (EV) in northern Italy from 01 August 2021 to 30 June 2023., Methods: Virological results of EVs were obtained from the regional sentinel surveillance database for influenza-like illness (ILI) in outpatients, and from the laboratory database of ten hospitals for inpatients with either respiratory or neurological symptoms. Molecular characterization of EVs was performed by sequence analysis of the VP1 gene., Results: In our ILI series, the rate of EV-positive specimens showed an upward trend from the end of May 2023, culminating at the end of June, coinciding with an increase in EV-positive hospital cases. The E-11 identified belonged to the D5 genogroup and the majority (83%) were closely associated with the novel E-11 variant, first identified in severe neonatal infections in France since 2022. E-11 was identified sporadically in community cases until February 2023, when it was also found in hospitalized cases with a range of clinical manifestations. All E-11 cases were children, with 14 out of 24 cases identified through hospital surveillance. Of these cases, 60% were neonates, and 71% had severe clinical manifestations., Conclusion: Baseline epidemiological data collected since 2021 through EV laboratory-based surveillance have rapidly tracked the E-11 variant since November 2022, alongside its transmission during the late spring of 2023., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The sensitivity of enteroviruses to disinfectants varies among genetically similar variants and coincides with amino acid changes in capsid proteins, although the effect of individual substitutions remains unknown. Here, we employed reverse genetics to investigate how amino acid substitutions in coxsackievirus B5 (CVB5) capsid proteins affect the virus' sensitivity to free chlorine and heat treatment. Of ten amino acid changes observed in CVB5 variants with free chlorine resistance, none significantly reduced the chlorine sensitivity, indicating a minor role of the capsid composition in chlorine sensitivity of CVB5. Conversely, a subset of these amino acid changes located at the C-terminal region of viral protein 1 led to reduced heat sensitivity. Cryo-electron microscopy revealed that these changes affect the assembly of intermediate viral states (altered and empty particles), suggesting that the mechanism for reduced heat sensitivity could be related to improved molecular packing of CVB5, resulting in greater stability or altered dynamics of virus uncoating during infection.

We report the presence of Echovirus 11 (E11) in wastewater in Sicily (Southern Italy), since August 2022. Overall, the 5.4 % of sewage samples (7/130) collected in 2022 were positives for E11 and then the percentage of E11-positive sewage samples reached the value of 27.27(18/66) in the first semester of 2023. Phylogenetic analysis of VP1 sequences showed for most E11-positive samples (16/25: 64 %) close genetic correlation (98.4-99.4 % nucleotide identity) to E11 lineage 1 strains involved in recently reported severe neonatal infections., Competing Interests: Declaration of competing interest Giovanni Maurizio Giammanco reports financial support was provided by AOUP Paolo Giaccone University Hospital Palermo. If there are other authors, they 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 B.V. All rights reserved.)

Enteroviruses cause a wide range of neurological illnesses such as encephalitis, meningitis, and acute flaccid paralysis. Two types of enteroviruses, echovirus E4 and E9, have recently been detected in South Africa and are known to be associated with meningitis and encephalitis. The objective of this study was to characterize enterovirus strains detected in cerebrospinal fluid specimens of hospitalized patients in the private and public sector to identify genotypes associated with meningitis and encephalitis. From January 2019 to June 2021 enterovirus positive nucleic acid samples were obtained from a private (n = 116) and a public sector (n = 101) laboratory. These enteroviruses were typed using a nested set of primers targeting the VP1 region of the enterovirus genome, followed by Sanger sequencing and BLASTn analysis. Forty-two percent (91/217) of the strains could be genotyped. Enterovirus B species was the major species detected in 95% (86/91) of the specimens, followed by species C in 3% (3/91) and species A in 2% (2/91) of the specimens. Echovirus E4 and E9 were the two major types identified in this study and were detected in 70% (64/91) and in 10% (9/91) of specimens, respectively. Echovirus E11 has previously been identified in sewage samples from South Africa, but this study is the first to report Echovirus E11 in cerebrospinal fluid specimens from South African patients. The genotypes identified during this study are known to be associated with encephalitis and meningitis. The predominant detection of echovirus E4 followed by E9 corresponds with other studies conducted in South Africa., (© 2024 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Aims: Enteroviruses are significant human pathogens associated with a range of mild to severe diseases. This study aims to understand the diversity and genetic characterization of enteroviruses circulated in southwest China's border cities by using environmental surveillance., Methods and Results: A total of 96 sewage samples were collected in three border cities and a port located in Yunnan Province, China from July 2020 to June 2022. After cell culture and VP1 sequencing, a total of 590 enterovirus isolates were identified, belonging to 21 types. All PV strains were Sabin-like with ≤6 nucleotide mutations in the VP1 coding region. Echovirus 6, echovirus 21 (a rare serotype in previous studies), and coxsackievirus B5 were the predominant serotypes, which accounted for 21.19%, 18.31%, and 13.39% of the total isolates, respectively. The prevalence of the common serotypes varied across different border cities and periods. Phylogenetic analysis revealed the presence of multiple evolutionary lineages for E21, E6, and E30, some of which formed distinct branches., Conclusions: High diversity of enteroviruses and distinct lineages of predominant serotypes circulated in southwest China's border cities., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Coxsackievirus B3 (CVB3) is known to cause acute myocarditis and pancreatitis in humans. We investigated the microRNAs (miRNAs) that can potentially govern the viral life cycle by binding to the untranslated regions (UTRs) of CVB3 RNA. MicroRNA-22-3p was short-listed, as its potential binding site overlapped with the region crucial for recruiting internal ribosome entry site trans -acting factors (ITAFs) and ribosomes. We demonstrate that miR-22-3p binds CVB3 5' UTR, hinders recruitment of key ITAFs on viral mRNA, disrupts the spatial structure required for ribosome recruitment, and ultimately blocks translation. Likewise, cells lacking miR-22-3p exhibited heightened CVB3 infection compared to wild type, confirming its role in controlling infection. Interestingly, miR-22-3p level was found to be increased at 4 hours post-infection, potentially due to the accumulation of viral 2A protease in the early phase of infection. 2A pro enhances the miR-22-3p level to dislodge the ITAFs from the SD-like sequence, rendering the viral RNA accessible for binding of replication factors to switch to replication. Furthermore, one of the cellular targets of miR-22-3p, protocadherin-1 (PCDH1), was significantly downregulated during CVB3 infection. Partial silencing of PCDH1 reduced viral replication, demonstrating its proviral role. Interestingly, upon CVB3 infection in mice, miR-22-3p level was found to be downregulated only in the small intestine, the primary target organ, indicating its possible role in influencing tissue tropism. It appears miR-22-3p plays a dual role during infection by binding viral RNA to aid its life cycle as a viral strategy and by targeting a proviral protein to restrict viral replication as a host response.IMPORTANCECVB3 infection is associated with the development of end-stage heart diseases. Lack of effective anti-viral treatments and vaccines for CVB3 necessitates comprehensive understanding of the molecular players during CVB3 infection. miRNAs have emerged as promising targets for anti-viral strategies. Here, we demonstrate that miR-22-3p binds to 5' UTR and inhibits viral RNA translation at the later stage of infection to promote viral RNA replication. Conversely, as host response, it targets PCDH1, a proviral factor, to discourage viral propagation. miR-22-3p also influences CVB3 tissue tropism. Deciphering the multifaced role of miR-22-3p during CVB3 infection unravels the necessary molecular insights, which can be exploited for novel intervening strategies to curb infection and restrict viral pathogenesis., Competing Interests: The authors declare no conflict of interest.

Echovirus 25 (E25), a member of the Enterovirus B (EV-B) species, can cause aseptic meningitis (AM), viral meningitis (VM), and acute flaccid paralysis (AFP). However, systematic studies on the molecular epidemiology of E25, especially those concerning its evolution and recombination, are lacking. In this study, 18 strains of E25, isolated from seven provinces of China between 2009 and 2018, were collected based on the Chinese hand, foot, and mouth disease (HFMD) surveillance network, and 95 sequences downloaded from GenBank were also screened. Based on the phylogenetic analysis of 113 full-length VP1 sequences worldwide, globally occurring E25 strains were classified into 9 genotypes (A-I), and genotype F was the dominant genotype in the Chinese mainland. The average nucleotide substitution rate of E25 was 6.08 × 10 -3 substitutions/site/year, and six important transmission routes were identified worldwide. Seventeen recombination patterns were determined, of which genotype F can be divided into 9 recombination patterns. A positive selector site was found in the capsid protein region of genotype F. Recombination analysis and pressure selection analysis for genotype F showed multiple recombination patterns and evolution characteristics, which may be responsible for it being the dominant genotype in the Chinese mainland. This study provides a theoretical basis for the subsequent prevention and control of E25., (© 2024. The Author(s).)

Breast cancer's immunosuppressive environment hinders effective immunotherapy, but oncolytic viruses hold promise for addressing this challenge by targeting tumor cells and altering the microenvironment. Yet, neutralizing antibodies and immune clearance impede their clinical utility. This study explored microRNA-modified coxsackievirus B3 (miR-CVB3), an innovative oncolytic virus, and its potential in breast cancer treatment. It investigated miR-CVB3's impact on immune-related proteins and utilized exosomes as both protective shields and delivery carriers. Results demonstrated miR-CVB3's capacity to reshape immune-related protein profiles toward a more immunostimulatory state and enhance exosome-mediated immune cell activation. Notably, cancer cell-released exosomes encapsulating miR-CVB3 (ExomiR-CVB3) maintained its antitumor cytotoxicity and bolstered its immunostimulatory effects. Moreover, ExomiR-CVB3 shielded miR-CVB3 from neutralizing antibodies and rapid immune clearance when it was systemically administered. Building on these findings, ExomiR-CVB3 was engineered with the AS1411 aptamer and doxorubicin (ExomiR-CVB3/DoxApt), enhancing therapeutic efficacy. This notable approach, combining genomic modification, aptamer surface decoration, and doxorubicin addition, demonstrated safe delivery of CVB3 to cancer cells. Comprehensive in vitro and in vivo analyses revealed selective breast cancer cell targeting, cell death induction, and significant immune cell infiltration within the tumor microenvironment while sparing healthy organs. In summary, this study highlights ExomiR-CVB3/DoxApt as a pioneering breast cancer treatment strategy adaptable for diverse cancer types, offering a potent and versatile approach to reshaping cancer immunotherapy.

Evidence is emerging on the roles of long noncoding RNAs (lncRNAs) as regulatory factors in a variety of viral infection processes, but the mechanisms underlying their functions in coxsackievirus group B type3 (CVB3)-induced acute viral myocarditis have not been explicitly delineated. We previously demonstrated that CVB3 infection decreases miRNA-21 expression; however, lncRNAs that regulate the miRNA-21-dependent CVB3 disease process have yet to be identified. To evaluate lncRNAs upstream of miRNA-21, differentially expressed lncRNAs in CVB3-infected mouse hearts were identified by microarray analysis and lncRNA/miRNA-21 interactions were predicted bioinformatically. MEG3 was identified as a candidate miRNA-21-interacting lncRNA upregulated in CVB3-infected mouse hearts. MEG3 expression was verified to be upregulated in HeLa cells 48 h post CVB3 infection and to act as a competitive endogenous RNA of miRNA-21. MEG3 knockdown resulted in the upregulation of miRNA-21, which inhibited CVB3 replication by attenuating P38-MAPK signaling in vitro and in vivo. Knockdown of MEG3 expression before CVB3 infection inhibited viral replication in mouse hearts and alleviated cardiac injury, which improved survival. Furthermore, the knockdown of CREB5, which was predicted bioinformatically to function upstream of MEG3, was demonstrated to decrease MEG3 expression and CVB3 viral replication. This study identifies the function of the lncRNA MEG3/miRNA-21/P38 MAPK axis in the process of CVB3 replication, for which CREB5 could serve as an upstream modulator., Competing Interests: Declaration of Competing Interest All authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Enteroviruses (EVs), single-stranded, positive-sense RNA viruses, can be classified into four species (A-D), which have previously been linked to a diverse range of disease manifestations and infections affecting the central nervous system. In the Enterovirus species B (EV-B), Echovirus type 11 (E11) has been observed to occasionally circulate in Taiwan, which was responsible for an epidemic of enterovirus infections in 2018. Here, 48 clinical specimens isolated in 2003, 2004, 2009, and 2018 were collected for the high-throughput sequencing. Notably, we identified 2018 Taiwanese strains having potential recombinations in the 3D gene, as well as one 2003 strain having a double recombination with E6 and Coxsackievirus B5 in the P2 and P3 regions, respectively. Additionally, one amino acid signature mutated from the Histidine (H) in throat swab specimens to the Tyrosine (Y) in cerebral spinal fluid specimens was detected at position 1496 (or 57) of the genomic coordinate (or 3A gene) to further demonstrate intra-host evolution in different organs. In conclusion, this study identifies potential intertypic recombination events and an intra-host signature mutation in E11 strains, isolated during a 2018 neurological disease outbreak in Taiwan, contributing to our understanding of its evolution and pathogenesis., (© 2023 Wiley Periodicals LLC.)

Importance: This study is the first report of echovirus 5 (E5) associated with severe acute respiratory infection and obtained the first E5 whole-genome sequence in China. Combined with the sequences available in the GenBank database, the first genotyping, phylogenetic characteristics, recombination, and genetic evolutionary analysis of E5 was performed in this study. Our findings providing valuable information on global E5 molecular epidemiology., Competing Interests: The authors declare no conflict of interest.

Viral myocarditis is an inflammatory disease of the myocardium, often leads to cardiac dysfunction and death. PARKIN (PRKN) and PINK1, well known as Parkinson's disease-associated genes, have been reported to be involved in innate immunity and mitochondrial damage control. Therefore, we investigated the role of parkin and PINK1 in coxsackievirus B3 (CVB3)-induced viral myocarditis because the etiology of myocarditis is related to abnormal immune response to viral infection and mitochondrial damage. After viral infection, the survival was significantly lower and myocardial damage was more severe in parkin knockout (KO) and PINK1 KO mice compared to wild-type (WT) mice. Parkin KO and PINK1 KO showed defective immune cell recruitment and impaired production of antiviral cytokines such as interferon-gamma, allowing increased viral replication. In addition, parkin KO and PINK1 KO mice were more susceptible to CVB3-induced mitochondrial damage than WT mice, resulting in susceptibility to viral-induced cardiac damage. Finally, using publicly available RNA-seq data, we found that pathogenic mutants of the PRKN gene are more common in patients with dilated cardiomyopathy and myocarditis than in controls or the general population. This study will help elucidate the molecular mechanism of CVB3-induced viral myocarditis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Long noncoding RNAs (lncRNAs) modulate many aspects of biological and pathological processes. Recent studies have shown that host lncRNAs participate in the antiviral immune response, but functional lncRNAs in coxsackievirus B5 (CVB5) infection remain unknown. Here, we identified a novel cytoplasmic lncRNA, LINC1392, which was highly inducible in CVB5 infected RD cells in a time- and dose-dependent manner, and also can be induced by the viral RNA and IFN-β. Further investigation showed that LINC1392 promoted several important interferon-stimulated genes (ISGs) expression, including IFIT1, IFIT2, and IFITM3 by activating MDA5, thereby inhibiting the replication of CVB5 in vitro. Mechanistically, LINC1392 bound to ELAV like RNA binding protein 1 (ELAVL1) and blocked ELAVL1 interaction with MDA5. Functional study revealed that the 245-835 ​nt locus of LINC1392 exerted the antiviral effect and was also an important site for ELAVL1 binding. In mice, LINC1392 could inhibit CVB5 replication and alleviated the histopathological lesions of intestinal and brain tissues induced by viral infection. Our findings collectively reveal that the novel LINC1392 acts as a positive regulator in the IFN-I signaling pathway against CVB5 infection. Elucidating the underlying mechanisms on how lncRNA regulats the host innate immunity response towards CVB5 infection will lay the foundation for antiviral drug research., (Copyright © 2023 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Appendix has a distinct abundance of lymphatic cells and serves as a reservoir of microbiota which helps to replenish the large intestine with healthy flora. And it is the primary site of IgA induction, which shapes the composition of the intestinal microbiota. Recent population-based cohort studies report that appendectomy is associated with an increased risk of acute myocardial infarction and ischemic heart disease. Here, whether appendectomy has an effect on the occurrence and development of coxsackievirus B3 (CVB3)-induced viral myocarditis is studied. 10 3 TCID 50 CVB3 was inoculated i.p. into appendectomized and sham-operated mice. RNA levels of viral load and pro-inflammatory cytokines in the hearts and the intestine were detected by RT-PCR. Compared to sham-operated mice, appendectomized mice exhibited attenuated cardiac inflammation and improved cardiac function, which is associated with a systemic reduced viral load. Appendectomized mice also displayed a reduction in cardiac neutrophil and macrophage infiltration and pro-inflammatory cytokine production. Mechanistically, we found that CVB3 induced an early and potent IL-10 production in the cecal patch at 2 days post infection. Appendectomy significantly decreased intestinal IL-10 and IL-10 + CD4 + Treg frequency which led to a marked increase in intestinal (primary entry site for CVB3) anti-viral IFN-γ + CD4 + T and IFN-γ + CD8 + T response and viral restriction, eventually resulting in improved myocarditis. Our results suggest that appendix modulates cardiac infection and inflammation through regulating intestinal IL-10 + Treg response.

Background: Echovirus 30 is prone to cause hand-foot-and-mouth disease in infants and children. However, molecular epidemiologic information on the spread of E30 in southwestern China remains limited. In this study, we determined and analyzed the whole genomic sequences of E30 strains isolated from the stools of patients with hand-foot-and-mouth disease in Yunnan Province, China, in 2019., Methods: E30 isolates were obtained from fecal samples of HFMD patients. The whole genomes were sequenced by segmented PCR and analyzed for phylogeny, mutation and recombination. MEGA and DNAStar were used to align the present isolates with the reference strains. The VP1 sequence of the isolates were analyzed for selection pressure using datamonkey server., Results: The complete genome sequences of four E30 were obtained from this virus isolation. Significant homologous recombination signals in the P2-3'UTR region were found in all four isolates with other serotypes. Phylogenetic analysis showed that the four E30 isolates belonged to lineage H. Comparison of the VP1 sequences of these four isolates with other E30 reference strains using three selection pressure analysis models FUBAR, FEL, and MEME, revealed a positive selection site at 133rd position., Conclusions: This study extends the whole genome sequence of E30 in GenBank, in which mutations and recombinations have driven the evolution of E30 and further improved and enriched the genetic characteristics of E30, providing fundamental data for the prevention and control of diseases caused by E30. Furthermore, we demonstrated the value of continuous and extensive surveillance of enterovirus serotypes other than the major HFMD-causing viruses., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Coxsackievirus B3 (CVB3) is one of the major pathogens of viral myocarditis, lacking specific anti-virus therapeutic options. Increasing evidence has shown an important involvement of the miR-17-92 cluster both in virus infection and cardiovascular development and diseases, while its role in CVB3-induced viral myocarditis remains unclear. In this study, we found that miR-19a and miR-19b were significantly up-regulated in heart tissues of CVB3-infected mice and exerted a significant facilitatory impact on CVB3 biosynthesis and replication, with a more pronounced effect observed in miR-19b, by targeting the encoding region of viral RNA-dependent RNA polymerase 3D (RdRp, 3D pol ) to increase viral genomic RNA stability. The virus-promoting effects were nullified by the synonymous mutations in the viral 3D pol -encoding region, which corresponded to the seed sequence shared by miR-19a and miR-19b. In parallel, treatment with miR-19b antagomir not only resulted in a noteworthy suppression of CVB3 replication and infection in infected cells, but also demonstrated a significant reduction in the cardiac viral load of CVB3-infected mice, resulting in a considerable alleviation of myocarditis. Collectively, our study showed that CVB3-induced cardiac miR-19a/19b contributed to viral myocarditis via facilitating virus biosynthesis and replication, and targeting miR-19a/19b might represent a novel therapeutic target for CVB3-induced viral myocarditis., Competing Interests: Declaration of competing interest The authors declared no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Enteroviruses are a common cause of seasonal childhood infections. The vast majority of enterovirus infections are mild and self-limiting, although neonates can sometimes develop severe disease. Myocarditis is a rare complication of enterovirus infection. Between June 2022 and April 2023, twenty cases of severe neonatal enteroviral myocarditis caused by coxsackie B viruses were reported in the United Kingdom. Sixteen required critical care support and two died. Enterovirus PCR on whole blood was the most sensitive diagnostic test. We describe the initial public health investigation into this cluster and aim to raise awareness among paediatricians, laboratories and public health specialists.

Echoviruses belong to the genus Enterovirus in the Picornaviridae family, forming a large group of Enterovirus B (EV-B) within the Enteroviruses. Previously, Echoviruses were classified based on the coding sequence of VP1. In this study, we performed a reliable phylogenetic classification of 277 sequences isolated from 1992 to 2019 based on the full-length genomes of Echovirus. In this report, phylogenetic, phylogeographic, recombination, and amino acid variability landscape analyses were performed to reveal the evolutional characteristics of Echovirus worldwide. Echoviruses were clustered into nine major clades, e.g., G1-G9. Phylogeographic analysis showed that branches G2-G9 were linked to common strains, while the branch G1 was only linked to G5. In contrast, strains E12, E14, and E16 clustered separately from their G3 and G7 clades respectively, and became a separate branch. In addition, we identified a total of 93 recombination events, where most of the events occurred within the VP1-VP4 coding regions. Analysis of amino acid variation showed high variability in the a positions of VP2, VP1, and VP3. This study updates the phylogenetic and phylogeographic information of Echovirus and indicates that extensive recombination and significant amino acid variation in the capsid proteins drove the emergence of new strains., (© 2023. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Enterovirus B (EVB) is a common species of enterovirus, mainly consisting of Echovirus (Echo) and Coxsackievirus B (CVB). The population is generally susceptible to EVB, especially among children. Since the 21st century, EVB has been widely prevalent worldwide, and can cause serious diseases, such as viral meningitis, myocarditis, and neonatal sepsis. By using cryo-electron microscopy, the three-dimensional (3D) structures of EVB and their uncoating receptors (FcRn and CAR) have been determined, laying the foundation for the study of viral pathogenesis and therapeutic antibodies. A limited number of epitopes bound to neutralizing antibodies have also been determined. It is unclear whether additional epitopes are present or whether epitope mutations play a key role in molecular evolutionary history and epidemics, as in influenza and SARS-CoV-2. In the current study, the conformational epitopes of six representative EVB serotypes (E6, E11, E30, CVB1, CVB3 and CVB5) were systematically predicted by bioinformatics-based epitope prediction algorithm. We found that their epitopes were distributed into three clusters, where the VP1 BC loop, C-terminus and VP2 EF loop were the main regions of EVB epitopes. Among them, the VP1 BC loop and VP2 EF loop may be the key epitope regions that determined the use of the uncoating receptors. Further molecular evolution analysis based on the VP1 and genome sequences showed that the VP1 C-terminus and VP2 EF loop, as well as a potential "breathing epitope" VP1 N-terminus, were common mutation hotspot regions, suggesting that the emergence of evolutionary clades was driven by epitope mutations. Finally, footprints showed mutations were located on or near epitopes, while mutations on the receptor binding sites were rare. This suggested that EVB promotes viral epidemics by breaking the immune barrier through epitope mutations, but the mutations avoided the receptor binding sites. The bioinformatics study of EVB epitopes may provide important information for the monitoring and early warning of EVB epidemics and developing therapeutic antibodies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wang 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.)

Coxsackievirus belongs to the Picornaviridae family and is one of the major pathogens that cause hand, foot and mouth disease (HFMD) in infants and children with potential serious complications and even deaths. The pathogenesis of this virus is not fully elucidated and no vaccine or antiviral drug has been approved. In this study, a full-length infectious cDNA clone of coxsackievirus B5 virus was assembled and the recombinant virus displayed similar growth kinetics and ability to cause cytopathic effects as the parental virus. Luciferase reporter was then incorporated to generate both full-length and subgenomic replicon (SGR) reporter viruses. The full-length reporter virus is suitable for high-throughput antiviral screening, while the SGR is a useful tool to study viral-host interactions. More importantly, the full-length reporter virus has also been shown to infect the suckling mouse model and the reporter gene could be detected using an in vivo imaging system, thus providing a powerful tool to track viruses in vivo. In summary, we have generated coxsackievirus B5 reporter viruses and provided unique tools for studying virus-host interactions in vitro and in vivo as well as for high-throughput screenings (HTS) to identify novel antivirals., (Copyright © 2023 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Picornaviruses infect a wide variety of cell types in vitro , with rapid replication kinetics and pronounced cytopathic effect. Coxsackievirus B3 (CVB3) can also establish a persistent infection in vivo that can lead to pathology, including dilated cardiomyopathy and myocarditis. One model system to study persistent infection is the pancreatic ductal cell line PANC-1, which CVB3 infects and is maintained indefinitely. We have characterized this model for CVB3 infection to study persistent infection for over 6 months. We find that CVB3 rapidly replicates within PANC-1 cells without robust cytopathic effect, and after 1 month in culture, titers stabilize. We find that infection does not significantly affect cellular viability. Persistent virus reverts to lytic infection when transferred to Huh7 or Vero cells. We find that persistent CVB3 adapts to PANC-1 cells via mutation of its capsid proteins and, curiously, the viral polymerase (3Dpol) to generate a high-fidelity polymerase. Persistent infection is associated with reduced cleavage of eIF4G, reduced plaque size, and decreasing particle infectivity. We further find that polyamine metabolism is altered in persistently infected cells, with the rate-limiting enzyme ornithine decarboxylase (ODC1) reduced in translation. We further find that targeting polyamine synthesis reduces persistent infection without affecting the viability of the PANC-1 cells. Finally, we find that viral fidelity is essential to maintaining CVB3 infection, and targeting viral fidelity reduces persistent virus infection. Together, these data highlight a novel role for polyamines and fidelity in persistent CVB3 infection and suggest avenues for therapeutic development to target persistent infection. IMPORTANCE Enteroviruses are significant human pathogens that can cause severe disease, including cardiomyopathies. Viruses like coxsackievirus B3 (CVB3) can cause tissue damage by lytically infecting cells; however, CVB3 can also persistently infect, which has been associated with several pathologies. Studying persistent infection in vitro is challenging, as CVB3 lytically infects most cellular model systems. Here, we show that CVB3 establishes persistent infection in pancreatic ductal cells in vitro , similar to prior studies on other coxsackieviruses. We also show that this infection results in adaptation of the virus to these cells, as well as changes to cellular metabolism of polyamines., Competing Interests: The authors declare no conflict of interest.

We report nine severe neonatal infections caused by a new variant of echovirus 11. All were male, eight were twins. At illness onset, they were 3-5 days-old and had severe sepsis and liver failure. This new variant, detected in France since April 2022, is still circulating and has caused more fatal neonatal enterovirus infections in 2022 and 2023 (8/496; 1.6%, seven associated with echovirus 11) compared with 2016 to 2021 (7/1,774; 0.4%). National and international alerts are warranted.

Echovirus 11 (E11) has recently been associated with a series of nine neonatal cases of severe hepatitis in France. Here, we present severe hepatitis caused by E11 in a pair of twins. In one of the neonates, the clinical picture evolved to fulminant hepatitis. The E11 genome showed 99% nucleotide identity with E11 strains reported in the cases in France. Rapid genome characterisation using next generation sequencing is essential to identify new and more pathogenetic variants.

The genus Enterovirus (family Picornaviridae) contains numerous viruses, most of which have been identified in humans. Among them, the three serotypes of poliovirus, coxsackieviruses A and B, echoviruses, rhinoviruses and other enteroviruses (EVs) responsible in humans for a wide spectrum of clinical manifestations. There are also 60 identified EVs in different mammals. Some have been found in both humans and animals, demonstrating the possibility of zoonotic transmission of certain EVs. Compared to human EVs, genetic and epidemiological data for animal EVs are scarce. However, the detection of EV in various species of mammals and their presence on all continents suggest that the number of EV still to be discovered is very important. Some EVs found in animals have characteristics never seen in human EVs. Furthermore, the unique phylogenetic relationships observed between animal EVs raise interesting questions about the rules that govern the evolution of these viruses. The aim of this review is to present the salient data on animal EVs and to highlight the questions they raise.